protein-kinase inhibitors as potential leishmanicidal drugs
TRANSCRIPT
UNIVERSIDAD COMPLUTENSE DE MADRID FACULTAD DE FARMACIA
TESIS DOCTORAL
Protein-kinase inhibitors as potential leishmanicidal drugs
Inhibidores de proteín-quinasas como potenciales fármacos leishmanicidas
MEMORIA PARA OPTAR AL GRADO DE DOCTOR
PRESENTADA POR
Paula Martínez de Iturrate Sanz
Directores
Luis Ignacio Rivas López Carmen Belén Gil Ayuso-Gontán
Madrid
© Paula Martínez de Iturrate Sanz, 2021
UNIVERSIDAD COMPLUTENSE DE MADRID
FACULTY OF PHARMACY/FACULTAD DE FARMACIA
DEPARMENT OF MICROBIOLOGY II/DEPARTAMENTO DE
MICROBIOLOGÍA II
DOCTORAL THESIS/TESIS DOCTORAL
PROTEIN-KINASE INHIBITORS AS POTENTIAL
LEISHMANICIDAL DRUGS
INHIBIDORES DE PROTEÍN-QUINASAS COMO
POTENCIALES FÁRMACOS LEISHMANICIDAS
PhD DISSERTATION BY/ MEMORIA PARA OPTAR AL
GRADO DE DOCTOR PRESENTADA POR
Paula Martínez de Iturrate Sanz
SUPERVISORS/DIRECTORES:
Dr. Luis Ignacio Rivas López
Dr. Carmen Belén Gil Ayuso-Gontán
Madrid, 2020
Agradecimientos
Estos años de tesis han supuesto una experiencia vital que ha influido mucho en mi
desarrollo personal. Una experiencia que ha formado parte de un puente entre una etapa más
estudiantil e ingenua, y otra más madura y encarada con la vida adulta. Un trabajo que he
podido llevar a cabo gracias a la financiación del Fondo de Garantía Juvenil y a la
financiación del proyecto SAF2015-65740-R (MINECO). En las siguientes líneas, me
gustaría dedicar mis agradecimientos a las distintas personas que me han acompañado en
este viaje.
Me gustaría agradecer en primer lugar a mis directores de tesis, Luis Rivas y Carmen
Gil, por haberme brindado esta oportunidad y haberme dirigido a lo largo del camino.
Muchas gracias por todo lo que me habéis enseñado, y por vuestra paciencia y continua
motivación.
A Mariángeles y Montse quiero agradecerles la figura mentora que fueron para mí
cuando llegué al laboratorio, enseñándome con gran paciencia lo primero que aprendí sobre
Leishmania y técnicas de biología molecular. A Montse quiero agradecerle además su gran
compañerismo, y el enorme apoyo que supuso para mí durante su último año en el
laboratorio. En ella encontré una mentora, compañera y amiga. De la misma manera quiero
darle las gracias a Lorena, por haber sido un pilar clave de apoyo tanto personal como laboral
cuando más lo necesitaba. Tampoco quiero olvidar a aquellas personas que he conocido a su
paso por el laboratorio en estos años, como Sara, Sergio, Javi, Yulieth, Vicky, Iñaqui y
especialmente Cathy, en quien he encontrado una amiga para toda la vida. A todos vosotros
y más, por vuestro compañerismo, por vuestra amistad… muchas gracias.
También quiero agradecerles a aquellas personas que, quizá no lo saben, pero se
convirtieron en un pequeño refugio, una bocanada de aire, especialmente durante mi último
año de trabajo experimental. Por un lado, a Ana María y Concha, así como Gloria, Jose,
Adrián, Ana y Sofi (quien espero que me vuelva a llevar a clases de swing en un futuro tan
cercano como sea posible), con quienes he compartido numerosas conversaciones en la
comida. Y por otro lado, a “los del voley”, por esos buenos ratos de desconexión y deporte,
y en especial a Guille y Ana, que siempre han tenido palabras de ánimos cuando me han
encontrado en un momento bajo. Gracias.
A Ana Martínez, porque gracias a ella tuve la oportunidad de trabajar con Carmen Gil
y Luis Rivas, y a su grupo por haber generado la biblioteca química que he podido emplear
en este trabajo. Especialmente quiero agradecerle a Víctor Sebastián-Pérez su esencial papel
en la pre-selección virtual de los compuestos y sus estudios computacionales, así como en
la síntesis de los mismos. Muchas gracias.
A mis amigos Nuria y Adrián por haber estado siempre a mi lado, por aguantar esos
audios de Whatsapp interminables y mandarme otros tantos de vuelta, porque nos encanta
irnos por las ramas y sentir que estamos ahí, aun en la distancia. Al “grupo de amigos de
Madrid”, aunque algunos sean de otras ciudades e incluso estén en otros países. Por esos
días de “pizzina”, esos concursos de tapas, esas noches de cine y por más picnics en Debod
(cuando el Sr. Covid lo permita…). Por las risas, las lágrimas, las filosofadas, las tonterías.
Muchas gracias.
A toda mi familia, porque pase lo que pase siempre han estado ahí cuando lo he
necesitado. Gracias a mi prima Ana, porque fue ella quien pensó en mí cuando se enteró de
la convocatoria del CSIC de Garantía Juvenil gracias a la cual tuve mi primer contacto con
el mundo laboral, aquí en el CIB, haciendo lo que más me gusta. Gracias a mi Yaya por
llevarnos siempre a todos en su mente y corazón, sin importar las distancias. Gracias a mis
padres, Mª Pilar y Jesús, por haberme apoyado en todo lo que he necesitado, cada uno a su
propia manera, siempre a mi lado. Gracias a mi hermano Adrián, porque su apoyo fraternal
cala con más fuerza que ningún otro. Gracias. Os quiero infinito.
Finalmente, a mi pareja, Curro. Gracias por estar en lo bueno y en lo malo. Por tu
paciencia y tu comprensión. Por preocuparte. Por animarme. Por apoyarme. Te quiero.
A todos vosotros, una vez más, muchas gracias.
Index
Abbreviations ....................................................................................................................... 1
Resumen ............................................................................................................................... 7
Summary ............................................................................................................................ 11
1. Introduction ................................................................................................................... 17
1.1. Leishmaniasis ............................................................................................................. 17
1.1.1. Taxonomy of Leishmania ....................................................................................... 18
1.1.2. Clinical manifestations of leishmaniasis .................................................................. 20
1.1.3. Life cycle of Leishmania ........................................................................................ 21
1.1.4. Control of leishmaniasis ......................................................................................... 23
1.1.4.1. Prevention of leishmaniasis infection: vaccines, vector and reservoir control ..... 23
1.1.4.2. Diagnosis ....................................................................................................... 24
1.1.5. Biology of Leishmania ........................................................................................... 25
1.1.5.1. Metabolic characteristics of Leishmania .......................................................... 25
1.1.5.1.1. Carbon metabolism ................................................................................... 25
1.1.5.1.2. Oxidative phosphorylation ......................................................................... 27
1.1.5.1.3. Other metabolic peculiarities: Folate synthesis, ergosterol and trypanothione.... 29
1.1.6. Signal transduction in Leishmania .......................................................................... 29
1.1.6.1. The kinome of Leishmania ............................................................................... 31
1.1.7. State of the art of the chemotherapy for leishmaniasis .............................................. 39
2. Objectives ...................................................................................................................... 47
3. Materials and Methods ................................................................................................. 51
3.1. Chemical compounds, media, reagents and laboratory equipment ............................ 51
3.2. Cell cultures and cell harvesting ................................................................................ 51
3.3. Leishmanicidal activity of the compounds on axenic parasites ................................. 52
3.4. Macrophage cytotoxicity of leishmanicidal compounds ........................................... 53
3.5. Leishmanicidal activity against intracellular amastigotes ......................................... 54
3.6. Extraction and purification of LdGSK-3s .................................................................. 54
3.7. Measurement of LdGSK-3s activity .......................................................................... 56
3.8. Bioenergetic assays .................................................................................................... 57
3.8.1. In vivo monitoring of intracellular ATP levels ......................................................... 57
3.8.2. Assessment of plasma membrane permeabilization .................................................. 57
3.8.3. Mitochondrial membrane potential (ΔΨm) of Leishmania ......................................... 58
3.8.4. Evaluation of the O2 consumption rate of the parasite .............................................. 58
3.8.5. Assessment of programed cell death induced by the compounds ............................... 59
4. Results and discussion .................................................................................................. 63
4.1. LdGSK-3s extraction and purification ....................................................................... 65
4.2. Selection of the different compounds tested .............................................................. 70
4.3. Assessment of the biological and inhibitory activities of the selected compounds ... 71
4.3.1. Reported inhibitors of human protein kinases (Sets 1 and 1.1) .................................. 72
4.3.2. In silico inhibitors of LmjGSK-3s (Set 2), LmjCK1.2 (Set 3) and LmxMAPK4 (Set 4) ..
............................................................................................................................ 89
4.3.3. Leishmanicidal activity of LdGSK-3s inhibitors on intracellular amastigotes ........... 114
4.4. Off-target effects of protein kinase inhibitors .......................................................... 117
4.4.1. Energy metabolism of Leishmania as an off-target effect of PKIs ........................... 118
4.4.1.1. Variation of the intracellular levels of ATP in L. donovani promastigotes by the
leishmanicidal LdGSK-3s inhibitors .......................................................................... 119
4.4.1.2. Inhibition of the electrochemical potential of the Leishmania mitochondrion (ΔΨm)
................................................................................................................... 122
4.4.1.3. Inhibition of O2 consumption ......................................................................... 124
4.4.1.4. Identification of the PKI target inside the respiratory chain ............................ 127
4.4.1.5. Induction of programmed cell death in L. infantum promastigotes ................... 134
5. Conclusions .................................................................................................................. 139
Bibliography ..................................................................................................................... 143
Results dissemination ...................................................................................................... 167
Abbreviations
1
Abbreviations
α-GP α-glycerophosphate
αKG α-ketoglutarate
ΔΨm mitochondrial electrochemical potential
λEM excitation wavelength
λEX emission wavelength
5-Me-6-BIO 6-bromo-5-methylindirubin-3’-oxime
6-BIO 6-bromoindirubin-3’-oxime
Ac acetate
AC adenylate cyclase
AcCoA acetyl-CoA
ADP adenosine diphosphate
AGC kinase group that includes the protein kinase A, G and C
families
AIDS acquired immune deficiency syndrome
AIRK aurora kinase
ALM autoclaved-killed L. major vaccine prototype
AmB amphotericin B
AmBd amphotericin B deoxycholate
Asc ascorbate
ATP adenosine triphosphate
CAMK Ca2+/calmodulin-dependent kinase
CAMK1 Ca2+/calmodulin-dependent kinase 1
CAMKL CAMK-like kinase
cAMP cyclic adenosine monophosphate
cGMP cyclic guanosine monophosphate
CDK/CRK cyclin-dependent kinase/cdc2-related kinase
CDPK Ca2+-dependent protein kinase
Cit citrate
CK1 casein kinase 1
CK2 casein kinase 2
2
CL cutaneous leishmaniasis
CLK cell division control (CDC)-like kinase
CMGC kinase group that includes the CDK, MAPK, GSK and CLK
families
CoQ Coenzyme Q
CytC cytochrome c
DALY disability-adjusted life years
Dig digitonin
DMNPE D-luciferin D-Luciferin 1-(4,5-dimethoxy-2-nitrophenyl)ethyl ester
DNDi Drugs for Neglected Diseases initiative
DYRK dual-specificity tyrosine-regulated kinase
EDTA ethylenediaminetetraacetic acid
ePK eukaryotic protein kinase
FAD flavin adenine dinucleotide
FCCP carbonyl cyanide-p-trifluoromethoxyphenylhydrazone
FDA Food and Drug Administration
FFAA fatty acids
FRD fumarate reductase
G3P glyceraldehyde 3-phosphate
Glc6P glucose 6-phosphate
GALM gentamycin-attenuated L. major vaccine prototype
GPCR G-protein-coupled receptor
GS2 phosphoglycogen synthase peptide-2
GSK-3 glycogen-synthase kinase-3
HBSS Hank’s balanced salt solution
HePc hexadecylphosphocholine (miltefosine)
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
HIFCS heat inactivated fetal calf serum
HIV Human Immunodeficiency Virus
HMK halomethylketone
hPK human protein-kinase
HPLC high-performance liquid chromatography
Abbreviations
3
IC50 compound concentration that induces 50% inhibition
IC80 compound concentration that induces 80% inhibition
i.m. intramuscular
i.v. intravenously
IPTG isopropyl β-D-1-thiogalactopyranoside
ITDZ iminothiadiazole
kDNA kinetoplast
LAmB liposomal amphotericin B
LAMP loop-mediated isothermal amplification
LRRK2 leucine-rich repeat kinase 2
M199 medium 199
M199-HIFCS M199 supplemented with 20% HIFCS
MA meglumine antimoniate
Mal malate
MAPK mitogen-activated protein kinase
MAPKK mitogen-activated protein kinase kinase
MAPKKK mitogen-activated protein kinase kinase kinase
MAPKKKK mitogen-activated protein kinase kinase kinase kinase
MBC medicinal and biological chemistry
MCL mucocutaneous leishmaniasis
MPM murine peritoneal macrophages
MTT 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium
bromide
MW molecular weight
NAD nicotinamide adenine dinucleotide
NDR nuclear DBF2-related kinase
NEK never in mitosis gene A(NimA)-related kinase
OAA oxaloacetate
OD optical density
Omy oligomycin
PBS phosphate-buffered saline
PCR polymerase chain reaction
4
PDK1 phosphoinositide-dependent protein kinase 1
PEP phosphoenolpyruvate
PI propidium iodide
PK protein kinase
PKDL post-kala azar dermal leishmaniasis
PKA protein kinase A; cAMP-dependent protein kinase
PKB protein kinase B; Akt kinase
PKC protein kinase C
PKG protein kinase G; cGMP-dependent protein kinase
PKI protein kinase inhibitor
PLK polo-like kinase
PPP pentose phosphate pathway
psi pounds per square inch
Pyr pyruvate
RCK ROS-cross-hybridizing kinase
RGC Receptor guanylate cyclase group
RNS reactive nitrogen species
ROS reactive oxygen species
RPMI 1640 Roswell Park Memorial Insitute 1640 medium
RPMI-HIFCS RPMI 1640 medium supplemented with 10% HIFCS
RPMIØRED RPMI 1640 medium without red phenol
RPMIØRED-HIFCS RPMIØRED medium supplemented with 10% HIFCS
RSK ribosomal S6-kinase
SAR structure-activity relationship
SCoA succinyl-CoA
SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
SI selectivity index
SRPK SR-rich protein kinase
SSG sodium stibogluconate
STE kinase homolog of yeast sterile
STE7 kinase homolog of yeast sterile 7
STE11 kinase homolog of yeast sterile 11
Abbreviations
5
STE20 kinase homolog of yeast sterile 20
Succ succinate
TCA tricarboxylic acid cycle
TDZD thiadiazolidindione
TK tyrosine kinase
TKL tyrosine kinase-like
TMPD N,N,N',N'-tetramethyl-p-phenylenediamine
TPP target product profile
TTBK tau tubulin kinase
TX-100 Triton X-100
VL visceral leishmaniasis
WEE1 Wee1-like protein kinase 1
WHO World Health Organization
Resumen
7
Resumen
Título: Inhibidores de proteín-quinasas como potenciales fármacos leishmanicidas.
Introducción
La leishmaniasis es una enfermedad parasitaria de transmisión vectorial causada por
hasta 20 especies del género Leishmania, un protozoo con un ciclo de vida caracterizado por
tener dos formas morfológicas adaptadas a cada uno de sus dos hospedadores: el
promastigote (extracelular) en el vector invertebrado, y el amastigote (intracelular) en el
hospedador vertebrado. Aunque la transmisión de la leishmaniasis es principalmente
zoonótica, el ciclo antroponótico de la enfermedad tiene lugar en zonas endémicas con alta
densidad humana. Su prevalencia ha aumentado en las últimas décadas debido a diversos
factores incluyendo el aumento de viajes por todo el mundo y el aumento de la distribución
geográfica del vector flebotomo por el calentamiento global.
Aunque hay vacunas humanas en desarrollo, el tratamiento principal es la
quimioterapia. Sin embargo, los fármacos en uso presentan problemas de costes, toxicidad
y aparición de resistencias. En la búsqueda de nuevos tratamientos farmacológicos se están
llevando a cabo dos estrategias: desarrollo de tratamientos combinados con los actuales
fármacos disponibles, y descubrimiento de nuevos compuestos con potencial leishmanicida
mediante tres posibles acercamientos: reposicionamiento de fármacos, cribados fenotípicos
y cribados dirigidos a diana.
En Leishmania hay pocas dianas validadas como esenciales para el parásito, entre ellas
las proteín-quinasas GSK-3s, CK1.2 y MAPK4. Las proteín-quinasas conforman una de las
familias de proteínas más numerosas en la mayoría de organismos eucariotas, incluida
Leishmania, dada su participación en numerosos procesos celulares, muchos de ellos
esenciales. Por ello, los cribados enfocados en una proteín-quinasa validada son de gran
interés, especialmente si se combinan con un cribado fenotípico que aseguren su efectividad
en el parásito, y permitan la búsqueda de actividades leishmanicidas más allá de la diana
específica objeto del cribado.
8
Objetivos generales
El principal objetivo de esta tesis se ha centrado en el descubrimiento de nuevos
agentes leishmanicidas con un mecanismo de acción definido. Para ello, se han evaluado
distintos grupos de compuestos orgánicos de bajo peso molecular mediante la combinación
de un cribado enfocado en la inhibición de la isoforma corta de la GSK-3 de L. donovani
(LdGSK-3s) y un cribado fenotípico en promastigotes y amastigotes axénicos. Además, se
evaluó el efecto de los compuestos activos en distintos parámetros bioenergéticos para
definir su efecto en la homeostasis intracelular de parásito y explorar posibles dianas
adicionales.
Resultados
Tras expresar y purificar la GSK-3s de L. donovani a partir de E. coli BL21(D3)
transfectadas con un plásmido pET28a(+) con el gen de dicha proteín-quinasa, se
seleccionaron 4 grupos de compuestos (Sets 1 a 4). El Set 1 se compuso con inhibidores de
proteín-quinasas humanas, mientras que los Sets 2, 3 y 4 se originaron a partir de hits
obtenidos de cribados virtuales enfocados en la GSK-3s y la CK1.2 de L. major y la MAPK4
de L. mexicana, respectivamente. Adicionalmente, se formaron los Sets 1.1 y 2.1 con
compuestos seleccionados a partir de la optimización de los mejores compuestos activos de
los Sets 1 a 4.
En total fueron evaluados 136 compuestos, y se identificaron 14 hits con actividad
inhibidora en LdGSK-3s y actividad leishmanicida, pertenecientes a tres familias químicas
diferentes (tiadiazolidindionas, halometilcetonas y quinonas). De estos 14 compuestos, 5
mostraron un buen índice de selectividad (IS >10). Adicionalmente, 4 quinonas identificadas
(compuestos 72, 75, 76 y 80) son los primeros inhibidores específicos de la GSK-3s de
Leishmania, sin inhibición cruzada con la GSK-3β humana, su homólogo humano más
cercano.
Mediante el uso de herramientas computacionales, se identificó el modo de unión de
las tiadiazolidindionas (compuestos 1 y 2) y las halometilcetonas (compuestos 6, 28, 29, 32
y 33) activas en LdGSK-3s a través de unión covalente a la Cys169. Por otro lado, se evaluó
Resumen
9
la efectividad de dichos compuestos en macrófagos infectados con amastigotes, como
modelo más cercano al fisiológico. De los 14 hits, las halometilcetonas 6 y 32 y las quinonas
72, 76 y 79 disminuyeron significativamente la carga parasitaria de los macrófagos.
Finalmente, se analizó el efecto de los inhibidores de LdGSK-3s leishmanicidas en el
metabolismo energético de promastigotes de Leishmania. La halometilcetona 6 y las
quinonas 72, 75 y 76 produjeron colapso bioenergético en el parásito, con despolarización
de la membrana mitocondrial, inhibición de la respiración con disminución de los niveles
intracelulares de ATP, y desencadenando finalmente la apoptosis del parásito.
Específicamente, se identificó al complejo III de la cadena transportadora de electrones
como la diana de inhibición del compuesto 6, mientras que las quinonas 72, 75 y 76
mostraron un doble efecto desacoplador e inhibidor de la cadena respiratoria de Leishmania.
Conclusiones
La combinación de un cribado enfocado en la GSK-3s de Leishmania con un cribado
fenotípico en el parásito ha llevado a la identificación de nuevos compuestos de interés para
el desarrollo de nuevos fármacos leishmanicidas. Además, para algunos de estos compuestos
se ha identificado una diana adicional en la cadena respiratoria del parásito. Esta naturaleza
multi-diana es una característica ventajosa que reduce la probabilidad de que emerjan
resistencias causadas por mutaciones en la diana inicial.
Summary
11
Summary
Title: Protein-kinase inhibitors as potential leishmanicidal drugs.
Introduction
Leishmaniasis is a parasitic vector-borne disease caused by more than 20 species from
the genus Leishmania, a protozoan with two distinct morphological forms throughout its life
cycle adapted to each one of its two hosts: the promastigote (extracellular) in the invertebrate
vector, and the amastigote (intracellular) in the vertebrate host. Although it is mostly a
zoonotic disease, anthroponotic transmission occurs in endemic areas with high human
density. Moreover, its prevalence increased in the last years due to several factors including
an increase in worldwide travelling as well as an increase in the geographical distribution of
the phlebotomine vector due to global warming.
Despite the efforts to develop human vaccines, the main treatment against
leishmaniasis relies on chemotherapy. However, all the current clinical drugs available have
drawbacks due to toxicity, high-cost and/or resistance. Two approaches are leading the
search for new pharmacological treatments: combination therapy using current drugs
available, and searching for new compounds with a leishmanicidal potential through three
major strategies: drug repurposing, phenotypic screenings and target-based screenings.
Protein kinases GSK-3s, CK1.2 and MAPK4 are among the scarce validated targets in
Leishmania. Owing to their participation in many and diverse cellular processes, protein
kinases are one of the largest protein families in most eukaryotic organisms, including
Leishmania. Hence, target-based screenings focused on a validated protein kinase are of
great interest, especially if combined with a phenotypic screening to ensure effectiveness on
living parasites, as well as to uncover off-target effects.
12
Objectives
The main objective of this thesis was to find new leishmanicidal agents with a defined
mechanism of action. For this purpose, different groups of small-size organic compounds
have been evaluated by combination of a target-based approach on the inhibition of the short
isoform of the GSK-3 of L. donovani (LdGSK-3s), together with phenotypic screenings on
axenic promastigotes and amastigotes. Furthermore, the effect of active compounds on
different bioenergetic parameters was assessed as a subtle approach to gauge their effect on
the intracellular homeostasis of the parasite and explore feasible off-targets.
Results
The GSK-3s of L. donovani was successfully expressed and purified from E. coli
BL21(D3) transfected with a pET28a(+) plasmid encoding the gene for this protein kinase.
Then, 4 groups of compounds were gathered (Sets 1 to 4). Set 1 comprised human protein
kinase inhibitors, whereas Sets 2, 3 and 4 originated from virtual screenings focused on the
GSK-3s and CK1.2 of L. major, and the MAPK4 of L. mexicana, respectively. Additionally,
Sets 1.1 and 2.1 comprehended compounds selected as a refinement of the best active
compounds from Sets 1 to 4.
Among the 136 compounds evaluated, 14 leishmanicidal hits with inhibitory activity
on LdGSK-3s were identified. They belonged to three different chemical families with
different scaffolds (thiadiazolidindiones, halomethylketones and quinones). Out of these 14
hits, 5 showed a good selectivity index (SI >10). In addition, 4 quinones (compounds 72, 75,
76 and 80) are the first GSK-3 inhibitors of Leishmania, devoid of cross-inhibition on the
human GSK-3β, the closest human homologue for this kinase.
Through computational tools, the interactions of the thiadiazolidindiones (compounds
1 and 2) and halomethylketones (compounds 6, 28, 29, 32 and 33) with LdGSK-3s were
analysed. Formation of a covalent bond with Cys169 was identified as the binding mode of
these hits. Additionally, all 14 hits were evaluated on infected macrophages in order to assess
their effectivity against Leishmania in an intracellular model. Halomethylketones 6 and 32,
and quinones 72, 76 and 79 significantly reduced the parasite load of infected macrophages.
Summary
13
Finally, the effect of these leishmanicidal LdGSK-3s inhibitors on the energy
metabolism of Leishmania promastigotes was assessed. Halomethylketone 6 and quinones
72, 75 and 76 induced the bioenergetic collapse of the parasite, with mitochondrial
depolarization, inhibition of respiration and decrease of intracellular ATP levels, with
apoptosis ensuing. Their specific targets on the respiratory chain of Leishmania were
complex III for compound 6, while quinones 72, 75, and 76 showed both uncoupling and
inhibitory effects on the respiration of the parasite.
Conclusions
The combination of a target-based screening focused on the GSK-3s of Leishmania
with a phenotypic screening in the parasite has led to the identification of new compounds
with appealing activities for their further development as new leishmanicidal drugs.
Moreover, some of these compounds have an additional target in the respiratory chain of the
parasite. The multi-target nature of these compounds is an advantageous trait to confront a
feasible raise of resistance caused by mutation in a single target.
INTRODUCTION
Introduction
17
1. Introduction
1.1. Leishmaniasis
Leishmaniasis is a term that encompasses the group of vector-borne parasitic diseases
caused by more than 20 protozoan species belonging to the genus Leishmania. The parasite
is transmitted by phlebotomine sandflies, and leads to one of three major groups of
leishmaniasis with diverse clinical manifestations: cutaneous (CL), mucocutaneous (MCL),
and visceral leishmaniasis (VL) or kala-azar. It is mostly a zoonotic disease where feral and
domestic mammals act as the main reservoirs of the parasite, but anthroponotic transmission
of Leishmania can take place in areas of high human density.1,2 The World Health
Organization’s (WHO) latest report3 states that, as of 2018, leishmaniasis is endemic in 98
countries across Latin America, the Indian Subcontinent, Central and South America, as well
as South Sudan, Ethiopia, Kenya and European countries of the Mediterranean Region, with
sporadic outbreaks every 10-15 years due to the loss of herd immunity, which is especially
relevant in anthroponotic areas.4 Two of the latest and most relevant sporadic outbreaks in
Southern Europe took place in Fuenlabrada (Spain, 2012)5,6 and Modena (Italy, 1997-2016),7
with more than 400 and 35 immunocompetent patients affected respectively.
Like other poverty-associated diseases, leishmaniasis is one of the so-called Neglected
Tropical Diseases due to its impact on human health and poor economical investment for its
control and treatment. In fact, the highest prevalence of this disease occurs in low-income
populations from developing countries, where people suffer from hunger, poor living
conditions and faulty health facilities. On a global scale, over 1 billion people are at risk each
year, with an estimation of 700,000-1,000,000 new cases of leishmaniasis per year and
26,000-65,000 deaths annually.8 Furthermore, the economic impact of the disease is
estimated in approximately 1 million disability-adjusted life years (DALYs), this is,
1 million years of productive life lost due to disability.9 This number increases up to ca
18
2.2 million additional DALYs when major depressive disorders associated are included,
accounting for the psychosocial impact of facial mutilation or scarring on CL and MCL
patients.10 Moreover, the impact of leishmaniasis is worsened by co-morbidity with other
pathologies, including malnutrition and infections that compromise the immune system.11,12
Leishmania-HIV coinfection is especially relevant, as this virus targets macrophages and
CD4+ lymphocytes ―the axis of immunity against Leishmania13― with synergetic effects
on the outcome of both infections.14,15
In the last years, the prevalence of leishmaniasis has increased due to several factors
such as “imported leishmaniasis”, associated with the increase in worldwide travelling
(including animal reservoirs and undetected transport of vectors),16-18 as well as human
migrations such as those forced by war conflicts.19,20 It is also inevitably linked to global
warming, which increases the geographical distribution of sandfly species towards higher
latitudes,21,22 as well as loss of efficacy of current treatments due to resistance, side-effects
and/or high costs.23-25
There are several potential human vaccines against leishmaniasis under different
phases of clinical trials, but none are commercially available so far.26 Therefore, the fight
against the spread and persistence of Leishmania is, nowadays, almost exclusively
dependent on chemotherapy despite treatment failures and costs. Aiming to improve this
scenario, the WHO, big pharma companies, and other institutions including academia, are
currently collaborating to make their large collections of compounds available to the
scientific community, as well as to supply current drugs at cost for underdeveloped
countries.27,28
1.1.1. Taxonomy of Leishmania
The taxonomy of Leishmania has undergone multiple reviews,29 from pure
morphological and clinical criteria to genomic comparisons. Currently, the genus
Leishmania belongs to the Trypanosomatidae family, and is divided into three subgenera:
L. (Leishmania), L. (Viannia) and L. (Sauroleishmania). The latter only infects reptiles,
Introduction
19
whereas different species within L. (Leishmania) and L. (Viannia) are grouped in complexes
associated with specific clinical forms of leishmaniasis30 (Figure 1).
Figure 1. Taxonomical classification of Leishmania (Modified from van der Auwera et al.31).
20
1.1.2. Clinical manifestations of leishmaniasis
The diversity of symptoms for leishmaniasis is mostly dependent on the
immunological status of the host and the infecting Leishmania species. From a clinical
perspective, the different pathologies are grouped under three major forms:8
1) Visceral leishmaniasis is the deadliest form of leishmaniasis if left untreated.32 It is
caused by L. donovani and L. infantum in the Old World, and L. chagasi (infantum) in
the New World. Incubation period ranges from 10 days to over 1 year, and symptoms
and clinical signs usually appear gradually. VL is characterised by irregular fever
episodes, weight loss, hepatosplenomegaly and anaemia. For a certain percentage of
immunocompetent VL patients, infection may course as asymptomatic.33 There are
two forms of VL:
Anthroponotic VL or kala-azar is caused by L. donovani in India, Nepal,
Bangladesh, Sudan and Ethiopia.32 Occasionally, 6-12 months or more after an
apparent recovery from VL, patients may suffer a cutaneous complication
denominated post-kala azar dermal leishmaniasis (PKDL). PKDL is
characterised by hypo-pigmented macular, maculopapular, and nodular rash.34 In
Sudan, this relapse may occur earlier or even concurrently to VL, and while PKDL
frequently heals spontaneously in Africa, this rarely happens in India.
Zoonotic VL is caused by L. infantum in Central and South America, and the
Mediterranean Region.8
2) Cutaneous leishmaniasis is the most common form of leishmaniasis. It is caused by
L. aethiopica, L. major, and L. tropica in the Old World, and L. amazonensis and L.
mexicana in the New World. It is characterised by a macule at the site of inoculation
that evolves into a papule.35,36 In a later stage, the papule frequently turns into an ulcer
that leaves a permanent scar after healing. Sometimes, standard CL results in more
complicated forms:37
Diffuse CL derives from CL caused by L. aethiopica in the Old World, and from
L. mexicana and L. amazonensis in the New World. It is characterised by widely
Introduction
21
dispersed skin lesions, usually without ulceration. Diffuse CL does not heal
spontaneously and relapses are usual.
Disseminated CL originates in the New World from CL caused by L. braziliensis,
L. panamensis, L. guyanensis and L. amazonensis. Disseminated CL cases show 20
to hundreds of nodular or ulcerated lesions, with or without mucosal damage.
3) Mucocutaneous leishmaniasis occurs exclusively in the New World. It is caused by
species from the braziliensis complex, especially L. braziliensis and L. panamensis.
Leishmania migrates from a primary cutaneous lesion to the mucosal tissues of the
mouth and upper respiratory tract via lymphatic or haematogenous dissemination,
where it causes the complete or partial destruction of the oropharyngeal mucosa and
cartilage.36,38
As stated before, the concurrence of leishmaniasis and AIDS is of special relevance.
Leishmania-HIV co-infected individuals have a higher chance to suffer acute and fatal forms
of leishmaniasis because of their compromised immune system. VL-HIV patients show
atypical organ-infections such as the gastrointestinal tract, peritoneal space, lungs, pleural
space, and skin. In the New World, CL-HIV patients suffer numerous polymorphic and
relapsing lesions.37
1.1.3. Life cycle of Leishmania
Leishmania is a unicellular parasite with a digenetic life cycle and two major
morphological forms, adapted for two extremely different environments.39 The elongated
and flagellated extracellular promastigote faces slightly alkaline conditions and
environmental temperature inside the gut of the sandfly vector. In contrast, the ovoid
non-motile intracellular amastigote dwells in an acidic environment inside the
parasitophorous vacuole of its vertebrate host, full of different hydrolases and reactive
species of oxygen and nitrogen (ROS and RNS respectively), with temperatures ranging
from 32 ºC (CL and MCL) to 37 ºC (VL)40,41 (Figure 2).
22
Figure 2. Life Cycle of Leishmania (adapted from Hurrel et al.).42
Leishmania vectors are female sandflies from the genus Phlebotomus in the Old World
and Lutzomyia in the New World. Phlebotomine sandflies feed on plant juices and sugary
secretions, but females also blood feed on vertebrate hosts to produce eggs.43 The infected
female sandflies inject metacyclic Leishmania promastigotes from her proboscis into the bite
site during the blood meal44 (1). In the vertebrate host, these metacyclic promastigotes are
phagocytised by neutrophils. Leishmania survives in the neutrophil preventing the fusion of
the phagosome with the azurophil granules, while inducing apoptosis in these cells (2). The
apoptotic neutrophil ensures the uptake of the parasite by the macrophage without promoting
its activation. Alternatively, the metacyclic promastigotes enter the macrophage by
opsonization through the binding of their surface proteins with the C3 factor of the
complement (3). Promastigotes end up in a phagosome that eventually fuses with lysosomes
and finally becomes a parasitophorous vacuole.45 Afterwards, the promastigotes transform
into amastigotes, with an oval shape and a short and afunctional flagellum (4). Amastigotes
Introduction
23
multiply within the parasitophorous vacuole until they lyse the macrophage, and continue to
infect and replicate in new macrophages46 (5). When a sandfly feeds on a Leishmania-
infected vertebrate, they ingest infected macrophages and/or circulating amastigotes (6).
Amastigotes revert to promastigotes in the gut of the vector and, after several rounds of
replication, undergo metacyclogenesis (7). Through metacyclogenesis, initial low-infective
procyclic promastigotes migrate into the proboscis as highly infective metacyclic
promastigotes, closing the cycle.47
1.1.4. Control of leishmaniasis
The epidemiology of leishmaniasis is determined by many different factors that
influence vector and human behaviours, such as climate change and war conflicts.48,49
Specific protocols in each regional context have been established to address the persistence
and prevalence of leishmaniasis. While early diagnosis and chemotherapy are essential to
alleviate the severity of the pathology and prevent death, vaccines as well as vector and
reservoir control are useful to prevent infection and reduce the transmission of Leishmania.50
1.1.4.1. Prevention of leishmaniasis infection: vaccines, vector and reservoir control
Although it is well known that immunoprotection is achieved after recovery from
leishmaniasis, a human vaccine has not been approved yet. Three generations of vaccines
have been developed over the past few decades. First generation vaccines consist of whole
parasites physically/genetically attenuated or killed. Second generation vaccines are
developed with specific protein antigens and the third generation includes genetic
immunization through an expression vector encoding parasite antigens to be expressed by
the host.50-52 So far, few vaccines have entered human clinical trials, and none have reached
Phase III yet (Table 1). The latest vaccine to enter clinical trials was the third-generation
vaccine ChAd63-KH, which was designed for prevention and treatment of VL and PKDL
caused by L. donovani,26 and consists of simian adenovirus ChAd63 carrying two
Leishmania genes that encode two parasite proteins (KMP11 and HASPB).50
24
Table 1. State-of-art of human vaccines against leishmaniasis under clinical trials.26
Clinical Phase I Clinical Phase II
First generation
vaccine -
Autoclaved-killed L. major (ALM)
Gentamycin-attenuated L. major (GALM)
Second generation
vaccine
LEISH-F1
LEISH-F3
LEISH-F2
SMT+NH
Third generation
vaccine - ChAd63-KH
Despite the current efforts to develop human vaccines, nowadays prevention of
leishmaniasis essentially relies upon the control of reservoirs and vectors.48,53 Sandfly
control encompasses physical and chemical barriers: use of indoor and outdoor insecticide
sprays, insecticide-impregnated curtains and bed nets, topical insect repellents, and
minimization of skin exposure. Options for reservoir control ranges from dog collar
repellents, canine drugs and vaccines for domestic dogs, to destruction or chemical treatment
of burrows of wild animals, as well as euthanasia and culling.54-56
1.1.4.2. Diagnosis
Early diagnosis is key to mitigating the development of a more severe pathology and
even death. Current standardized protocols include clinical, parasitological, serological and
antigen-detection tests depending on the type of leishmaniasis and resources available on the
spot.57,58 Most tests require special equipment only available in hospitals and research
centres, but there are two diagnostic tests available on the field: the direct agglutination test,
and the rK39 dip-stick test. The immunochromatographic rK39 test has proven to be
essential for early diagnosis of VL despite its poor performance in VL-HIV and PKDL
patients and its inability to discriminate asymptomatic infections.52 New methods in the
pipeline for early detection of leishmaniasis include the loop-mediated isothermal
amplification (LAMP) assay, a PCR assay that works at environmental temperature.59
Introduction
25
1.1.5. Biology of Leishmania
In comparison to other eukaryotic cells, trypanosomatids show some peculiarities
concerning their intracellular organelles, such as glycosomes, acidocalcisomes, and a single
well-developed mitochondrion, accounting for significant metabolic differences:60
― Glycosomes are peroxisome-like organelles found in trypanosomatids that harbour the
enzymes involved in the first seven steps of glycolysis, as well as enzymes for other
metabolic pathways such as the pentose phosphate pathway (PPP), gluconeogenesis and
part of the β-oxidation of fatty acids.61
― Acidocalcisomes are membrane-bound metal-storage organelles implicated in
polyphosphate and pyrophosphate metabolism, also found in Apicomplexa parasites.62
― All Kinetoplastida including Trypanosomatidae possess a single mitochondrion with
extensive ramification that accounts for up to 12% of the volume of the parasite. In
contrast, mammalian cells can encompass hundreds of mitochondria in as much as 20%
of their cell volume. Furthermore, the trypanosomatid mitochondrion contains a unique
well-developed mitochondrial disk-shaped DNA called kinetoplast (kDNA) that
accounts for about 30% of the total DNA of these parasites.63,64
1.1.5.1. Metabolic characteristics of Leishmania
In trypanosomatid parasites, adaptive mechanisms to survive in their hosts include
energy and redox balance, regulated by the compartmentalization of different metabolic
pathways within the cytoplasm and the aforementioned organelles.65 Moreover, Leishmania
has stage-specific metabolic adaptations aimed to ensure an adequate supply of nutrients and
evade different stresses in each of its two hosts. In fact, the promastigote stage mainly
depends on oxidative phosphorylation for ATP synthesis,66 whereas the amastigote stage has
a higher dependence on glycolysis.41
1.1.5.1.1. Carbon metabolism
Leishmania constitutively expresses most of the main enzymes involved in the carbon
metabolism throughout its life cycle, including those belonging to catabolic pathways for
26
glucose, amino acids, and fatty acids. It is auxotrophic for purines, vitamins, heme and some
amino acids. Glucose is the preferred carbon source. However, the carbon metabolism is
reprogrammed in the amastigote stage for intracellular growth and survival in the vertebrate
host.65,67,68
The carbon metabolism of the Leishmania promastigote is characterized by a surplus
uptake of glucose, preferentially catabolized via glycolysis and succinate fermentation in the
glycosomes. End-products of glycolysis and succinate fermentation are further catabolized
via the tricarboxylic acid (TCA) in the mitochondrion, where CO2, reducing agents (NADH
and FADH2) and anabolic precursors are produced.67 NADH and FADH2 generated in the
TCA cycle are used to replenish reducing agents at the inner membrane of the
mitochondrion, where they are required for ATP synthesis through oxidative
phosphorylation.66,69 The excess of glucose is managed through an overflow metabolism that
secretes partially oxidized glucose subproducts and avoids overload of the TCA cycle, thus
preventing intracellular stress. Leishmania promastigotes also utilize non-essential amino
acids (aspartate, alanine, and glutamate) besides glucose as a carbon source67,68 (Figure 3,
panel A).
In contrast, the amastigote stage shows a semi-quiescent state with a stringent
metabolism. These metabolic changes are associated with differentiation signals such as
temperature and pH, and not dependent on nutrient limitations in the phagolysosome.68 It is
surmised that the stringent metabolism of the Leishmania amastigote confers resistance
against nutrient depravation and other stresses (oxidative, nitrosative, pH, temperature)
within the parasitophorous vacuole, thus being essential for intracellular survival and
virulence.65 Glucose and amino acid uptake are significantly lower in comparison to the
promastigote stage, but both carbon sources are utilized much more efficiently, showing
scarce overflow metabolism.68 Major energy-consuming processes are specifically repressed
and the flux through the TCA is decreased to avoid overproduction of endogenous ROS due
to electron leaks from the respiratory chain with a high mitochondrial NADH/NAD+ ratio.70
Instead of amino acids, amastigotes resort to fatty acid oxidation as an additional carbon
source besides glucose. Moreover, the metabolic intermediaries of the TCA cycle for
glutamate, glutamine and aspartate biosynthesis are replenished through β-oxidation of fatty
acids65 (Figure 3, panel B).
Introduction
27
Figure 3. Central carbon metabolism of promastigotes (A) and amastigotes (B) of L. mexicana.68
Major carbon sources and overflow metabolites are in blue and green boxes respectively. Fluxes through
dotted pathways are down-regulated relative to the opposite stage. Abbreviations: αKG, α-ketoglutarate;
AcCoA, acetyl-CoA; Cit, citrate; Fum, fumarate; FFAA, fatty acid; Glc6P, glucose 6-phosphate; G3P,
glyceraldehyde 3-phosphate; Mal, malate; OAA, oxaloacetate; Ac, acetate; PEP, phosphoenolpyruvate;
PPP, pentose phosphate pathway; Pyr, pyruvate; SCoA, succinyl-CoA; Suc, succinate.
1.1.5.1.2. Oxidative phosphorylation
The mitochondrion of Leishmania promastigotes has a fully functional TCA cycle68
and a regular eukaryotic respiratory chain in their inner mitochondrial membrane, with four
electron transport complexes and a mitochondrial F0/F1 ATP-synthase. Oxidative
phosphorylation takes place due to the coupled activities of these elements: during electron
transport, complexes III and IV generate a transmembrane proton gradient that F0/F1
ATP-synthase (also referred to as complex V) uses for ATP synthesis63 (Figure 4). Although
it is present in the respiratory chain of Leishmania,41 complex I of trypanosomatids lacks
subunits essential for proton pumping, so it is thought that complex I does not play ―or
barely plays― a role in energy generation, and that the main function of this complex is to
maintain a balanced mitochondrial NADH/NAD+ ratio.71 Consequently, the main electron
donor in Leishmania promastigotes is succinate, as substrate for complex II.63 Arguably, a
mitochondrial NADH-dependent fumarate reductase (FRD) present in Leishmania may be
28
considered an alternative to complex I, as it oxidizes NADH (the natural substrate for
complex I) using fumarate as an electron acceptor to generate succinate, which is further
oxidized by complex II.72
Figure 4. Respiratory chain of Leishmania promastigotes. It consists of complex I (NADH-quinone
oxidoreductase, E.C. 7.1.1.2), complex II (succinate-ubiquinone oxidoreductase, E.C. 1.3.5.1), complex
III (ubiquinol-cytochrome c3 oxidoreductase, E.C. 7.1.1.8) and complex IV (cytochrome c2 oxidase, E.C.
1.9.3.1). Ubiquinone (Coenzyme Q; CoQ) and cytochrome c (CytC) are mobile electron carriers between
the complexes. Complex V is an ATP-synthase that generates mitochondrial ATP using the
transmembrane proton gradient generated by the electron transport chain.63 FRD: fumarate reductase.
Otherwise, as previously mentioned, the amastigote stage of Leishmania adopts a
stringent metabolism that entails reduction of highly energetic cellular processes with
avoidance of oxidative stress.65 Since a surplus of glucose increases mitochondrial NADH
and ROS production, oxidative phosphorylation and hexose uptake are reduced, although
hexose uptake remains essential for survival.73,74 Switching from oxidative phosphorylation
to glycolysis as an energy source is a thriving mechanism shared with other intracellular
pathogens to decrease their susceptibility to exogenous and endogenous RNS/ROS.75
Chakraborty et al.41 observed a downregulation of mitochondrial electron transport in
L. donovani amastigotes with strong reduction of the respiration. Moreover, fumarate served
Introduction
29
as an electron sink in the anaerobic metabolism of the parasite, resulting in excretion of the
succinate excess generated. Interestingly, the parasite may couple succinate and proton
pumping as a metabolic energy source for nutrient and ion transport. Plasma membrane
electron transport has been described before in L. donovani promastigotes.76,77
1.1.5.1.3. Other metabolic peculiarities: Folate synthesis, ergosterol and trypanothione
Trypanosomatids also have specific metabolic differences with respect to their hosts.
Folate metabolism: While mammalian cells are auxotrophic for folic acid,
trypanosomatid parasites are capable of de novo folate synthesis. Folates work as
cofactors in the metabolism of amino acids, DNA and RNA synthesis and other essential
metabolic pathways. Interestingly, folate is produced from pteridines, which are
synthesized by insects and mammals. Hence, Leishmania salvages pteridines for folate
biosynthesis from their hosts.78
Ergosterol: Similar to fungi, the major sterol in the plasma membrane of trypanosomatid
protozoa is ergosterol, as opposed to cholesterol in mammals.79
Trypanothione: It is a low molecular mass thiol formed by two molecules of glutathione
conjugated to a single molecule of spermidine. Similar to glutathione in mammalian
cells, trypanothione governs the redox metabolism of Leishmania. It works as an electron
donor for the reduction of peroxides and the formation of deoxyribonucleotides, among
other redox tasks. Therefore, it plays an important role in the antioxidant defences of
intracellular Leishmania against nitrosative and oxidative stresses from the macrophage.
Trypanothione is also involved in the defence against some heavy metals and
xenobiotics, and is essential for the glyoxalase system, which removes toxic and
mutagenic intermediates.80,81
1.1.6. Signal transduction in Leishmania
Signal transduction enables cellular responses to external and internal stimuli,
allowing the cell to adapt to changes and maintain intracellular homeostasis. In parasites
such as Leishmania, the importance of signal transduction is emphasized by the drastic
30
environmental changes undergone by the parasite throughout its life cycle. Receptors for
these stimuli initiate signal cascades that trigger and regulate expression of certain genes,
activity of specific proteins, and/or changes in cell cycle progression involved in parasite
differentiation, survival, and growth. Members of signalling pathways in eukaryotes, such
as adenylate cyclases, phosphodiesterases, protein kinases and phosphatases among others,
are present in Leishmania.82
Adenylate cyclases (ACs) and phosphodiesterases mediate cAMP-dependent
signalling cascades by regulation of the cAMP intracellular levels through cAMP synthesis
and hydrolysis, respectively. Interestingly, in trypanosomatids it has been suggested that
some ACs play an essential role as signalling receptors due to their extracellular N-terminal
end, which is thought to contain a binding domain for an external ligand. Thus, it is surmised
that ACs in trypanosomatids compensate the lack of G-protein-coupled receptors (GPCRs)
found in other eukaryotes.
Protein kinases (PKs) and protein phosphatases regulate the phosphorylated state of
target proteins or peptides by phosphorylation and dephosphorylation, respectively. Protein
kinases are one of the largest protein families for most eukaryotic organisms, underlying
their relevance in the regulation of cellular growth and survival. Eukaryotic PKs are
characterized by a long-conserved domain (eukaryotic kinase domain) composed of 11
subdomains and a specific three-dimensional structure. They are largely distributed into
three categories according to the amino acid targeted for phosphorylation: Ser/Thr, Tyr, and
dual-specificity kinases. More specifically, eukaryotic PKs are additionally grouped by the
Manning classification into nine groups (Table 2) following phylogenetic criteria (Figure
5).83 Alternatively, protein phosphorylating enzymes devoid of the eukaryotic kinase domain
are labelled as atypical PKs and PK-like.84
Introduction
31
Table 2. Eukaryotic protein kinases groups according to Manning.83
Eukaryotic protein
kinase groups Name description
AGC Protein kinase A, G and C families
CAMK Ca2+/Calmodulin-dependent kinase group
CMGC CDK, MAPK, GSK, and CLK families
STE Homologs of yeast sterile 7, 11, and 20
CK1 Casein Kinase 1 group
TK Tyrosine kinase group
TKL Tyrosine kinase-like group
RGC Receptor guanylate cyclase group
“Other” Kinase families that do not fit elsewhere
1.1.6.1. The kinome of Leishmania
Comparative studies of the kinomes of L. major, Trypanosoma brucei, T. cruzi,
L. infantum and L. braziliensis identified 196, 176, 190, 224 and 221 putative protein kinases
respectively, and observed some remarkable peculiarities of the trypanosomatid
kinomes:85,86
The AGC and the CAMK groups are relatively poorly represented (13.8-15.6% of their
kinome), whereas the CMGC and the STE groups are fairly well represented (more than
40% of their PK repertoire), as compared to these paired PK groups in the human kinome
(26.4% and 20.8% respectively).83
Trypanosomatidae parasites are apparently devoid of protein kinases G or
cGMP-dependent (PKGs).82
Although trypanosomatids do not have any tyrosine kinase or tyrosine kinase-like
groups, it is well known that phosphorylation on tyrosine occurs. Parsons et al.85
32
proposed that atypical TKs and dual-specificity kinases encoded in the trypanosomatid
genomes carry out this task.
Figure 5. Phylogenetic classification and distribution of human protein kinases.87
Introduction
33
In addition, Borba et al.86 identified 157 Leishmania-specific protein kinases after
comparing the kinomes of L. infantum, L. braziliensis and humans. These
Leishmania-specific PKs were further categorized in functional groups; almost one third
(31%) of these enzymes were assigned to signal transduction, while the remainder were
distributed into cell growth and death processes (36%), and a variety of other functions
(metabolism, environmental adaptation, cell motility, morphogenesis…). Moreover,
phosphoproteomic studies in Leishmania endorsed the regulation of PKs as essential for
promastigote-to-amastigote differentiation, since phosphorylation of most proteins in the
parasite occurs in a stage-specific manner (promastigote or amastigote) rather than
constitutively (in both stages).88,89
Therefore, protein kinases are appealing targets for Leishmania chemotherapy due to
their variety of essential roles in the parasite. In fact, some of them have already been
validated as pharmacological targets for the development of new leishmanicidal drugs
(Table 3), although the roles of most PKs identified in Leishmania are still unclear.
3
4 34 Table 3. Eukaryotic protein kinases (ePKs) described in the kinomes of Leishmania spp.85,86,90
ePK
groups
PK families in
Leishmania Function
Validated target
in Leishmania
AGC
Protein kinase A or
cAMP-dependent (PKA)
Phosphorylation of downstream protein targets in response to an increase in cAMP
intracellular levels.91 In Leishmania spp., PKA is almost exclusively active in the metacyclic
promastigote, hence it is likely relevant for the primary invasion of the macrophage.82
No
Protein kinase C (PKC) PKC-like activity in Leishmania has been reported,92 although its encoding gene has not been
identified in any of the trypanosomatid genomes analysed so far. No
Protein kinase B (PKB)
or Akt
An Akt-like kinase is upregulated in Leishmania under stress conditions, such as nutrient
deficiency and heat shock.93,94 No
Nuclear DBF2-related
kinases (NDR)
Kinome analyses support the existence of NDR kinases in Leishmania, but there is no further
knowledge about them. Two NDR kinases in T. brucei, PK50 and PK53, are essential for a
successful cytokinesis of its bloodstream stage.95
No
Phosphoinositide-
dependent protein kinase
1 (PDK1)
Involved in the regulation of relevant processes of metabolism, growth, proliferation and
survival, driven by lipid signalling in higher eukaryotic organisms.96,97 There is no further
information on specific PDK1 kinases of Leishmania.
No
Ribosomal S6 kinase
(RSK)
Downstream in the mTOR pathway in mammals. S6K1 is responsible for the phosphorylation
of the ribosomal protein S6, with increase of protein synthesis and proliferation ensuing.98
There are no reports regarding the functionality of any RSK of Leishmania.
No
35
Intro
ductio
n
3
5
Table 3 (continued). Eukaryotic protein kinases (ePKs) described in the kinomes of Leishmania spp.85,86,90
ePK
groups
PK families in
Leishmania Function
Validated target
in Leishmania
CAMK
CAMK1
Member of the calmodulin-dependent protein kinase cascade. The gene of CAMK1 has been
identified in the Leishmania kinome without further notes on its functionality. CAMKs in
other eukaryotic organisms are involved in gene transcription, translation, ion channel
regulation, and cell death processes.99
No
CAMK-like kinase
(CAMKL)
Genes for CAMK-like kinases have been described in Leishmania but are yet to be studied.
A CAMK-like protein of the Apicomplexa Toxoplasma gondii is surmised to be a virulence
factor through the phosphorylation of host proteins.100
No
Ca2+-dependent protein
kinase (CDPK)
CDPKs of Leishmania have not been studied. Their role in other Protozoa, such as the
apicomplexan Plasmodium falciparum, is the regulation of cytosolic Ca2+.101 No
CMGC
Cyclin-dependent kinase
(CDK) or cdc2-related
kinase (CRK)
Crucial for the control of the cell cycle of the parasite. CRK1 and CRK3 are essential for G1/S
transition. CRK3 is also key for G1 and G2/M phase progression together with mitotic cyclins
CYC2 and CYC6, respectively.102,103 CRK12 is essential for growth and survival although its
precise role is unclear.104
CRK1, CRK386,105
CRK12104
Mitogen-activated
protein kinase (MAPK)
MAPKs of Leishmania spp. are involved in flagellar growth regulation (LMAPK3,
LmxMAPK9, LmxMAPK13, LmxMAPK14), stage-differentiation (LmxMAPK1,
LmxMAPK2, LMAPK3, LmxMAPK4), parasite growth (LmjMAPK7), morphology
(LmxMAPK6), and intracellular survival (LmxMAPK1, LmxMAPK2).106,107 LdMAPK1 also
regulates the activity of heat shock proteins.108
MAPK3107
MAPK4109
36 Table 3 (continued). Eukaryotic protein kinases (ePKs) described in the kinomes of Leishmania spp.85,86,90
ePK
groups
PK families in
Leishmania Function
Validated target in
Leishmania
CMGC
Glycogen synthase
kinase-3 (GSK-3)
Mammalian GSK-3 is a multi-task signalling protein involved in energy metabolism,
proliferation, differentiation, immunity, and cell death and survival.110
Leishmania has short and long GSK-3 isoforms, and the former is crucial for parasite cell
growth.111
LdGSK-3111
Cell division control
(CDC)-like kinase
(CLK),
SR-rich protein kinase
(SRPK),
Dual-specificity tyrosine-
regulated kinase (DYRK)
In higher eukaryotes, CLKs and SRPKs are involved in RNA processing and splicing by
regulating the cellular distribution and activity of SR proteins,112 whereas DYRK1 is known
to pre-phosphorylate substrates of GSK-3.113
Additionally, as dual-specific protein kinases, CLKs and DYRKs are involved in tyrosine
phosphorylation in trypanosomatids.86
No
Casein Kinase 2 (CK2)
CK2 in Leishmania is secreted, but also distributed within the cytoplasm and across the
external surface of the plasma membrane of the parasite. It is involved in promastigote
proliferation and infectivity.114
No
ROS-cross-hybridizing
kinase (RCK)
RCKs have been described as MAPK-related in other protozoa and higher organisms. They
are involved in cilia length and intraflagellar transport of protein complexes along the
axoneme.115-117 Thus, it may be inferred that RCKs in Leishmania are likely involved in
processes of the flagellum, like other MAPKs.
No
Intro
ductio
n
37
Table 3 (continued). Eukaryotic protein kinases (ePKs) described in the kinomes of Leishmania spp.85,86,90
ePK
groups
PK families in
Leishmania Function
Validated target
in Leishmania
CK1
CK1
CK1 kinases are involved in the regulation of the cell cycle, cellular transport, receptor
signalling, transcription, DNA repair and apoptosis.
In Leishmania, CK1 isoform 2 (CK1.2) is secreted and phosphorylates host substrates,
promoting infection of the host cell.118,119
CK1.2118
Tau tubulin kinase
(TTBK)
TTBKs of Leishmania have not been studied. In other organisms TTBKs phosphorylate tau
microtubule-associated protein and tubulin.120 No
STE
STE7/MAPKK
MAPKKs are dual-specific86 protein kinases homologs of yeast sterile 7 (STE7). They
phosphorylate MAPKs; as such, they participate in the regulation of parasite differentiation
and the maintenance of the flagellum length,85 as well as tyrosine phosphorylation.86
No
STE11/MAPKKK
The MAPKKKs, homologs of yeast sterile 11 (STE11), are abundant in trypanosomatids.
They activate MAPKKs by phosphorylation upstream the MAPK signalling pathways.85,121 In
T. brucei, STE11 was associated with cell growth and stage differentiation.122
No
STE20/MAPKKKK
Homologs of the yeast sterile 20 (STE20) are rare in trypanosomatids.85 They can function as
scaffolds for pathway assembly and MAPKKK self-activation; otherwise they can directly
phosphorylate MAPKKKs.121 Although Leishmania STE20 has not been studied, STE20 of
T. brucei is essential for NDR kinase activation.95
No
3
8
38 Table 3 (continued). Eukaryotic protein kinases (ePKs) described in the kinomes of Leishmania spp.85,86,90
ePK
groups
PK families in
Leishmania Function
Validated target
in Leishmania
“Other”
Aurora kinase (AIRK) Aurora kinases in Leishmania are surmised to regulate chromosome segregation and
cytokinesis, as in other eukaryotic cells.123
Aurora kinase 1
(AIRK)123
Polo-like kinase (PLK)
PLK genes were found in Leishmania, but their functionality has not been described yet.
In T. brucei, PLKs are essential during mitosis, playing a role in kDNA segregation, basal
body duplication and cytokinesis.124
No
Never in mitosis gene A
(NimA)-related kinase
(NEK)
NEKs are present in Leishmania genomes but have not been studied in detail.
RDK2 is a NEK essential for parasite proliferation in T. brucei,122 accordingly to the role
played by NEKs in cell cycle progression and cytoskeletal functions.85,123
No
Wee1-like protein kinase
(WEE1)
No specific studies on Leishmania WEE1 have been conducted. Nevertheless, WEE1 kinase
is known to participate in tyrosine phosphorylation86 and it is involved in cell division,85 as it
has been described for T. brucei.125
No
Introduction
39
1.1.7. State of the art of the chemotherapy for leishmaniasis
As previously stated, the only current treatment available for leishmaniasis is
chemotherapy.126,127 However, the existing drug repertoire is scarce, with downsides such as
toxicity, high cost, parenteral administration and emergence of resistance.128 Two
approaches are being used to fix this scenario. In a long-term strategy, the pipeline for
leishmanicidal drugs is fed with new compounds to broaden chemotherapeutic options.127 In
a short-term range, combination therapy of current antileishmanial drugs successfully
reduces dosing, treatment duration, toxicity and drug resistance.25
Current drugs for leishmaniasis chemotherapy approved by the FDA129 encompasses
pentavalent antimonials, amphotericin B, miltefosine and paromomycin as first-line options,
and pentamidine as a second-line alternative (Figure 6, Table 4).
Figure 6. Chemical structures of the current drugs approved for the treatment of human
leishmaniasis.
40 Table 4. Main current drugs available for chemotherapeutic treatment of human leishmaniasis. (i.v.: intravenously, i.m.: intramuscularly)
Leishmanicidal drugs Adm. route Mechanism of action Side effects and downsides Resistance References
PENTAVALENT
ANTIMONIALS
Meglumine antimoniate
(MA)
Sodium stibogluconate (SSG)
i.v.
or
i.m.
Interferes with the redox
metabolism by targeting the
trypanothione system of the
Leishmania amastigote.
Severe side effects such as
hepatic and renal toxicity and
cardiac abnormalities. Requires
extended hospitalization with
higher costs.
Emerging resistance
especially in the
Indian subcontinent.
128,130
AMPHOTERICIN B
Deoxycholate
(AmBd)
i.m
Causes plasma membrane
permeabilization by selective
binding to ergosterol of the
parasite membrane.
Highly toxic, frequently causes
electrolyte abnormalities, ne-
phrotoxicity and anaemia.
There are sparse
cases of clinical
AmB resistance.
127,128 AMPHOTERICIN B
liposomal formulation
(LAmB)
(Ambisome)
Significantly lower toxicity than
AmBd, but its implementation is
restricted due to its higher cost
and cold-chain transport require-
ment.
PAROMOMYCIN
i.m. for VL,
topical for
CL
Selectively interferes in
Leishmania ribosomal activity,
inhibiting protein synthesis.
Affects mitochondrial membrane
potential causing inhibition of
respiration.
Injection-site pain, hepato- and
ototoxicity, and renal dys-
function.
Laboratory-induced
resistance has been
described.
127,128,131
Intro
ductio
n
41
Table 4 (continued). Main current drugs available for chemotherapeutic treatment of leishmaniasis. (i.v.: intravenously, i.m.: intramuscularly)
Leishmanicidal drug Adm. route Mechanism of action Side effects and downsides Resistance References
MILTEFOSINE Oral
Modifies cell membrane com-
position by interfering with
phosphatidylcholine biosynthesis.
Significantly reduces mitochon-
drial membrane potential and
cytochrome c oxidase activity.
It may promote apoptosis through
Akt inhibition.
Immunomodulatory activity in
macrophages.
Teratogenic. Causes gastro-
intestinal discomfort.
Broad range of sensitivity among
Leishmania species, with low
efficacy in HIV co-infection
cases.
Its long half-life favours drug
resistance.
Easily induced in
vitro.
Growing reports of
resistant parasites in
clinical settings.
127,132,133
PENTAMIDINE
i.v.
or
i.m.
The mitochondrion is likely an
important target, but more targets
are suspected.
Severe adverse effects, from
cardiotoxicity to several
metabolic and electrolyte
disturbances (hypo- or
hyperglycaemia, type 1 diabetes
mellitus, hyperkalaemia,
hypocalcaemia…)
Unresponsiveness in
Indian patients.
49,127,131
42
Additional approaches in the search for new leishmanicidal treatments involve drug
repositioning as well as target-based and phenotypic screenings.134
Target-based methods aim to find new inhibitors against a well-defined target, e.g. a
specific enzyme. Identified hits are compounds with a known mechanism of action that
need further validation in a cellular model.
Phenotypic approaches rely on the screening of compounds directly against the parasite.
Hit compounds inhibit cellular growth, but further target deconvolution is desirable in
order to unveil their mechanism of action.
In drug repositioning, compounds initially developed against a specific pathology are
assayed for leishmanicidal activity. An important asset of this method is that most of the
toxicological and preclinical data of the compounds are already available.135 Some of the
current drugs used in the treatment of leishmaniasis are, in fact, repositioned drugs:
miltefosine was originally developed as an anticancer drug, paromomycin as a
bactericidal antibiotic and amphotericin B is an antifungal.127
Some years ago, the Drugs for Neglected Diseases initiative (DNDi) initiated a search
for novel leishmanicidal agents and established target product profiles (TPPs) in close
association to academic groups. TPPs define the parameters and criteria of each stage of
drug development: administration route, dosing regimen, safety and tolerability levels, cost
and shelf life.136 Nowadays, different institutions and big pharma companies collaborate with
DNDi in phenotypic and target-based screenings of their chemical libraries.28 In recent years,
five new lead series have reached preclinical stages of development such as the
aminopyridazole DNDI-5561, and even clinical phase I like the oxaborole DNDI-6148, the
nitroimidazole DNDI-0690 or the pyrazolopyrimidine GSK3186899/DDD853651
(Figure 7). Although their mechanism of action is not fully understood, some clues about the
biochemical targets in Leishmania have been envisaged.137
Introduction
43
Figure 7. Leishmanicidal compounds of the DNDi pipeline that have reached Phase I.
OBJECTIVES
Objectives
47
2. Objectives
Due to the urgent need for new effective treatments for leishmaniasis, the main
objective of this PhD thesis is the discovery of new leishmanicidal agents with a known
mechanism of action. For this purpose, we have evaluated different sets of compounds under
a target-based approach focused on the inhibition of the short isoform of GSK-3 of
Leishmania, a validated drug target against the parasite, in combination with a phenotypic
screening on axenic promastigotes and amastigotes. Moreover, as energy metabolism is a
reliable parameter of cell viability, bioenergetic parameters were studied as a beacon to
assess the physiological homeostasis of the parasite and explore off-target effects.
The specific objectives set for this purpose were as follows:
1.- To select human protein kinase inhibitors (hPKIs) with representative scaffolds
from our in-house chemical library as potential LdGSK-3s inhibitors.
2.- To select virtual screening hits that putatively bind to pharmacologically validated
kinases in Leishmania.
3.- To test the selected compounds against LdGSK-3s in a target-based approach.
4.- To assess the leishmanicidal activity and cytotoxicity on murine macrophages of
the selected compounds under a phenotypic screening strategy.
5.- To establish structure-activity correlations for those PKIs with leishmanicidal
activity and LdGSK-3s inhibition, identified as hits.
6.- To evaluate the capability of our hits to decrease the parasite load in peritoneal
murine macrophage infections.
7.- To test inhibition of the energy metabolism of Leishmania as an additional
off-target mechanism of our hits.
MATERIALS AND METHODS
Materials and Methods
51
3. Materials and Methods
3.1. Chemical compounds, media, reagents and laboratory equipment
This work reports the evaluation of 136 heterocyclic compounds with low molecular
weight (MW <500) and purity 95 % by HPLC from our in-house chemical library, named
MBC chemical library.138 The tested compounds differ in their chemical scaffolds and
substitution patterns. In this work, each compound was labelled with a number from 1 to 136
(Tables 7, 8, 10, 11, 12 and 13).
Reagents, materials, media and equipment used in cell cultures, buffers and assays
were purchased from Sigma-Aldrich unless stated otherwise.
3.2. Cell cultures and cell harvesting
The Leishmania cell lines used in this project were Leishmania infantum JPC
(MCAN/ES/98/LLM-722) and 3-LUC L. donovani (MHOM/SD/00/1S-2D) promastigotes,
as well as L. pifanoi (MHOM/VE/60/Ltrod) and mCherry-L. pifanoi axenic amastigotes.
Elicited murine peritoneal macrophages (MPM) from Balb/C mice (9 to 14-week-old males)
were also used.
L. infantum promastigotes were grown at 26 ºC in RPMI 1640 medium supplemented
with 10% heat inactivated fetal calf serum (RPMI-HIFCS; Table 5). L. donovani 3-LUC
promastigotes, a modified strain of L. donovani promastigotes that expresses a mutated
Phothinus pyralis luciferase gene, were grown in the same conditions with addition of
30 µg/mL geneticin in the medium.139
L. pifanoi axenic amastigotes were grown at 32 ºC in M199 supplemented with
20% HIFCS (M199-HIFCS; Table 5). mCherry-L. pifanoi axenic amastigotes, a strain
52
transfected with an expression vector encoding the gene for the red fluorescent protein
mCherry, were cultured in the same conditions plus 50 µg/mL blasticidin hydrochloride in
the medium.
Table 5. Media and buffer composition.
Medium/Buffer Composition
RPMI-HIFCS
RPMI 1640 medium (Gibco) supplemented with 10% Heat Inactivated Fetal
Calf Serum (HIFCS), 5 mM HEPES (Biowest), 1.7 mM NaHCO3, 2 mM
L-glutamine, 20 U/mL unicilin (ERN, S.A.) and 24 µg/mL gentamicin
(NORMON, S.A.), pH 6.8-6.9.
M199-HIFCS
M199 medium (Gibco) supplemented with 20% HIFCS, 0.5% trypticase
peptone (BD Biosciences), 13.9 mM D-glucose, 76.7 µM hemin, 5.1 mM
glutamine and 40 µg/mL gentamicin; pH 7-7.2.
DMEM-HIFCS DMEM medium (Gibco) supplemented with 10% HIFCS, 2 mM
L-glutamine and 100 U/mL penicillin-streptomycin.
HBSS-Glc
Hank’s Balanced Salt Solution (137 mM NaCl, 5.4 mM KCl, 0.4 mM
KH2PO4, 4.2 mM NaHCO3, 0.4 mM Na2HPO4; pH 7.2-7.3) supplemented
with 1% D-glucose.
Leishmania parasites were harvested at late exponential growth phase (~107 cells/mL)
by 10 min centrifugation (10 min, 1,600 ×g, 4 ºC).
MPM harvesting was carried out in mice by elicitation with thioglycollate
(intraperitoneal injection, 1 mL 10% thioglycollate, 72 h). Elicited macrophages were
extracted by peritoneal wash (×2) with 10 mL HBSS-Glc (Table 5) using 25G and 21G
needles (BD MicrolanceTM), followed by centrifugation (10 min, 400 ×g, 4 ºC).
3.3. Leishmanicidal activity of the compounds on axenic parasites
The 136 compounds from Sets 1 to 4 and Sets 1.1 and 2.1 were tested for
leishmanicidal activity by phenotypic screening on axenic promastigotes and amastigotes
Materials and Methods
53
through a colorimetric assay based on MTT reduction to formazan. Under these conditions,
inhibition of MTT reduction accounts for the inhibition of parasite proliferation.
Parasites resuspended in their respective growth medium were aliquoted into
96-microwell plates (2×106 parasites/mL, 200 µL/well) and incubated with the
corresponding compound (72 h at 24 ºC for L. infantum promastigotes; 96 h at 32 ºC for
L. pifanoi axenic amastigotes).
In order to avoid interference in the readout of formazan, L. infantum promastigotes
were prepared in RPMI-HIFCS using RPMI-1640 without phenol red (Gibco)
(RPMIØRED-HIFCS). L. pifanoi axenic amastigotes were prepared in M199-HIFCS, and
removal of the medium was required due to colorimetric interference of hemin. For this, the
content of each well plus an additional wash with 200 µL HBSS-Glc were collected in
1.5 mL Eppendorf tubes. Amastigotes were further diluted with additional 800 µL
HBSS-Glc. After centrifugation (4 min, 13,000 ×g, 4 ºC), pellets were resuspended in
200 µL HBSS-Glc and transferred back into a 96-microwell plate.
For all axenic parasites, MTT was added to each well (0.5 mg/mL, final concentration).
MTT reduction by viable parasites was stopped within 2 h of incubation with 10% SDS
(50 µL/well). Afterwards, the solubilized formazan was read at 595 nm in a 680 BioRad
Microplate-reader.
Each sample was made by triplicate and assays were repeated at least twice. The IC50s
(concentration required for 50% inhibition in vitro) of leishmanicidal compounds were
calculated with SigmaPlot v11.0.
3.4. Macrophage cytotoxicity of leishmanicidal compounds
MPMs in DMEM-HIFCS (Table 5) were seeded in 96-well plates (1×105 cells/well).
Cells were allowed to adhere to the bottom of the wells (24 h at 37 ºC, 5% CO2), followed
by removal of the medium and addition of compounds in RPMIØRED-HIFCS. After 48 h
of incubation, MTT reduction was carried out as stated in Section 3.3.
54
Experiment design and IC50 calculations were conducted as previously described (see
Section 3.3). The Selectivity Index (SI) of each compound was defined as the ratio IC50 axenic
amastigote/IC50 macrophage.
3.5. Leishmanicidal activity against intracellular amastigotes
MPMs resuspended in DMEM-HIFCS (Table 5) were transferred into a 24-microwell
plate (2×105 cells/well) with a sterile round 22 mm diameter coverslide at the bottom of each
well. Cells were allowed to adhere to the glass surface of the coverslides for 24 h (37 ºC,
5% CO2).
Afterwards, the medium was replaced with a 0.5 mL suspension of mCherry-L. pifanoi
axenic amastigotes (6×105 cells/well; 3:1 ratio parasite:macrophage) in RPMI-HIFCS.
Phagocytosis was allowed to proceed for 4 h at 32 ºC. Non-phagocytosed amastigotes were
removed by gentle washing of the wells (×3) with 1 mL of HBSS at 32 ºC. Next, 1 mL/well
RMPI-HIFCS was added and the infection was left to proceed for 24 h at 32 ºC. Finally, the
infected macrophages were incubated with 1 mL of compound at its corresponding
concentration in new RPMI-HIFCS for 16-20 h, at 32 ºC. The parasite index (number of
amastigotes per macrophage) was evaluated with optical and fluorescence microscopy
(Leica DIALUX 20). At least six different fields gathering up to 100 macrophages were
counted in each preparation.
Each sample was made by duplicate and experiments were repeated at least twice.
3.6. Extraction and purification of LdGSK-3s
E. coli BL21(D3) was transfected by electroporation with the expression vector
pET28a(+) containing the gene of the short isoform of L. donovani GSK-3 (LdGSK-3s),
kindly donated by Dr. Smirlis (Hellenic Pasteur Institute).111 The LdGSK-3s gene was
C-terminally fused to a poly-His tag and placed at the restriction site of the polylinker Nco I
and Xho I under the T7lac promoter. The process of expression, extraction and purification
Materials and Methods
55
of LdGSK-3s is compiled in Figure 8, whereas the buffers used in each stage of the process
are described in Table 6. Fractions and samples of each purification step were analysed for
their protein pattern by SDS-PAGE electrophoresis on a 10% polyacrylamide gel. Fractions
with high purity and protein content of LdGSK-3s were pooled and dialysed with an Amicon
cell filtration (30 kDa cut-off membrane) using Kinase PBS as washing buffer. Lastly,
LdGSK-3s was quantified after ultracentrifugation (147,000 ×g, 1 h, 4 ºC) and filtration
through a 0.22 μm nitrocellulose filter, and preserved at -80 ºC.
Figure 8. Flowchart of the expression, extraction and purification of LdGSK-3s. French Press:
Thermo Scientfic; Ni2+ column: HisTrapTM FF crude 5 mL (Merck, Ref. 17-5286-01).
56
Table 6. Buffers used for LdGSK-3s purification, dialysis, and activity evaluation.
Buffer Composition
Kinase PBS
150 mM NaCl, 1.5 mM H2KPO4, 2.7 mM KCl, 8.3 mM HNa2PO4, 60 mM
β-glycerophosphate disodium, 1 mM Na3VO3, 1 mM NaF, 1 mM disodium
phenyl phosphate; pH 7.5
Lysis/Binding
Buffer
Kinase PBS with 10mM imidazole, plus protease inhibitors cocktail (Roche
Ref. 1697498); pH 7.5
Washing Buffer
Kinase PBS plus additional 150 mM NaCl (300 mM NaCl final concentration),
30 mM imidazole, 1% Triton X-100 (TX-100), and protease inhibitors cocktail;
pH 7.5
Elution Buffer Kinase PBS with 300 mM imidazole plus protease inhibitors cocktail; pH 7.5
Kinase Assay Buffer 50 mM HEPES, 1 mM EGTA, 1 mM EDTA, 15 mM magnesium acetate, 0.1
mg/mL BSA; pH 7.5
3.7. Measurement of LdGSK-3s activity
Enzymatic activity of selected compounds was tested using 20 ng of purified
LdGSK-3s in the presence of 25 μM phosphoglycogen synthase peptide-2 (GS-2; Millipore,
Ref. 12-241) and 1 μM ATP (Sigma-Aldrich, Ref. A7699) in 40 μL Kinase Assay Buffer in
black 96-microwell plates. The reaction proceeded for 30 min at 30 ºC, followed by addition
of 40 μL Kinase Glo reagents (Promega, Ref. V6712). After 10 min of incubation at room
temperature, the remaining ATP from the kinase reaction was measured by an end-point
luminescence readout obtained in a Varioskan Flash microplate reader.
Each sample was made by duplicate and assays were repeated at least twice. The
following controls were included: i) samples with GS-2 and ATP (without LdGSK-3s) to
obtain the maximum luminescent signal, ii) samples with GS-2, ATP and LdGSK-3s to
obtain the minimum luminescent signal due to maximal LdGSK-3s activity, iii) samples with
GS-2, ATP, LdGSK-3s and a commercial inhibitor (10 μM indirubin-3’-monoxime-5-
sulphonic acid)140 to demonstrate the specific activity of LdGSK-3s. The IC50s against
Materials and Methods
57
LdGSK-3s activity were calculated with the statistical programs included in SigmaPlot
v11.0.
3.8. Bioenergetic assays
3.8.1. In vivo monitoring of intracellular ATP levels
Real-time variation of Leishmania intracellular ATP levels was studied using 3-LUC
L. donovani promastigotes.139 This strain was obtained by electroporation of the parental
strain with a pLEXSY expression vector encoding a cytoplasmic luciferase from Phothinus
pyralis, mutated at the import signal for glycosomes located at the C-terminal tripeptide,
preventing its transport into this organelle.
Promastigotes (22×106 parasites/mL) in HBSS-Glc plus 50 μM DMNPE D-luciferin
(GOLDBIO, USA) were immediately aliquoted into a black 96-microwell plate
(90 μL/well). Luminescence was monitored in a POLARstar Galaxy microplate reader
(BMG LABTECH). Once a steady readout of luminescence was obtained, 10 µL of the
corresponding compounds in HBSS-Glc at 10-fold final concentration were added, and
changes in luminescence were followed up for at least 30 min.
Samples were made by duplicate and assays were repeated at least twice. Positive
controls for plasma membrane permeabilization (0.1% TX-100) and inhibition of oxidative
phosphorylation (1.5 µM 1,4-naphthoquinone) were also included.
3.8.2. Assessment of plasma membrane permeabilization
Leishmania parasites (22×106 parasites/mL) in HBSS-Glc plus 1µM SYTOXTM Green
(Invitrogen) were dispensed into a 96-microwell black plate (90 μL/well). Increase in the
fluorescence of SYTOXTM Green was monitored in a POLARstar Galaxy microplate reader
(λEX = 485 nm, λEM = 520 nm). After an initial reading of the basal fluorescence, 10 µL of
the corresponding compounds at 10-fold final concentration in HBSS-Glc were added.
58
Variation of the fluorescence was followed up for ~30 min. Afterwards, maximal
permeabilization value for each well (100%) was established by the addition of 10 µL
1% TX-100.141
Samples were prepared by duplicate and assays were repeated at least twice. TX-100
(0.1%, final concentration) was used as a positive control of plasma membrane
permeabilization.
3.8.3. Mitochondrial membrane potential (ΔΨm) of Leishmania
Variation of mitochondrial membrane polarization was evaluated through the
intracellular accumulation of the fluorescent dye rhodamine 123, which is driven by the
electrochemical potential of the mitochondrion. Leishmania parasites in HBSS-Glc
(20×106 parasites/mL, 100 µL final volume) were incubated with the compounds in 1.5 mL
eppendorf tubes for 4 h at the optimal temperature for each parasite (26 ºC for promastigotes,
32 ºC for amastigotes). The medium was removed after centrifugation (4 min, 13,000 ×g,
4 ºC) and the pellet resuspended in 100 µL 0.3 µg/mL rhodamine 123 and incubated for
10 min at room temperature in darkness. After washing the samples with HBSS-Glc to
remove extracellular dye, the pellet was resuspended in 900 µL HBSS-Glc and transferred
to cytometry tubes. Rhodamine 123 fluorescence was measured in a Coulter XL EPICS flow
cytometer (λEX = 488 nm, λEM = 520 nm).141
Samples were prepared by duplicate and assays were repeated at least twice. A
40 min-treatment with 20 mM KCN was used as a depolarization control.
3.8.4. Evaluation of the O2 consumption rate of the parasite
Variation in the O2 consumption rate of L. infantum promastigotes was measured in a
Clark electrode (Hansatech Instruments) at 1×108 cells/mL in 600 μL of Respiration Buffer
(10 mM Tris-HCl, 125 mM sucrose, 65 mM KCl, 1 mM MgCl2, 2.5 mM NaH2PO4, 0.3 mM
EGTA, 5 mM succinic acid; pH 7.2). Compounds were added with a Hamilton pipette at
Materials and Methods
59
100-fold final concentration to whole parasites, and oxygen consumption was monitored for
8-10 min.
In order to identify the targets of the selected compounds, they, as well as substrates
and inhibitors of the respiratory chain, were also tested on the mitochondrion of Leishmania
promastigotes permeabilized with 60 µM digitonin. Before the addition of the pertinent
treatment, Leishmania mitochondria were supplemented with 100 μM ADP to ensure O2 was
the limiting substrate for respiration.
Each assay was repeated at least twice. Untreated parasites and mitochondrion were
used as a control for basal respiration. Malonate (10 mM) and oligomycin (12.6 μM) were
used as controls for the inhibition of Complexes II and V respectively. FCCP (10 μM) was
used as an uncoupler control. Substrates used included 6.7 mM α-glycerophosphate for
Complex I, and 0.1 mM TMPD-1.7 mM ascorbate for Complex IV.
3.8.5. Assessment of programed cell death induced by the compounds
L. infantum promastigotes (2×106 cells/mL) in RPMIØRED-HIFCS were incubated
with the compounds in 24-well plates (1 mL/well) for 72 h at 26 ºC. Next, samples were
transferred to 1.5 mL Eppendorf tubes and centrifuged (15,700 ×g, 4 min, 4 ºC). Each pellet
was resuspended in 1 mL HBSS-Glc and centrifuged again in order to further wash away
the compounds. Next, the pellets were resuspended in 15 μL of the supernatant. Fixation and
permeabilization of the parasites were carried out by careful addition of 200 μL 70% cold
ethanol to each resuspended pellet and overnight incubation at 4 ºC. Ethanol was removed
by centrifugation and washing of the samples (×2) with 1 mL HBSS-Glc. Finally, each pellet
was resuspended in 500 μL HBSS-Glc with 20 μg/mL propidium iodide (PI) and 3 mg/mL
Ribonuclease A, and incubated in darkness at room temperature in cytometry tubes for
30 min. Fluorescence of PI was measured in a Coulter XL EPICS flow cytometer
(λEX = 488 nm, λEM = 620 nm).
60
Each assay was repeated at least twice. Untreated cells were used as control for a
standard cell cycle histogram, and miltefosine (15 μM) was used as control for an
apoptosis-inducing respiratory inhibitor.
RESULTS AND DISCUSSION
Results and discussion
63
4. Results and discussion
Since no vaccines against human leishmaniasis are commercially available yet, the
treatment against Leishmania exclusively relies on chemotherapy despite its loss of
effectiveness over the last decades due to side effects, high costs, and emergence of
resistance.23-25 Few leads have been recruited in the pipeline for drug discovery against
leishmaniasis,142 but none of them have reached clinical phase II yet, and the mechanism of
action of many remains unveiled. The lack of preclinical candidates renders the pipeline
extremely fragile to cope with the inherent attrition of any drug discovery and development
program. Thus, it is imperative to find new hits.
One of the best approaches to feed the pipeline is the screening of chemical collections
with potential leishmanicidal activity. These screenings can be phenotypic or target-based.
Despite the importance of target identification for later stages in drug development, the
DNDi guidelines do not prioritize target-based screenings over phenotypic ones, prompted
by the urgent need for new hits and the scarce number of validated targets for kinetoplastid
parasites. Thus, the current pipeline for novel antileishmanial drugs is mostly sustained by
hits selected from phenotypic screenings.28 In this work, we focused our interest in new
leishmanicidal hits with a presumed mechanism of action without dismissing the feasibility
of off-side targets. To this end, phenotypic and target-based screenings were concurrently
carried out, with selected compounds from our in-house chemical library assayed for
inhibition of both Leishmania proliferation and Leishmania GSK-3s (LGSK-3s) activity.
GSK-3 is a protein kinase highly conserved throughout eukaryotic organisms, from
yeast and protozoa to plants and mammals. It is a ubiquitous multi-task serine-threonine PK
of the CMGC family. More than 100 substrates for this enzyme have been reported,143 with
over 40 identified as bona fide. In higher eukaryotes, GSK-3 has two isoforms, GSK-3α and
GSK-3β, and participates in a large number of cellular processes including Wnt, Notch and
Hedgehog signalling pathways, insulin regulation of glucose, neural development, cell
64
division, transcription, and cell survival and death.144 GSK-3 in Leishmania spp. has a long
and a short isoform. The latter (GSK-3s) (Figure 9) is the closest homologue to the Homo
sapiens GSK-3β (HsGSK-3β), sharing 49% sequence identity. LGSK-3 is essential for the
progression of the cellular cycle, and its inhibition leads to the programmed cell death of the
parasite.111 Due to its localization in the flagellum and the basal body, it was recently
suggested that the short isoform of LGSK-3 additionally participates in the regulation of
flagellum morphogenesis and/or motility besides cell division, as it was surmised for
T. brucei GSK-3s.145 Moreover, Xingi et al.111 reported that the expression of L. donovani
GSK-3s (LdGSK-3s) in amastigotes (both axenic and isolated from spleen) is 3-fold lower
than in the promastigote stage. Since the amastigote form undergoes metabolic reprograming
and adopts a semi-quiescent state,68 the lower expression of LdGSK-3s in amastigotes may
be related to the overall downregulation of its metabolism.
Figure 9. Crystal structure of LmjGSK-3s (PDB code: 3E3P). The α-helical and β-sheet domains are
coloured in cyan and blue, respectively. Yellow colours within the active site stand for the hinge and
activation loop. The glycine-rich loop, disordered in the crystal structure, is represented with magenta-
coloured dots.140
Results and discussion
65
The GSK-3s of Leishmania became an attractive target for drug development after its
pharmacological validation by Xingi et al.111 using indirubin analogues, which are described
as potent inhibitors of mammalian CDKs and GSK-3. Among them, 5-Me-6-BIO showed
leishmanicidal activity and inhibited LdGSK-3s. This work endorsed the feasibility of
repositioning HsGSK-3β inhibitors against LGSK-3s. Since then, only two additional studies
documented a target-based approach focused on LGSK-3s; one tested other indirubin
analogues146 whereas the other evaluated commercial protein kinase inhibitors.140 Two
additional studies addressed the importance of efficient computational methods for the
virtual pre-selection of GSK-3 inhibitors for their subsequent screening in vitro.147,148
In this work, we aimed to expand the diversity of chemical scaffolds with
leishmanicidal properties and find new alternatives with a different mechanism of action
from those of current antileishmanial drugs in clinical use. For this purpose, we used small
heterocyclic molecules from diverse chemical families, some of which formerly developed
as potential human PKIs, to be screened against a recombinant GSK-3s of L. donovani.
4.1. LdGSK-3s extraction and purification
LdGSK-3s was obtained from E. coli BL21(D3) transfected with a pET28a(+) plasmid
containing the gene for LdGSK-3s. The expression vector pET28(+) is regulated by a lac
operon that includes the resistance gene to kanamycin. Insertion of the LdGSK-3s gene
within the polylinker region between Nco I and Xho I sites provides the expressed enzyme
with a C-terminal poly-(His)6 tag. The poly-His(6) tag is a standard peptide tag used for
single-step purification of recombinant proteins on immobilized metal ion affinity
chromatography, using Ni2+ or Co2+-loaded resins.149 This procedure has been used for the
purification of other Leishmania kinases besides LdGSK-3s,111 such as LmjCK1.2,118
LmxMAPK4 and LmxMKK5,150 LdAIRK1,123 LmxCRK3,151 and more recently
LdMAPK3.107 Expression and purification of recombinant LdGSK-3s was carried out
according to the work of Rachidi et al.118 with two modifications: i) disruption by French
press instead of sonication, owing to the easier control of the temperature of the process; and
ii) purification via Ni2+-column over the Co2+-column due to its slightly higher performance.
66
The transfected E. coli BL21(D3) bacteria were selectively grown in 1 L of
Luria-Bertani broth with 50 μg/mL kanamycin. Expression of LdGSK-3s was induced at an
OD595nm = 0.6 by addition of 1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG)
(Figure 10, panel A). Cells were harvested by centrifugation 3 h later, and LdGSK-3s was
subsequently purified by cellular disruption of the pellet with Lysis Buffer and extrusion
through a French press, followed by solubilization of the enzyme from the lysate with 0.1%
TX-100. Afterwards, the insoluble debris from the lysate was removed by centrifugation.
The soluble LdGSK-3s was purified by affinity chromatography on a 5 mL Ni2+-column
profiting from its poly-(His)6 tag. Non-bound material and weak unspecific binding of
contaminants were removed by 15 column volumes of Washing Buffer containing 30 mM
imidazole, which competes with His residues in the binding to the Ni2+-column. Finally,
LdGSK-3s was eluted with 4 column volumes of Elution Buffer containing 300 mM
imidazole, and collected in fractions of 1.8 mL of volume. The SDS-PAGE protein patterns
of fractions and samples of each purification step are compiled in Figure 10 (panels B and
C). In these polyacrylamide gels, LdGSK-3s appears as a wide band centered at ~40 kDa.111
Results and discussion
67
Figure 10. SDS-PAGE electrophoresis of the samples from each purification step. LdGSK-3s is
highlighted in a red box. M: molecular weight marker. A) Protein pattern of E. coli
BL21(D3)-pET28a(+)LdGSK-3s before and after 3 h induction with 1mM IPTG. B) Protein pattern for
each purification step. I: cell culture (pellet) solubilized in Lysis Buffer. II: Lysate solution after French
press lysis. III: Supernatant obtained after 30 min of solubilization with 0.1% TX-100 and centrifugation
of the lysate solution. IV: Non-bound material from the supernatant. V-VIII: Samples from the
exhaustive washing of the column with Washing Buffer. C) SDS-PAGE pattern of the fractions (1-18)
eluted from the Ni2+ column with Elution buffer.
In order to remove the imidazole, the highly purified LdGSK-3s from fractions 3-18
was pooled and dialyzed in an Amicon filtration device with a 30 kDa membrane cut-off
against Kinase PBS. Next, non-soluble material was removed from the protein solution by
centrifugation. Finally, the enzyme was filtered through a 0.22 μm pore filter, and the
resulting LdGSK-3s (Figure 11; sample d, circled in green) was quantified with
bicinchoninic acid, using bovine serum albumin for the standard curve. One litre of E. coli
BL21(D3)-pET28a(+)LdGSK-3s culture (OD595nm = 0.6) after induction yielded 11.86 mg
68
of LdGSK-3s. For the sake of comparison, the reported yield of recombinant LdMAPK3 was
around 2-4 mg/L.107
Figure 11. Electrophoretic pattern of the different steps of the concentration and dialysis of
LdGSK-3s. M: molecular weight marker. a: Pooled fractions (3-18) from imidazole elution. b: Dialyzed
protein retained by the Amicon, 30 kDa cut-off membrane. c: Ultracentrifuged protein (147,000 ×g, 1 h).
d: Resulting LdGSK-3s after filtration through a 0.22 μm nitrocellulose filter. e-h: Eluted samples from
the four consecutive washings in the Amicon device. i: Pellet from the ultracentrifugation step.
Next, we checked the kinase activity of the purified LdGSK-3s through a
luminescence-based activity test using the Kinase-Glo® kit (Promega), which allows the
measurement of ATP consumption by the kinase. The reagents of this kit consist of a
thermostable luciferase plus D-luciferin that emit luminescence in the presence of Mg2+, O2
and ATP (Figure 12). Addition of the Kinase-Glo® reagents to a kinase reaction quenches
the reaction and allows indirect measuring of the kinase activity through the end-point
luminescence generated by the luciferase with the remaining ATP. This method avoids less
safer alternatives, such as radioactive assays based on the incorporation of γ-32P or γ-33P into
proteins or peptide substrates, as well as more expensive and time consuming assays based
on phosphorylation-dependent changes in fluorescence or luminescence of tagged peptide
substrates and/or substrate-specific antibodies.152
Results and discussion
69
Figure 12. Mechanism of ATP measurement in the Kinase-Glo® method.153
The use of the Kinase-Glo® kit was first described for the study of HsGSK-3β
activity,153 and has been used with the GSK-3 of other organisms145 including
Leishmania,111,146 as well as for other PKs.154,155 The main limitation of this method is that
contamination with other ATP-hydrolysing enzymes leads to overestimation of the kinase
activity.152 To avoid this issue, GSK-3 specific activity was achieved using
phosphoglycogen synthase peptide 2 (GS-2, sometimes named GS-1;
YRRAAVPPSPSLSRHSSPHQ(pS)EDEEE) as substrate for LdGSK-3s (Figure 13, panel
A), as described for HsGSK-3β,156 TbGSK-3s145 and Plasmodium falciparum GSK-3.157 The
use of peptide substrates instead of proteins not only provides higher solubility, but also a
single and specific phosphorylation sequence demanded by the kinase. In fact, GSK-3
preferably recognizes and phosphorylates substrates with a prior phosphorylation under a
S/TXXXpS/T pattern,143 and the commercial GS-2 peptide fulfils this pattern, presenting a
21pS residue phosphorylated by CK-2.156
Moreover, inhibition of kinase activity with indirubin-3’-monoxime-5-sulphonic acid,
a known inhibitor of GSK-3,140 was used to confirm that the measured ATP consumption
was specific to LdGSK-3s (Figure 13, panel B). The experimental IC50 on LdGSK-3s
determined in our assays for this inhibitor (IC50 = 2.39 ± 0.22 μM) was similar to that
described on the short isoform of GSK-3 from L. infantum (LiGSK-3s,
70
IC50 = 2.22 ± 0.07 μM),140 as expected from the 100% sequence identity between
L. infantum and L. donovani GSK3-s.111
Figure 13. Validation of the LdGSK3-s kinase activity. Panel A: Controls for GSK-3 kinase activity.
Panel B: IC50 for the inhibitor indirubin-3’-monoxime-5-sulphonic.
4.2. Selection of the different compounds tested
In a first step, four different sets of compounds from our in-house chemical library138
were assessed. Set 1 consisted of 27 described inhibitors of human PKs such as GSK-3β,
CK1 or LRRK2. Remarkably, besides GSK-3β, CK1 also has a corresponding counterpart
in Leishmania, named CK1.2. Set 2 to 4 encompassed compounds selected from our
chemical library after performing virtual screening on the GSK-3s,111 CK1.2118 and
MAPK4109 of Leishmania, all three validated as pharmacological targets against the parasite.
Virtual screening is a powerful tool for the selection of chemical structures with putative
binding capability to a specific given locus in the targeted protein. In Leishmania, a recent
work has already profited from this strategy to primarily identify putative inhibitors of the
Akt-like kinase of this parasite,94 while other studies used these tools to study enzyme-
inhibitor interactions in the AIRK123 and CDK12104 protein kinases of the parasite. In this
work, virtual screening was extensively used for a primary screening as well as to infer the
feasible location of the binding site inside the targeted protein. For the virtual screening on
Results and discussion
71
the GSK-3s of Leishmania, the search was focused on allosteric pockets of the enzyme using
the crystal structure of LmjGSK-3s,140 leading to Set 2 with 24 compounds. With regard to
the selection of potential inhibitors of LmjCK1.2 and LmxMAPK4, as no crystal structures
were available, homology models of both enzymes were built prior to performing a virtual
screening focused on their catalytic site. As a result, 14 compounds (Set 3) were selected as
in silico hits for the LmjCK1.2 model and 28 (Set 4) for the LmxMAPK4 model.158,159
Two additional subsets of compounds, Set 1.1 and Set 2.1, were also included in this
work, selected as a refinement of those compounds within Set 1 to 4 with the highest
performance in the initial assays for leishmanicidal and LdGSK-3s inhibition, as well as
cytotoxicity.
4.3. Assessment of the biological and inhibitory activities of the selected
compounds
The phenotypic screening and IC50 estimations for Leishmania parasites were
specifically carried out with axenic cultures of promastigotes and amastigotes in exponential
phase. Promastigotes in exponential growth phase (procyclic promastigotes) were used to
better study the effect of our compounds on the active metabolism of this proliferative stage,
in contrast from those in stationary growth phase, which are mostly constituted by non-
dividing metacyclic promastigotes with a low metabolic rate.160 The use of an established
line of axenic amastigote culture of L. (amazonensis) pifanoi ensured homogeneity and
afforded a large production of amastigotes while adhering to the 3R principle to spare animal
lives, which is not fulfilled with lesion-derived amastigotes. Axenic amastigote cultures are
obtained by induction of amastigote differentiation in axenic promastigote cultures via
stepwise increase of temperature and acidic pH besides the use of specific media. Axenic
amastigote cultures have been achieved for many Leishmania species including L. donovani,
L. tropica, L. mexicana, L. braziliensis, L. panamensis, L. amazonensis and the more
recently discovered L. orientalis.161-164 These axenic amastigotes are closely similar to
lesion-derived amastigotes, mimicking several different morphological, biological,
molecular, biochemical and immunochemical features, including their metabolic profile,
72
with upregulation of stage-specific molecules and conservation of their infectivity.68,163 With
regard to their pharmacological profile, amphotericin B showed similar activity on both
axenic and lesion-derived amastigotes, whereas the axenic amastigote model lacks this
translational aspect when the activity of the drug is dependent on its activation by the
macrophage (as reported for pentavalent antimonials)165 and/or modulation of the
macrophage (as described for miltefosine).166 The amastigote axenic model was used in this
work for a primary phenotypic screening to ensure the effectivity of selected compounds
against this form of the parasite. Those hits with proliferation and LdGSK-3s inhibitory
activities were further evaluated on amastigote-infected macrophages to assess the relevance
of the host cell for their physiological leishmanicidal activity.
4.3.1. Reported inhibitors of human protein kinases (Sets 1 and 1.1)
Compounds from Set 1 (1-27) are reported inhibitors of human kinases. Compounds
1 to 11 are human GSK-3 inhibitors from different chemical families (including
thiadiazolidindione,167 iminothiadiazole,168 quinoline,169 halomethylketone,170 maleimide171
and thiazole172 derivatives) and different enzyme-binding modes. Compounds 12 to 20 are
ATP-competitive inhibitors of human CK1 bearing a benzothiazole173 or imidazole174
scaffold in their structure, and compounds 21 to 27 are indolinone-like175 LRRK2 inhibitors.
The biological activities of these compounds on Leishmania promastigotes, amastigotes, and
MPMs are compiled in Table 7.
From the phenotypic assays carried out as described in Material and Methods, only 9
out of the 27 compounds from Set 1 (compounds 1, 2, 5, 6, 8, 10, 19, 25 and 26) resulted
active on Leishmania. Their leishmanicidal activities were consistently higher on axenic
amastigotes than on promastigotes, except for the indolinone 25, with a similar activity on
both stages. For compounds 5 and 8, leishmanicidal activity was limited to the amastigote
form. Within the 9 leishmanicidal compounds, cytotoxicity on MPMs was always lower than
their respective leishmanicidal activity on amastigotes except for the benzothiazole 19.
Moreover, the highest selectivity index values (SI) were found in the thiadiazolidindione 1,
the maleimide 8 and the indolinone 26 (SIs = 16.5, >20.0 and 23.8, respectively).
Results and discussion
73
With regard to the enzymatic activity on LdGSK-3s, no inhibitory effect was observed
for the human CK1 and LRRK2 inhibitors from this set. In contrast, within the human
GSK-3 inhibitors, the thiadiazolidindiones (TDZDs) 1 and 2, the iminothiadiazoles
(ITDZs) 3 and 4, and the halomethylketones (HMKs) 6 and 7 showed inhibition of
LdGSK-3s at micro- and nanomolar range.
In order to get a deeper insight into the putative structure-activity relationship (SAR)
of active compounds from Set 1, a focused set of 20 compounds (Set 1.1) was gathered
(Table 8). This second selection included ten derivatives of the GSK-3 inhibitor 6 and
maleimide 7 (compounds 28 to 37) and ten benzothiazole derivatives of the CK1 inhibitor
19 (compounds 38 to 47). From this set, eight compounds (compounds 28-30, 32, 33, 36, 43
and 46) showed significant leishmanicidal activity (IC50s <25 μM). Only the benzothiazole
43 was more active on promastigotes than amastigotes. The best selectivity profiles were
those of compounds 28, 36 and 46 (SI = 14.6, ~11.6 and >25 respectively).
As observed for Set 1, none of the compounds from Set 1.1 that were analogues of the
CK1 inhibitor 19 had activity on the GSK-3s of Leishmania. Among the HsGSK-3
inhibitors from this set, those that act through an irreversible binding mode on the human
enzyme (compounds 28 to 33) were inhibitors of LdGSK-3s, except for compounds 30 and
31. When inhibition of LdGSK-3s was compared to the leishmanicidal activity within each
compound, only compound 33 showed a higher inhibitory effect on the enzyme than on the
parasite.
Altogether, only the TDZDs 1 and 2 and the HMK 6 from Set 1 and the HMK
derivatives 28, 29, 32 and 33 from Set 1.1 showed both leishmanicidal activity and inhibition
of LdGSK-3s. In contrast, the ITDZs 3 and 4 were potent LdGSK-3s inhibitors with IC50s at
submicromolar range, but lacked leishmanicidal activity.
74 Table 7. Enzymatic and biological characterization of Set 1 (selected human protein kinase inhibitors).
Compound Chemical formula LdGSK-3s
IC50 (μM)
L. infantum
promastigotes
IC50 (μM)
L. pifanoi
axenic
amastigotes
IC50 (μM)
Murine
peritoneal
macrophages
IC50 (μM)
Selectivity
index (SI)
1
1.1 ± 0.2 10.9 ± 0.4 2.0 ± 1.9 32.9 ± 3.5 16.5
2
0.32 ± 0.05 17.6 ± 2.3 7.1 ± 1.8 >50 >7.0
3
0.248 ± 0.004 >25 >50 - -
4
0.174 ± 0.001 >25 >50 - -
5
>10 >50 3.6 ± 1.3 9.9 ± 0.9 2.8
S
N
N
NNO
2HBr
Resu
lts and d
iscussio
n
75
Table 7 (continued). Enzymatic and biological characterization of Set 1 (selected human protein kinase inhibitors).
Compound Chemical formula LdGSK-3s
IC50 (μM)
L. infantum
promastigotes
IC50 (μM)
L. pifanoi
axenic
amastigotes
IC50 (μM)
Murine
peritoneal
macrophages
IC50 (μM)
Selectivity
index (SI)
6
1.8 ± 0.3 4.6 ± 0.2 2.2 ± 0.6 6.3 ± 1.2 2.9
7
17.7 ± 2.7 >50 >50 - -
8
>10 >50 2.5 ± 2.7 >50 >20.0
9
>10 >50 >50 - -
10
>10 >25 14.4 ± 2.6 32.0 ± 3.1 2.2
HN
O O
O
NHBr
HN
O O
O
NH
Me
MeO
NH
NH
O
S
N
Me
O
Me
MeO
NH
NH
O
S
N
Me
O
Br
76 Table 7 (continued). Enzymatic and biological characterization of Set 1 (selected human protein kinase inhibitors).
Compound Chemical formula LdGSK-3s
IC50 (μM)
L. infantum
promastigotes
IC50 (μM)
L. pifanoi
axenic
amastigotes
IC50 (μM)
Murine
peritoneal
macrophages
IC50 (μM)
Selectivity
index (SI)
11
>10 >50 >50 - -
12
>10 >50 >50 - -
13
>10 >50 >50 - -
14
>10 >50 >50 - -
MeO
NH
NH
O
S
N
Me
O
Br
Br
N
SNH
O
Cl
F3C
N
SNH
O
F3C
N
SNH
O
MeO
Cl
Resu
lts and d
iscussio
n
77
Table 7 (continued). Enzymatic and biological characterization of Set 1 (selected human protein kinase inhibitors).
Compound Chemical formula LdGSK-3s
IC50 (μM)
L. infantum
promastigotes
IC50 (μM)
L. pifanoi
axenic
amastigotes
IC50 (μM)
Murine
peritoneal
macrophages
IC50 (μM)
Selectivity
index (SI)
15
>10 >50 >50 - -
16
>10 >50 >50 - -
17
>10 >50 >50 - -
18
>10 >50 >50 - -
N
SNH
O
EtO
Cl
N
SNH
O
F3C
Cl
N
SNH
O
F3C
OMe
N
SNH
O
F3C
Cl
Cl
78 Table 7 (continued). Enzymatic and biological characterization of Set 1 (selected human protein kinase inhibitors).
Compound Chemical formula LdGSK-3s
IC50 (μM)
L. infantum
promastigotes
IC50 (μM)
L. pifanoi
axenic
amastigotes
IC50 (μM)
Murine
peritoneal
macrophages
IC50 (μM)
Selectivity
index (SI)
19
>10 32.8 ± 3.9 20.3 ± 1.9 >12.5 >0.6
20
>10 >25 >50 - -
21
>10 >25 >25 - -
22
>10 >25 >25 - -
N
SNH
O
Cl
Cl
N
N
Resu
lts and d
iscussio
n
79
Table 7 (continued). Enzymatic and biological characterization of Set 1 (selected human protein kinase inhibitors).
Compound Chemical formula LdGSK-3s
IC50 (μM)
L. infantum
promastigotes
IC50 (μM)
L. pifanoi
axenic
amastigotes
IC50 (μM)
Murine
peritoneal
macrophages
IC50 (μM)
Selectivity
index (SI)
23
>10 >50 >50 - -
24
>10 >50 >50 - -
25
>10 4.1 ± 0.1 4.5 ± 1.5 >25 >5.6
26
>10 22.9 ± 2.6 2.1 ± 0.2 ~50* ~23.8
27
>10 >50 >50 - -
* The viability of macrophages was 55.2 ± 6.1 % for compound 26 at 50 μM
NH
N
O
NH
Me
80 Table 8. Enzymatic and biological characterization of Set 1.1 (second selection of human protein kinase inhibitors).
Compound Chemical formula LdGSK-3s
IC50 (μM)
L. infantum
promastigotes
IC50 (μM)
L. pifanoi
axenic
amastigotes
IC50 (μM)
Murine
peritoneal
macrophages
IC50 (μM)
Selectivity
index (SI)
28
7.5 ± 1.4 6.6 ± 0.5 0.5 ± 0.1 7.3 ± 1.0 14.6
29
10.3 ± 2.0 4.4 ± 0.1 2.4 ± 0.4 3.6 ± 0.5 1.5
30
>10 14.2 ± 1.0 1.2 ± 0.2 3.2 ± 0.4 2.7
31
>10 >50 >50 - -
32
3.0 ± 0.4 8.6 ± 0.3 1.2 ± 0.2 7.9 ± 0.6 6.6
33
1.6 ± 0.2 >50 6.5 ± 2.0 >25 >3.8
HN
O O
O
N
MeBr
Resu
lts and d
iscussio
n
81
Table 8 (continued). Enzymatic and biological characterization of Set 1.1 (second selection of human protein kinase inhibitors).
Compound Chemical formula LdGSK-3s
IC50 (μM)
L. infantum
promastigotes
IC50 (μM)
L. pifanoi
axenic
amastigotes
IC50 (μM)
Murine
peritoneal
macrophages
IC50 (μM)
Selectivity
index (SI)
34
>10 >50 >25 - -
35
>10 >50 29.5 ± 9.8 >25 >0.8
36
>10 >50 4.3 ± 0.2 ~50* ~11.6
37
>10 >50 >50 - -
* The viability of macrophages was 51.0 ± 1.9 % for compound 36 at 50 μM.
HN
O O
O
NMe
Me
HN
O O
O
NH
NMe
HN
O O
O
NNMe
Me
HN
O O
O
NH
Me Me
82 Table 8 (continued). Enzymatic and biological characterization of Set 1.1 (second selection of human protein kinase inhibitors).
Compound Chemical formula LdGSK-3s
IC50 (μM)
L. infantum
promastigotes
IC50 (μM)
L. pifanoi
axenic
amastigotes
IC50 (μM)
Murine
peritoneal
macrophages
IC50 (μM)
Selectivity
index (SI)
38
>10 >50 >50 - -
39
>10 >50 >50 - -
40
>10 >50 >50 - -
41
>10 >50 >50 - -
N
SNH
O
MeO
OMe
N
SNH
O
EtO
OMe
N
SNH
O
F3C
OMe
N
SNH
O
F3C
OMe
Resu
lts and d
iscussio
n
83
Table 8 (continued). Enzymatic and biological characterization of Set 1.1 (second selection of human protein kinase inhibitors).
Compound Chemical formula LdGSK-3s
IC50 (μM)
L. infantum
promastigotes
IC50 (μM)
L. pifanoi
axenic
amastigotes
IC50 (μM)
Murine
peritoneal
macrophages
IC50 (μM)
Selectivity
index (SI)
42
>10 >50 >50 - -
43
>10 8.1 ± 1.6 13.3 ± 1.7 4.1 ± 2.8 0.3
44
>10 >50 >50 - -
N
SNH
NHO
F3C
OMe
N
SNH
NHO
F3C
Cl
N
SNH
NHO
Me
OMe
Me
O
84 Table 8 (continued). Enzymatic and biological characterization of Set 1.1 (second selection of human protein kinase inhibitors).
Compound Chemical formula LdGSK-3s
IC50 (μM)
L. infantum
promastigotes
IC50 (μM)
L. pifanoi
axenic
amastigotes
IC50 (μM)
Murine
peritoneal
macrophages
IC50 (μM)
Selectivity
index (SI)
45
>10 >50 >50 - -
46
>10 6.5 ± 0.7 1.0 ± 0.1 >25 >25.0
47
>10 >50 >50 - -
N
SNH
F3C
OMe
N
SNH
O
Me
EtO
O
N
S
Me
NH
EtO
O
OCl
Results and discussion
85
Additional computational studies were performed to decipher the binding mode of the
LdGSK-3s inhibitors from these sets and explain the lack of activity of some HsGSK-3β
inhibitors. TDZDs 1 and 2 are well-known non-ATP competitive HsGSK-3β inhibitors167
that bind covalently to the thiol group of Cys199 at the active site of GSK-3,176 as also
described for the HMK family171 (6, 28, 29, 32, as well as compound 33, a maleimide with
a HMK unit). Since the Cys199 is conserved in the Leishmania GSK-3s as Cys169, this
residue was expected to behave as the acceptor group for the covalent inhibition of
LdGSK-3s by compounds 1, 2, 6, 28, 29, 32 and 33. In contrast, the ITDZs 3 and 4 are
described substrate-competitive HsGSK-3β inhibitors.168 As the binding region for the
substrate in the GSK-3 in Leishmania is rather similar to the human one, compounds 3 and
4 were surmised to interact with LdGSK-3s through this site. These hypotheses were
subsequently confirmed by different molecular docking studies carried out on the crystal
structure of LmjGSK-3s140 with compounds 2, 3, 4 and 6 as representative molecules (Figure
14). The maleimide 34, an HsGSK-3β reversible inhibitor with nil inhibitory activity on
LdGSK-3s, was also included in the molecular docking studies for the sake of comparison
with the maleimide 33, an HsGSK-3β irreversible inhibitor with LdGSK-3s inhibition. Both
maleimides are reported as ATP-competitive HsGSK-3β inhibitors with almost identical
structure, differing in the presence of the HMK tail in compound 33 with respect to its
absence in compound 34.171 It was identified that the lack of LdGSK-3s inhibition by
maleimide 34 obeys the restricted access of this compound into the ATP binding cavity due
to the Met100 residue of LGSK-3s, which is a bulkier gatekeeper than Leu132 in HsGSK-3β
(Figure 15). Additionally, docking studies with quinoline 5, an HsGSK-3β allosteric
inhibitor, showed that its inability to inhibit LdGSK-3s was caused by the replacement of
the flexible and cationic Arg209 from the human kinase with the neutral and rigid Pro178 in
the leishmanial GSK-3s.177
86
Figure 14. Binding mode into the LmjGSK-3 enzyme for ITDZ, TDZD, and HMK representative
compounds.177 A) Blind docking poses obtained from the most representative clusters for 2 (TDZD) and
4 (ITDZ) in the ATP binding site and the substrate binding site, respectively. B) Superimposition of most
representatives regular docking results of ITDZ compounds 3 and 4. C) Superimposition of the best
covalent docking poses obtained for TDZDs (1 and 2) and HMK 6. D) Detailed view of the covalent
docking for compound 2.
Results and discussion
87
Figure 15. A) Superposition of the human (purple) and Leishmania (green) GSK-3 enzymes. Key
mutations in the ATP binding site are depicted as sticks. B) Superposition of the binding poses of
maleimide 34 to the human (purple) and Leishmania (green) GSK-3 enzymes.177
The reactivity of covalent inhibitors like TDZDs 1 and 2 as well as the HMKs 6, 28,
29, 32 and 33 towards thiol groups may cause cross-inhibition with thiol-dependent
enzymes. In Leishmania, such enzymes include the dimeric trypanothione reductase,
tryparedoxin and tryparedoxin peroxidase from the thiol-based redox system,80 adenosine
kinase from the purine savage pathway,178 carbonic anhydrases179 and cysteine proteases
among others. Remarkably, antimonials also have high affinity for thiol containing
biomolecules,180 suggesting a feasible synergy with GSK-3 inhibitors. As a downside, owing
to their potential unspecific-reactivity with nucleophilic residues, covalent inhibitors are
commonly defined as PAINs (pan-assay interference compounds). PAINs usually appear as
very promising hits by interfering in the readout of many biological assays,181 thus they are
usually removed from initial screenings. Nevertheless, the number of drugs acting through
covalent binding is far from scarce, and includes FDA approved drugs such as salicylic acid,
β-lactam antibiotics and several antitumoral drugs.182 A crucial aspect for the effectiveness
and safety of irreversible inhibitors is the turnover rate of their targets. This is especially
relevant in infectious diseases where the same compound can target both the pathogen
enzyme and its human counterpart, as proven by eflornithine (α-difluoromethylornithine;
88
DFMO). DFMO, an anti-trypanosomatid drug for African trypanosomiasis, is an irreversible
inhibitor of the ornithine decarboxylase (ODC) enzyme, a key regulator of the polyamine
biosynthesis pathway. DFMO has similar affinity and effective inhibition on both the human
and the T. brucei gambiense ODC, but the slower turnover rate (t1/2 over 6 h) of the parasitic
enzyme with respect to its human homolog (t1/2 <1 h) endows DFMO with inhibitory
specificity for the ODC of T. brucei.183 The turnover rate of the GSK-3s in Leishmania is
still unknown, whereas the reported half-life of GSK-3β in murine neurons is 41 (± 4)176 -
48184 h. Since intracellular amastigotes have higher generation time (33 h),185 achieving
selective effect on the GSK-3s of Leishmania amastigotes due to turnover rate differences
seems unlikely.
Nevertheless, when the in vitro inhibition of LdGSK-3s is compared with that of
HsGSK-3β (Table 9), the TDZDs 1 and 2, and the ITDZs 3 and 4 had higher inhibitory
activity on LdGSK-3s than on the human one, thus being appealing candidates for further
optimization.
Table 9. Inhibition of HsGSK-3β of the leishmanicidal LdGSK-3s inhibitors from Sets 1 and 1.1
(selections of human protein kinase inhibitors).
Compound HsGSK-3β
IC50 (μM)
LdGSK-3s
IC50 (μM)
1 2167 1.1 ± 0.2
2 0.7 ± 0.1186 0.32 ± 0.05
3 0.88168 0.248 ± 0.004
4 1.95168 0.174 ± 0.001
6 0.5170 1.8 ± 0.3
28 1.0170 7.5 ±1.4
29 5.0170 10.3 ±2.0
32 1.0170 3.0 ± 0.4
33 0.005 ± 0.001171 1.6 ± 0.2
Results and discussion
89
Furthermore, the TDZD 2 is also known as tideglusib, and is currently in clinical trials
as an HsGSK-3β inhibitor for neurological disorders (phase II), with little to no signs of
toxicity at daily doses ranging from 400 to 1,000 mg.187 Hence, owing to its toxicity profile
and higher activity on LdGSK-3s, tideglusib is an appealing candidate from drug
repurposing.
4.3.2. In silico inhibitors of LmjGSK-3s (Set 2), LmjCK1.2 (Set 3) and
LmxMAPK4 (Set 4)
As aforementioned, Xingi et al.111 first demonstrated the successful use of HsGSK-3β
inhibitors as a source for Leishmania GSK-3s inhibitors when they reported the finding of
LdGSK-3s inhibitors among some 6-bromo indirubins, which are strong inhibitors of
mammalian CDKs and GSK-3. While none of the compounds had higher affinity for
LdGSK-3s over the mammalian GSK-3β, Xingi and co-authors suggested focusing on amino
acid differences at the binding site of the enzyme as an approach for hit optimization. Ojo
and co-authors140 also demonstrated the appeal of drug repurposing by demonstrating
cross-inhibition of commercial HsGSK-3β inhibitors on the GSK-3s of different Leishmania
species. Only TDZDs 1 and 2 and ITDZs 3 and 4 from our screening of Set 1 had higher
activity on the Leishmania GSK-3s over HsGSK-3β (Table 9), thus we were prompted to
carry out virtual screenings of our chemical library (constituted by more than 2,000
compounds) for the selection of new groups of compounds. Virtual screening is a useful
technique to carry out massive target-based screenings of compounds on a targeted protein
when its structure, or at least the binding sites, are well defined.188 In Leishmania, this
approach was endorsed by the finding of two specific inhibitors in vitro of the Akt-like
kinase of L. panamensis, primarily selected from the virtual screening of a 600,000-
compound library focused on the regulatory pleckstrin domain of this modelled kinase.94
Thus, Sets 2 to 4 were selected from our in-house chemical library by virtual
screenings. To this end, the targets were expanded from the initial GSK-3s, to CK1.2 and
MAPK4, two additional pharmacologically validated protein kinases of Leishmania.109,118
90
Set 2 (compounds 48-71) was originated from the virtual screening against the
allosteric pockets of LmjGSK-3s,159 using the crystal structure of the enzyme provided by
Ojo et al.140 The lower conservation of allosteric pockets among protein kinases with their
active site opens a broader avenue for the finding of specific inhibitors. In Leishmania, the
successful exploration of allosteric pockets was reported through the identification of 4,6-
diamino substituted pyrazolopyrimidines as new inhibitors against L. major
methionyl-tRNA synthetase (MetRS), a key enzyme in protein synthesis.189
Our virtual screening on the allosteric pockets of LmjGSK-3s identified 24 compounds
with a variety of chemical scaffolds, and their biological activities were subsequently
evaluated (Table 10). From this set, only the quinone 48 showed a relatively significant
leishmanicidal activity on both stages besides LdGSK-3s inhibition (IC50s ca 10 µM for all
three activities).
Quinones are typical inhibitors of the respiratory chain, antagonizing the function of
mitochondrial ubiquinone and promoting oxidative stress.190,191 They are also considered
PAINs due to their redox-cycling properties and reactivity with nucleophilic residues.
Despite this, quinones such as the antineoplastic anthracyclines and the antitrypanosomatids
atavoquone and buparvaquone, among others, are nowadays in clinical use.181,191 Moreover,
naphthoquinones reportedly exhibit a wide variety of effects, including anticancer, antiviral,
immunomodulatory, trypanocidal and antimicrobial activities, due to inhibition of
respiration or covalent interaction with their targets.192
Hence, we were prompted to synthesize new naphthoquinone compounds to further
explore and optimize this scaffold.159 The biological activities of 23 new naphthoquinone
derivatives 72-94 (Set 2.1) are compiled in Table 11.
Resu
lts and d
iscussio
n
91
Table 10. Enzymatic and biological characterization of Set 2 (in silico LmjGSK-3s inhibitors).
Compound Chemical formula LdGSK-3s
IC50 (μM)
L. infantum
promastigotes
IC50 (μM)
L. pifanoi
axenic
amastigotes
IC50 (μM)
Murine
peritoneal
macrophages
IC50 (μM)
Selectivity
index (SI)
48
~10* 10.5 ± 1.2 11.2 ± 2.5 ND ND
49
>10 >50 >50 - -
50
>10 >50 >50 - -
51
>10 >50 >50 - -
* Compound 48 at 10 μM causes 45.7 ± 7.6 % inhibition of LdGSK-3s activity.
N
SNH
OO O
NH
Me
O
NH
O
HNO
N
HN
O
NHO
92 Table 10 (continued). Enzymatic and biological characterization of Set 2 (in silico LmjGSK-3s inhibitors).
Compound Chemical formula LdGSK-3s
IC50 (μM)
L. infantum
promastigotes
IC50 (μM)
L. pifanoi
axenic
amastigotes
IC50 (μM)
Murine
peritoneal
macrophages
IC50 (μM)
Selectivity
index (SI)
52
>10 >25 >25 - -
53
>10 >50 >50 - -
54
>10 >50 >50 - -
55
>10 >50 >50 - -
56
>10 >50 >50 - -
NH
NH
S N
N CF3
Resu
lts and d
iscussio
n
93
Table 10 (continued). Enzymatic and biological characterization of Set 2 (in silico LmjGSK-3s inhibitors).
Compound Chemical formula LdGSK-3s
IC50 (μM)
L. infantum
promastigotes
IC50 (μM)
L. pifanoi
axenic
amastigotes
IC50 (μM)
Murine
peritoneal
macrophages
IC50 (μM)
Selectivity
index (SI)
57
>10 >50 >50 - -
58
>10 >50 >50 - -
59
>10 >50 >50 - -
60
>10 >50 >50 - -
61
>10 >25 >50 - -
N
N
O
S
Et
F
F
Cl
94 Table 10 (continued). Enzymatic and biological characterization of Set 2 (in silico LmjGSK-3s inhibitors).
Compound Chemical formula LdGSK-3s
IC50 (μM)
L. infantum
promastigotes
IC50 (μM)
L. pifanoi
axenic
amastigotes
IC50 (μM)
Murine
peritoneal
macrophages
IC50 (μM)
Selectivity
index (SI)
62
>10 >50 >50 - -
63
>10 >50 >50 - -
64
>10 >50 >50 - -
65
>10 >50 20.8 ± 0.0 >25 >1.2
66
>10 >50 >50 - -
N
SNH
OO O
NH
OPh
OH
N
Me
O
NH
OHN
O6
OH
N
Me
O
NH
OHN
O
O
Ph
O
Resu
lts and d
iscussio
n
95
Table 10 (continued). Enzymatic and biological characterization of Set 2 (in silico LmjGSK-3s inhibitors).
Compound Chemical formula LdGSK-3s
IC50 (μM)
L. infantum
promastigotes
IC50 (μM)
L. pifanoi
axenic
amastigotes
IC50 (μM)
Murine
peritoneal
macrophages
IC50 (μM)
Selectivity
index (SI)
67
>10 >50 >50 - -
68
>10 >50 >50 - -
69
>10 >50 >50 - -
70
>10 >50 >50 - -
71
>10 >50 >50 - -
N
S
Me
NH
EtO
O
O
OMe
OMe
OMe
NH
NH
O
NOMe
OH
N
Me
O
NH
OHN
O
N
96 Table 11. Enzymatic and biological characterization of Set 2.1 (new quinone analogues).
Compound Chemical formula LdGSK-3s
IC50 (μM)
L. infantum
promastigotes
IC50 (μM)
L. pifanoi
axenic
amastigotes
IC50 (μM)
Murine
peritoneal
macrophages
IC50 (μM)
Selectivity
index (SI)
72
2.5 ± 0.1 1.51 ± 0.00 0.51 ± 0.00 2.6 ± 0.0 5.1
73
>10 1.97 ± 0.20 0.4 ± 0.0 >10 >25
74
>10 >25 10.6 ± 1.0 4.73 ± 0.60 0.4
75
3.7 ± 0.3 1.54 ± 0.00 0.15 ± 0.40 3.1 ± 0.1 20.6
76
2.5 ± 0.2 1.47 ± 0.00 0.4 ± 0.2 4.51 ± 0.30 11.2
Resu
lts and d
iscussio
n
97
Table 11 (continued). Enzymatic and biological characterization of Set 2.1 (new quinone analogues).
Compound Chemical formula LdGSK-3s
IC50 (μM)
L. infantum
promastigotes
IC50 (μM)
L. pifanoi
axenic
amastigotes
IC50 (μM)
murine
peritoneal
macrophages
IC50 (μM)
Selectivity
index (SI)
77
4.5 ± 0.6 3.8 ± 1.9 3.6 ± 0.6 8.1 ± 1.0 2.3
78
4.1 ± 0.1 2.9 ± 0.2 3.7 ± 1.1 7.4 ± 0.8 2.0
79
5.3 ± 1.0 5.3 ± 0.6 1.2 ± 0.4 13.0 ± 2.8 10.8
80
2.8 ± 0.1 4.9 ± 0.2 4.7 ± 0.8 9.8 ± 0.2 2.1
81
>10 19.8 ± 3.6 17.8 ± 1.1 17.0 ± 0.3 1.0
98 Table 11 (continued). Enzymatic and biological characterization of Set 2.1 (new quinone analogues).
Compound Chemical formula LdGSK-3s
IC50 (μM)
L. infantum
promastigotes
IC50 (μM)
L. pifanoi
axenic
amastigotes
IC50 (μM)
Murine
peritoneal
macrophages
IC50 (μM)
Selectivity
index (SI)
82
>10 1.4 ± 0.4 2.1 ± 0.6 1.3 ± 0.3 0.6
83
>10 16.9 ± 0.4 >25 - -
84
>10 19.1 ± 1.2 >25 - -
85
>10 >25 >25 - -
86
>10 >50 >50 - -
Resu
lts and d
iscussio
n
99
Table 11 (continued). Enzymatic and biological characterization of Set 2.1 (new quinone analogues).
Compound Chemical formula LdGSK-3s
IC50 (μM)
L. infantum
promastigotes
IC50 (μM)
L. pifanoi
axenic
amastigotes
IC50 (μM)
Murine
peritoneal
macrophages
IC50 (μM)
Selectivity
index (SI)
87
>10 >25 >25 - -
88
>10 4.5 ± 0.5 1.2 ± 0.1 4.0 ± 0.2 3.3
89
>10 5.9 ± 1.8 1.8 ± 0.3 4.3 ± 0.3 2.4
90
>10 7.1 ± 2.1 3.2 ± 0.1 7.5 ± 0.6 2.3
100
Table 11 (continued). Enzymatic and biological characterization of Set 2.1 (new quinone analogues).
Compound Chemical formula LdGSK-3s
IC50 (μM)
L. infantum
promastigotes
IC50 (μM)
L. pifanoi
axenic
amastigotes
IC50 (μM)
Murine
peritoneal
macrophages
IC50 (μM)
Selectivity
index (SI)
91
>10 >50 >50 - -
92
>10 >25 >25 - -
93
>10 4.1 ± 0.8 1.7 ± 0.2 3.8 ± 0.3 2.2
94
>10 4.5 ± 0.7 1.7 ± 0.1 4.2 ± 0.2 2.5
Results and discussion
101
Eighteen compounds (72-84, 88-90, 93 and 94) showed leishmanicidal activity in one
or both stages of the parasite. The trend towards a higher and preferential leishmanicidal
activity on the amastigote from previous sets was not fully maintained; quinones 77, 78, 80-
82 showed rather similar values for both stages, and activity on amastigotes was not
significant for compounds 83 and 84. The selectivity indexes were higher than 10 for
compounds 73, 75, 76 and 79 (SI= >25, 20.6, 11.2 and 10.8 respectively). Noteworthy,
quinones 72, 75-80 behaved as LdGSK-3s inhibitors with IC50s at low micromolar values.
While compound 48 (Set 2), a 6,7-dichloronapthoquinone, had IC50s around 10 μM
for LdGSK-3s inhibition and antileishmanial activity in both stages, its derivative
2-chloro-3-methoxy-1,4-naphthoquinone (compound 73, Set 2.1) achieved higher
leishmanicidal activities (IC50s = 1.97 ± 0.20 μM for promastigotes, 0.4 ± 0.0 μM for
amastigotes) and SI (>25), but was devoid of inhibitory activity on LdGSK-3s. From the
remaining 22 2-amino-1,4-naphthoquinones from Set 2.1, a trend for the substitution pattern
was observed. Quinones with an amine group (74, 81, 83-87, 91-92) were mostly inactive
against the parasite, whereas those with a urea (82), an amide (88-90), or a carbamate moiety
(72, 75-80, 93, 94) showed high leishmanicidal activities on both Leishmania stages. While
the presence of a chlorine atom at position 3 was not essential for leishmanicidal activity (72
vs 93 and 75 vs 94), its combination with a carbamate moiety (compounds 72, 75-80) was
strongly associated with LdGSK-3s inhibition. The LdGSK-3s inhibitors 72, 75, 76 and 80
did not show HsGSK-3β inhibition at 10 μM (19%, 30%, 25% and 19% of kinase inhibition,
respectively), with quinone 75 showing the highest SI (= 20.6). These findings were of
utmost importance because, to our knowledge, they are the first LdGSK-3 inhibitors with
scarce affinity for HsGSK-3β described so far.
Finally, Sets 3 (compounds 95-108) and 4 (compounds 109-136) were obtained from
virtual screenings on the catalytic site of homology models of LmjCK1.2 and LmxMAPK4
respectively.158,159
The CK1.2 of Leishmania was of special interest since inhibition of its human
counterpart, CK1δ, was reported for some compounds from our chemical library.173 The
search for feasible inhibitors of the Leishmania CK1.2 was exclusively guided by virtual
screening studies due to unsuccessful attempts to obtain a recombinant LmjCK1.2 in a
102
soluble and active form. The lack of crystal structures for LmjCK1.2 was sorted out by
building a homology model of the enzyme. The virtual screening on the ATP binding site of
the modelled LmjCK1.2 led to the identification of 14 potential inhibitors from different
chemical families (Set 3)158 that were subsequently assayed for their biological and
inhibitory activities (Table 12).
The MAPK4 is a genetically validated target against Leishmania that plays a vital role
in stage-differentiation. Thus, a virtual screening focused on the active site of an
LmxMAPK4 model was carried out, aiming to broaden the potentiality of our in-house
chemical library. Virtual screenings on modelled Leishmania MAPK4s have been reported
before, but no additional biological studies endorsed the theoretical results.106,193 Set 4 was
formed with 28 compounds from different chemical families identified in silico as
LmxMAPK4 inhibitors. Results from the subsequent enzymatic and biological assays are
compiled in Table 13.
Unsurprisingly, none of the compounds from Set 3 or 4 inhibited LdGSK-3s, although
a cross-inhibition with other PKs is especially feasible when the highly preserved ATP
binding site is targeted.194
Only the pteridine 100 out of the 14 compounds from Set 3 was significantly active
on the parasite, with an IC50 near 6.5 μM for both amastigote and promastigote stages and a
selectivity index of 8.1. As aforementioned, Leishmania salvages pteridines from its hosts
for its folate metabolism.78 Thus, pteridine 100 may interfere with folate biosynthesis by
inhibition of the pteridine reductase PTR1 of the parasite. PTR1 is a key enzyme in the
pteridine salvaging pathway that also substitutes the dihydrofolate reductase-thymidylate
synthase (DHFR-TS) in folate reduction when DHFR-TS is inhibited.195 As such, pteridine
100 may additionally inhibit PTR1 aside from its presumed inhibition of LmjCK1.2, and
may be synergetic with DHFR-TS inhibitors against the parasite.
Results and discussion
103
From Set 4, six compounds (112, 120, 121, 124, 131 and 133) showed leishmanicidal
activity. Only the benzothiophene 121 was more active on promastigotes than on
amastigotes, whereas compounds 112, 124 and 133 had no effect on the promastigote stage.
The thiazole 133 showed the highest SI (>11.5) of the set.
104
Table 12. Enzymatic and biological characterization of Set 3 (in silico LmjCK1.2 inhibitors).
Compound Chemical formula LdGSK-3s
IC50 (μM)
L. infantum
promastigotes
IC50 (μM)
L. pifanoi
axenic
amastigotes
IC50 (μM)
Murine
peritoneal
macrophages
IC50 (μM)
Selectivity
index (SI)
95
>10 >50 25.8 ± 5.9 >25 >0.6
96
>10 >50 >50 - -
97
>10 >50 >50 - -
98
>10 >50 25.1 ± 2.9 >25 >1.0
99
>10 >50 >50 - -
NH
N
O
O OMe
Resu
lts and d
iscussio
n
105
Table 12 (continued). Enzymatic and biological characterization of Set 3 (in silico LmjCK1.2 inhibitors).
Compound Chemical formula LdGSK-3s
IC50 (μM)
L. infantum
promastigotes
IC50 (μM)
L. pifanoi
axenic
amastigotes
IC50 (μM)
Murine
peritoneal
macrophages
IC50 (μM)
Selectivity
index (SI)
100
>10 6.6 ± 0.3 6.2 ± 0.0 >50 >8.1
101
>10 >50 >50 - -
102
>10 >50 >50 - -
103
>10 >50 >50 - -
104
>10 >50 >50 - -
OMe
MeO OMe
NH
O
O
O2N
Me
N
N
O
S
NH2
106
Table 12 (continued). Enzymatic and biological characterization of Set 3 (in silico LmjCK1.2 inhibitors).
Compound Chemical formula LdGSK-3s
IC50 (μM)
L. infantum
promastigotes
IC50 (μM)
L. pifanoi
axenic
amastigotes
IC50 (μM)
Murine
peritoneal
macrophages
IC50 (μM)
Selectivity
index (SI)
105
>10 >50 >25 - -
106
>10 >50 >50 - -
107
>10 >50 >50 - -
108
>10 >50 >50 - -
NH
NH
HN
ON
N
Me
O
N
N
N
O
S
N
Cl
Resu
lts and d
iscussio
n
107
Table 13. Enzymatic and biological characterization of Set 4 (in silico LmxMAPK4 inhibitors).
Compound Chemical formula LdGSK-3s
IC50 (μM)
L. infantum
promastigotes
IC50 (μM)
L. pifanoi
axenic
amastigotes
IC50 (μM)
Murine
peritoneal
macrophages
IC50 (μM)
Selectivity
index (SI)
109
>10 >50 >50 - -
110
>10 >50 >25 - -
111
>10 >50 >50 - -
112
>10 >50 15.2 ± 1.6 >25 >1.6
113
>10 >50 >50 - -
NH
NH
S
N
NH
O
S
NMe
Ph
MeO
NH
O
N
N
Cl
108
Table 13 (continued). Enzymatic and biological characterization of Set 4 (in silico LmxMAPK4 inhibitors).
Compound Chemical formula LdGSK-3s
IC50 (μM)
L. infantum
promastigotes
IC50 (μM)
L. pifanoi
axenic
amastigotes
IC50 (μM)
Murine
peritoneal
macrophages
IC50 (μM)
Selectivity
index (SI)
114
>10 >50 >50 - -
115
>10 >50 >50 - -
116
>10 >50 >50 - -
117
>10 >50 >50 - -
118
>10 >50 >25 - -
NNH
N
O
S
Resu
lts and d
iscussio
n
109
Table 13 (continued). Enzymatic and biological characterization of Set 4 (in silico LmxMAPK4 inhibitors).
Compound Chemical formula LdGSK-3s
IC50 (μM)
L. infantum
promastigotes
IC50 (μM)
L. pifanoi
axenic
amastigotes
IC50 (μM)
Murine
peritoneal
macrophages
IC50 (μM)
Selectivity
index (SI)
119
>10 >50 >50 - -
120
>10 11.0 ± 0.7 3.0 ± 0.3 14.3 ± 0.9 4.8
121
>10 3.7 ± 0.4 11.8 ± 0.7 >25 >2.1
122
>10 >50 >50 - -
NH
O
Cl
N
N
110
Table 13 (continued). Enzymatic and biological characterization of Set 4 (in silico LmxMAPK4 inhibitors).
Compound Chemical formula LdGSK-3s
IC50 (μM)
L. infantum
promastigotes
IC50 (μM)
L. pifanoi
axenic
amastigotes
IC50 (μM)
Murine
peritoneal
macrophages
IC50 (μM)
Selectivity
index (SI)
123
>10 >50 >50 - -
124
>10 >50 13.1 ± 4.3 >50 >3.8
125
>10 >50 >50 - -
126
>10 >50 >50 - -
N
SNH
HNO
Me
EtO
OO
Resu
lts and d
iscussio
n
111
Table 13 (continued). Enzymatic and biological characterization of Set 4 (in silico LmxMAPK4 inhibitors).
Compound Chemical formula LdGSK-3s
IC50 (μM)
L. infantum
promastigotes
IC50 (μM)
L. pifanoi
axenic
amastigotes
IC50 (μM)
Murine
peritoneal
macrophages
IC50 (μM)
Selectivity
index (SI)
127
>10 >50 >50 - -
128
>10 >50 >50 - -
129
>10 >50 >50 - -
130
>10 >50 >50 - -
OMe
MeO OMe
O
O
O
O2N
Cl
OMe
MeO OMe
O
O
O
Cl
Cl
OMe
MeO OMe
O
O
O
Cl
OH
N
Et
O
NH
OHN
O
OH
112
Table 13 (continued). Enzymatic and biological characterization of Set 4 (in silico LmxMAPK4 inhibitors).
Compound Chemical formula LdGSK-3s
IC50 (μM)
L. infantum
promastigotes
IC50 (μM)
L. pifanoi
axenic
amastigotes
IC50 (μM)
Murine
peritoneal
macrophages
IC50 (μM)
Selectivity
index (SI)
131
>10 9.1 ± 0.5 3.8 ± 0.6 8.8 ± 1.5 2.3
132
>10 >50 >50 - -
133
>10 >50 1.3 ± 0.1 >15 >11.5
134
>10 >50 >50 - -
OMe
MeO OMe
O
O
O
Cl
CF3
Resu
lts and d
iscussio
n
113
Table 13 (continued). Enzymatic and biological characterization of Set 4 (in silico LmxMAPK4 inhibitors).
Compound Chemical formula LdGSK-3s
IC50 (μM)
L. infantum
promastigotes
IC50 (μM)
L. pifanoi
axenic
amastigotes
IC50 (μM)
Murine
peritoneal
macrophages
IC50 (μM)
Selectivity
index (SI)
135
>10 >50 >50 - -
136
>10 >50 >50 - -
OMe
MeO OMe
O
O
O NO2
114
4.3.3. Leishmanicidal activity of LdGSK-3s inhibitors on intracellular
amastigotes
Aiming to assess their pharmacological value in a scenario closer to a real infection,
the 14 compounds with inhibitory activity on LdGSK-3s and leishmanicidal activity on
axenic amastigotes (TDZDs 1 and 2, HMKs 6, 28, 29 and 32, maleimide with HMK tail 33,
and quinones 72, 75-80; Tables 7, 8 and 11) were subsequently assayed on intracellular
parasites.
The activity of a given drug on the intracellular amastigote with respect to the axenic
model may differ not only due to traffic restrictions to reach the parasitophorous vacuole,
but also due to the physiology and response of the macrophage as a host cell. These effects
are of especial relevance for GSK-3 inhibitors, as cross-inhibition between the parasite and
mammalian homologs is observed for most of these inhibitors, as referred to in Sections
4.3.1 and 4.3.2.
The chosen intracellular model obeys two factors; firstly, the use of BALB/c MPMs
instead of a cellular monocytic line avoids an overestimation of the cytotoxicity. Some
human GSK-3 inhibitors have been reported as antitumoral agents.196,197 Thus, their toxicity
on tumoral monocytic cell lines is foreseeable, but avoided with primary cells. There are
reports on variation of drug effectiveness, initial infection levels, and infection development
according to the source of macrophages, including murine bone marrow macrophages and
human peripheral blood monocytes induced to macrophages198,199 besides MPMs. In our
case, MPMs were chosen for their macrophage yield, their quick parasite uptake and their
optimal support for amastigote survival and replication.200 Secondly, axenic amastigotes
from L. pifanoi (L. amazonensis) species were used due to their corroborated similarity to
those obtained from infected macrophages or animal lesions,163 affording a direct
comparison of a given drug on both amastigote types. In addition, the use of a recombinant
strain expressing the fluorescent protein mCherry facilitated quantification of the infection,
as shown in Figure 16 with representative images of infections treated with compound 6.
Results and discussion
115
Figure 16. Leishmanicidal activity of compound 6 on intracellular L. pifanoi amastigotes.
Representative fields of BALB/c murine peritoneal macrophages infected with mCherry-L. pifanoi axenic
amastigotes. Panels A and B: Untreated macrophages. Panels C and D: Infected macrophages treated with
compound 6 at 4 μM (near 2-fold its IC50 for axenic amastigotes). Panels A and C: Phase microscopy.
Panels B and D: Fluorescence microscopy (540-580 nm excitation filter). Red arrows: intracellular
amastigotes. Magnification bar: 20 μm.
Infected macrophages were treated with the compounds at an equipotent concentration
within their IC50 for amastigotes and their IC50 for MPM for 16-20 h. Statistical significance
was calculated by p values obtained with Student’s t-test.
The HMK 6, its analogue compound 32, and the quinones 72, 76 and 79 caused a
significant decrease in the parasite load beyond their respective IC50 with respect to axenic
amastigotes (near 2-fold their IC50 for compounds 6 and 72, 5-fold for compounds 32 and
76, and 10-fold for compound 79) (Figure 17).
116
Quinone 76 at 2 μM induced the highest decrease (76.3 %), followed by HMK 32
(65.7 % at 6 μM) and HMK 6 (63.3 % at 4 μM). Quinones 72 at 1 μM and 79 at 12 μM
caused a decrease of 39.1 and 37.9 % respectively.
Figure 17. Percentage variation of the parasite load of BALB/c murine peritoneal macrophages
infected with mCherry-L. pifanoi axenic amastigotes after treatment with the selected LdGSK-3s
inhibitors. Student’s t-test (*: p <0.05, **: p <0.01, ***: p <0.001).
Among these 5 compounds with statistically significant effects on intracellular
amastigotes, quinone 76 and HMK 32 had the highest effects and the best selectivity profiles
(SI = 11.2 and 6.6, respectively). Thus, these two compounds will be the best candidates for
further hit optimization.
The decrease and loss of effectiveness in intracellular infections for most of the
leishmanicidal compounds is a typical example of the high attrition experimented in the
pipeline for drug development in Leishmania. It is also a demonstration of how, as
Results and discussion
117
aforementioned, the macrophage takes part in the final outcome of a chemotherapeutic
treatment, not only by restrictions in the access of the drug to the parasitophorous vacuole,
but also by possible metabolic modifications of the drug, or through unexpected side effects
on its own physiology, already hijacked by the parasite.201
Nonetheless, leishmanicidal compounds with poor or nil in vivo activity can be
considered for nanoparticle encapsulation. Nanoencapsulation consists of the vehiculation
of drugs in particles of 10-1,000 nm of either organic or synthetic nature, such as liposomes,
polymer nanoparticles, solid lipid nanoparticles, and nanosuspensions. The endocytic system
of the macrophage captures the nanoparticles and drives their accumulation into the
parasitophorous vacuole, where Leishmania dwells. Besides increasing drug concentration
in the parasitophorous vacuole and decreasing off-side effects in the host cell,
nanoencapsulation also facilitates a preferential accumulation into the macrophages
reducing toxicity issues, and protects the drug from biological degradation. A variety of
vehicles and cargo leishmanicidal molecules has been used under this approach, including
liposome nanoencapsulation for meglumine antimoniate, and polymeric and solid lipid
nanoparticles for paromomycin and AmB. The liposomal formulation of AmB (Ambisome)
is probably the most well-known.202,203
4.4. Off-target effects of protein kinase inhibitors
Although target-based approaches are driven by the “one target, one drug” motto,
off-target effects from cross-inhibition are especially feasible for PK inhibitors, since many
of them bind to the active site of their targeted kinase, and the active site is highly conserved
among protein kinases.194 Indeed, some FDA-approved PKIs such as imatinib, sorafenib and
sunitinib are multi-kinase inhibitors.204 Compounds with a reactive group for nucleophilic
acceptors, such as TDZDs and HMKs, likely find off-targets on thiol-dependent
enzymes.80,178,179 Similarly, due to their chemical reactivity, quinones are also strong
candidates for a multi-target leishmanicidal mechanism involving the respiratory chain, as
reported for naphthoquinone derivatives in T. brucei.205
118
Despite increasing uncertainty about its mechanism of action, unexpected multi-target
effects may be essential for the effectiveness of a compound. Since effectiveness is the one
essential trait for drug development, multi-target compounds can be advantageous,
especially as they avoid resistance by target mutation.206,207 An example of a multi-target
drug in Leishmania is miltefosine, which primarily targets phospholipid metabolism, but
also causes direct inhibition of cytochrome c oxidase and Akt-like kinase.208 Although
miltefosine resistance is easily induced in vitro and reported in clinical settings, the
mechanisms identified describe a faulty accumulation of the drug, either by dysfunction of
its unique transporter (the aminophospholipid translocase Ldmt) or by overexpression of
efflux pumps, but not by target mutation.25
As drug resistances in Leishmania keep emerging, finding multi-target drug candidates
is a very desirable outcome that may increase the likelihood of avoiding some mutation-
induced resistances. Thus, our next objective was to explore feasible off-targets for our
leishmanicidal GSK-3 inhibitors.
4.4.1. Energy metabolism of Leishmania as an off-target effect of PKIs
The energy metabolism of the parasite is an excellent parameter to gauge its
intracellular homeostasis. In Leishmania, oxidative phosphorylation is the main source of
ATP synthesis, thus its inhibition leads to an irreversible bioenergetic collapse of the
parasite.63 In mammalian cells, GSK-3β is known to play a role in energy metabolism by
regulating the expression of metabolic proteins, the intermediary metabolism, and
mitochondrial function.209 GSK-3β is involved in the downregulation of mitochondrial
energy production through the modulation of the respiratory chain and the inhibition of
pyruvate dehydrogenase, among other substrates.210 Moreover, GSK-3β tips the balance
between the anabolic and catabolic metabolisms owing to its inhibitory activity on AMPK
(AMP-activated protein kinase), a crucial PK in the regulation of cellular energetics.
Hence, the effects of our leishmanicidal LdGSK-3s inhibitors on the energy
metabolism of Leishmania were evaluated. In order to prevent misleading conclusions and
Results and discussion
119
avoid variations in the energy metabolism caused as a secondary effect of the compounds,
assays were carried out at short incubation times.
4.4.1.1. Variation of the intracellular levels of ATP in L. donovani promastigotes by the
leishmanicidal LdGSK-3s inhibitors
The intracellular levels of ATP are the main parameter of energy metabolism of almost
any cell, including Leishmania parasites. Additionally, pyrophosphate is the main energy
coin across the exclusive acidocalcisome metabolism of Trypanosomatidae.211
In order to discriminate direct from indirect inhibition of ATP synthesis (e.g.
reprogramming of gene expression triggered by inhibition of GSK-3s, or other off-target
effects), variations in the intracellular ATP content of living parasites were monitored in
real-time after compound addition.
To this end, promastigotes of the 3-LUC L. donovani strain were used.139 This strain
is transfected with a cytoplasmic form of luciferase, which endows the parasite with in vivo
emission of luminescence in the presence of D-luciferin and ATP. The poor permeability of
D-luciferin across the plasma membrane at physiological pH was overcome by the use of a
caged luciferin analogue, DMNPE D-luciferin, making the cytoplasmic ATP the limiting
substrate of luminescence (Figure 18). Untreated parasites were used to monitor the intrinsic
decrease of luminescence during the assays. A decrease in the intracellular ATP levels can
be due to either inhibition of mitochondrial biosynthesis or permeabilization of the plasma
membrane by the compounds. Thus, 1,4-naphthoquinone (1.5 μM) was used as a positive
control for the inhibition of the respiratory chain, whereas 0.1% TX-100 provided the control
for full plasma membrane permeabilization139 (Figure 19, panels A and B).
120
Figure 18. Luminescence principle of 3-LUC L. donovani promastigotes. In the presence of
DMNPE-luciferin, the cytoplasmic luciferase expressed by 3-LUC L. donovani promastigotes produces
luminescence with consumption of intracellular ATP. Depletion of the intracellular ATP levels, caused
by either plasma membrane permeabilization or inhibition of mitochondrial of ATP synthesis, cuts out
the luminescence of this strain.
In order to rule out plasma membrane permeabilization as the cause of a decrease in
ATP levels, entrance of SYTOX Green into L. infantum promastigotes treated with the
chosen compounds was monitored. SYTOX Green is a cationic vital dye with poor
fluorescence in aqueous media that greatly increases after its intercalation in DNA.141 Since
SYTOX Green is non-permeable to an unscathed membrane (MW = 600), an increase in its
fluorescence can only occur after the severe disruption of the plasma membrane of the
parasite. As in the 3-LUC assay, 0.1% TX-100 was used as a positive control for full
permeabilization of the parasite (Figure 19, panels C and D).
For both assays, TDZDs 1 and 2, HMKs 6, 28, 29 and 32, and quinones 72, 75-80 were
assayed at their respective IC80; an equipotent concentration aimed to produce a large but
selective effect, as well as to facilitate comparison among the compounds. Compound 33
was excluded due to its nil leishmanicidal activity on the Leishmania promastigote.
Results and discussion
121
Time (min)
0 10 20 30
0
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40
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80
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0 10 20 30
72 (3 M)
75 (2 M)
76 (2 M)
77 (6 M)
78 (4.3 M)
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80 (7.5 M)
Untreated0.1% TX-100 0.1% TX-100
0
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1.5 M1,4-naphtoquinone
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32 (10 M)
Lum
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00)
A B
C D
Figure 19. Assessment of intracellular ATP variation and plasma membrane permeabilization by
the compounds selected. Panels A and B: Real-time monitoring of the luminescence of 3-LUC
L. donovani promastigotes after addition (t = 0) of the selected TDZDs, HMKs (Panel A) and quinones
(Panel B) at their respective IC80. Luminescence values were represented as percentage with respect to
the control of untreated parasites. 1,4-naphthoquinone (1.5 µM) and Triton X-100 (0.1%) were added as
controls for inhibition of oxidative phosphorylation and plasma membrane permeabilization respectively.
Panels C and D: Variation of SYTOX Green fluorescence after addition (t = 0) of the selected TDZDs,
HMKs (Panel C) and quinones (Panel D) to L. infantum promastigotes. Fluorescence settings:
λEX = 485 nm, λEM = 520 nm. Arrow: addition of 0.1% Triton X-100 (final concentration).
The quinones 72 (3 µM), 75 (2 µM) and 76 (2 µM) caused a rapid and irreversible
inhibition of the luminescence of 3-LUC L. donovani promastigotes, at a similar or even
more pronounced rate than the control treatment with 1,4-naphthoquinone at 1.5 µM (Figure
19, panel B). The decrease in luminescence caused by the HMK 6 (10 µM) was also quite
similar to 1.5 µM 1,4-naphthoquinone, while its analogue 32 (10 µM) barely induced a 20%
decrease in luminescence. Notably, the TDZD 1 induced a sharp but transitory drop in
luminescence that reverted to initial values by 10 minutes (Figure 19, panel A). None of
these compounds showed an increase of SYTOX Green fluorescence within the monitoring
122
time range (Figure 19, panels C and D). Consequently, membrane permeabilization was
ruled out as the mechanism of action of the compounds that caused the bioenergetic collapse
of the 3-LUC L. donovani promastigotes.
Once the severe disruption of the plasma membrane integrity was dismissed, the other
feasible target of the aforementioned compounds was the mitochondrial ATP synthesis. As
for compound 1, this TDZD most likely induces a transient leak of cytoplasmic ATP that we
may surmise to be due to a brief plasma membrane depolarization of the parasite.
4.4.1.2. Inhibition of the electrochemical potential of the Leishmania mitochondrion (ΔΨm)
In Leishmania, especially on the promastigote, oxidative phosphorylation is the main
source for ATP biosynthesis.66 This process is coupled to the respiratory chain with
generation of an electrochemical potential across the inner membrane of the mitochondrion
(ΔΨm). In fact, mitochondrial membrane depolarization is associated with inhibition of
oxidative phosphorylation, as well as activation of the intrinsic apoptotic pathway.141
The ΔΨm is the highest among the intracellular organelles, with a reported value
of -160 mV in L. major promastigotes.212 This accounts for the preferential accumulation of
hydrophobic cations into the mitochondrion under the Nernst equation.213 Hence, the
variation of the ΔΨm of Leishmania parasites can be easily monitored through the differential
accumulation of rhodamine 123, a fluorescent hydrophobic cation.141 The HMK 6 and the
quinones 72, 75 and 76 were selected from the previous assays to test their effect on the ΔΨm
of L. infantum promastigotes. The TDZD 1 was also selected in order to completely rule out
the mitochondrion of the parasite as its off-target. Compounds were incubated for 4 h with
the parasites at their respective IC80s prior to rhodamine 123 accumulation (described in
Materials and Methods). A 40 min incubation with 20 mM KCN was used as a control
inhibitor of oxidative phosphorylation (Figure 20).
Results and discussion
123
Figure 20. Rhodamine 123 accumulation in L. infantum promastigotes and its variation by the
selected compounds at their IC80. The number codes of the compounds are at the right top corner of
their respective panel. Parasites were incubated with the compounds as described in the Materials and
Methods section, followed by incubation with rhodamine 123. Rhodamine 123 accumulation was
measured by flow cytometry (λEX = 488 nm, λEM = 520 nm). Fluorescence values were expressed as a
percentage value with respect to the untreated control. Incubation with 20 mM KCN for 40 min was used
as positive control for mitochondrial membrane depolarization.
According to Figure 20, all the tested compounds decreased the accumulation of
rhodamine 123 in L. donovani promastigotes except for TDZD 1, as expected from its lack
of effect on the intracellular ATP content of 3-LUC L. donovani promastigotes. Compounds
6 (10 μM) and 72 (3 μM) induced the highest decrease of rhodamine 123 accumulation,
similar to the KCN-treated control (ca 70 %). Quinones 75 and 76 at 2 μM showed similar
effects, with 33 and 26.4 % decrease of rhodamine 123 fluorescence respectively.
124
Since lower rhodamine 123 accumulation is caused by mitochondrial membrane
depolarization, these results confirmed that HMK 6 and quinones 72, 75 and 76 inhibit the
oxidative phosphorylation of L. donovani.
For quinones 72¸ 75 and 76, their resemblance to ubiquinone is a feasible hypothesis
for their inhibitory activity on the respiratory chain of the parasite. Ubiquinone acts as a
membrane bound electron carrier among complexes I, II and III. The molecule is associated
with the inner mitochondrial membrane through its polyisoprenoid chain while its
benzoquinone ring accepts the electrons from complex I and II, and transfers them to
complex III.214 As the activity of complex I in trypanosomatids is rather poor,71 complex II
and III remain the most feasible targets for these compounds, as reported for other
quinones.191,215,216
4.4.1.3. Inhibition of O2 consumption
As mentioned before, electron transport throughout the respiratory chain is coupled to
mitochondrial ATP synthesis. Hence, inhibition of one of these processes results in the
inhibition of the other and vice versa. Consequently, the mitochondrial depolarizing
compounds 6 (Figure 22), 72, 75 and 76 (Figure 23) were assayed for inhibition of the
respiration of Leishmania promastigotes by polarographic monitoring using a Clark oxygen
electrode.
The Clark oxygen electrode consists of a silver anode and a platinum cathode
connected with a sealed and thermostatised jacketed-chamber through a Teflon membrane.
A magnetic stirrer ensures the rapid equilibration of the O2 content throughout the whole
fluid (Figure 21). The Teflon membrane allows diffusion of oxygen from the respiration
buffer within the chamber to the electrodes, where an electrochemical reaction creates a
voltage proportional to the oxygen concentration.217 The cellular density of the parasite
suspension for Clark electrode experiments was 5-fold higher with respect to the standard
one used for the other short-term functional experiments. This approach allowed speeding
up the time threshold for the observation of effects on parasite respiration before the O2
available was fully consumed.
Results and discussion
125
Figure 21. Clark-type oxygen electrode scheme.218 This device allows for the monitoring of O2
consumption of a cellular solution placed in the incubation chamber. The chamber is completely sealed
off during the experiment to avoid reconstitution of O2 levels. The magnetic stirrer bar at the bottom of
the chamber ensures homogeneous O2 levels across the cellular solution. Water at the needed temperature
flows through the “jacket” surrounding the incubation chamber to maintain the appropriate temperature
during the experiment. The electrodes that record the electrochemical changes in the solution associated
with the O2 levels are placed in a separate chamber in KCl solution, connected to the incubation chamber
through a Teflon membrane.
The halomethylketone 6 inhibited the respiration of L. infantum promastigotes in a
concentration dependent manner (Figure 22, panels B and C), reaching up to 51% inhibition
at 2-fold (20 μM) and 63% inhibition at 5-fold (50 μM) its IC80.
126
Figure 22. Oxygen consumption rates of living L. infantum parasites treated with the HMK 6.
Parasites were prepared in Respiration Buffer at 5-fold the usual cellular concentration established for
short-termed experiments (108 parasites/mL), and maintained at 26 ºC. Panel A: Untreated parasites.
Panels B and C: Parasites treated with HMK 6 at 2- and 5-fold its IC80 respectively. Values represent the
percentage (%) of O2 consumption rate. The different colours of the dotted lines account for the slope
measurements in each step of the experiment. The use of high concentrations of parasites and compounds
allowed for the observation of the effects of the compounds before O2 levels were fully depleted.
On the other hand, the L. infantum promastigotes treated with quinones 72, 75 and 76
at 3.3- to 5-fold their IC80 (10 μM) showed a double effect: the quinones initially induced an
acceleration of the O2 consumption rates, but the respiration of the parasites slowed down
afterwards (Figure 23, panels B, C and D). A feasible explanation is that the initial
acceleration entails a compensation mechanism to recover from the decrease in intracellular
ATP levels caused by the inhibition of oxidative phosphorylation by the quinones. Over
time, this increase is no longer feasible and the parasites undergo a steady loss of respiration
that also may be related to direct inhibition of the respiratory chain by the compounds.
Results and discussion
127
Figure 23. Oxygen consumption rates of L. infantum parasites with quinones 72, 75 and 76. The
addition of each compound is represented with a red arrow and its code number in bold black. Panel A:
untreated parasites. Panel B: treatment with compounds 72 at 3.3-fold its IC80. Panel C: treatment with
compound 75 at 5-fold its IC80. Panel D: treatment with compound 76 at 5-fold its IC80. Values represent
the percentage of O2 consumption rate with respect to the basal rate, before the addition of the compounds.
The different colours of the dotted lines account for the different slopes, whose respective percentage
values appear at the lower left corner of each panel.
4.4.1.4. Identification of the PKI target inside the respiratory chain
In Leishmania, since complex I is barely active,71 complex II is the main electron donor
for complex III through ubiquinone using succinate as substrate. Consequently, the proton
gradient across the membrane depends on complexes III and IV, with O2 as the final electron
acceptor at complex IV. The F0F1-ATPase (complex V) carries out ATP synthesis by
oxidative phosphorylation profiting from the H+ gradient created across the inner
membrane.63 Under this scenario, the site of inhibition for each compound was mapped by
using specific substrates and inhibitors for each complex on the mitochondrion of digitonin-
permeabilized L. infantum promastigotes.
128
The isolation of the single mitochondrion of trypanosomatids in an intact state is
unfeasible due to the large size of this organelle (ca 12% of the intracellular volume of the
parasite), along with the extreme resistance to mechanical disruption of the plasma
membrane due to its subpellicular layer of microtubules. This issue is solved by the use of
digitonin, a non-ionic detergent. At low concentrations, digitonin selectively disrupts the
plasma membrane through a preferential interaction with sterols, which are absent in the
inner mitochondrial membrane. This selective permeation of the plasma membrane affords
direct access of respiratory substrates and inhibitors to a functional mitochondrion with an
intact inner membrane.219
Figure 24 provides an explanation for this strategy. Panel A shows the variation of
control parasites after digitonin addition. The respiration increases after ADP addition,
accounting for the coupling degree between the respiratory chain and ATP synthesis.
Addition of FCCP, a protonophore molecule that dissipates the H+ gradient across the inner
membrane, ensures the maximal speed of the respiratory chain. Panel A’ provides a graphical
explanation of the polarographic pattern. Panel B shows the inhibition of respiration through
the inhibition of F0F1-ATPase by oligomycin (panel B’1), and the uncoupling of this
complex from the respiratory chain by FCCP addition (panel B’2).
Results and discussion
129
Figure 24. Oxygen consumption rates of digitonin-permeabilized L. infantum parasites. Panel A:
FCCP-treated mitochondria as a control of uncoupled respiration. Panel B: Control of uncoupled
respiration with oligomycin-inhibited F0F1-ATP synthase. Panels A’, B’1 and B’2: schemes of the effects
of FCCP and oligomycin on the respiratory chain of Leishmania. Final concentrations of substrates and
inhibitors: 60 μM digitonin (Dig), 100 μM ADP, 10 μM FCCP, 12.6 μM oligomycin (Omy).
Addition of TMPD-ascorbate, an electron donor for cytochrome c oxidase (complex
IV) led to a partial recovery of the respiration inhibited by HMK 6. This excluded complexes
IV and V as targets for this compound (Figure 25, panels A and A’). When the respiratory
chain of Leishmania is inhibited at complex II by malonate, the single source to feed
electrons into complex III is through complex I by addition of its substrate,
130
α-glycerophosphate (Figure 25, panels B and B’). As α-glycerophosphate did not improve
the respiration rate inhibited by compound 6, complex II was discarded as a target, leaving
complex III as the main inhibition site within the respiratory chain for this halomethylketone
(Figure 25, panels C and C’). Remarkably, the alkaline pH of the mitochondrion favours the
formation of thiolate, which increases the reactivity of nucleophilic compounds such as
halomethylketones.
The importance of cytochrome c reductase (complex III) as a leishmanicidal target is
endorsed by leishmanicidal compounds acting on this complex, including antimycin A and
other compounds in clinical use for other diseases caused by apicomplexans, such as
buparvaquone (hydroxynaphthoquinone) for theileriosis and tafenoquine
(8-aminoquinoline) for malaria.191,216 Other compounds of natural origin that act on the
cytochrome c reductase of Leishmania include chromanol derivatives, which are related to
vitamin E, and essential oil components, among others.220,221
Results and discussion
131
Figure 25. Oxygen consumption rates of digitonized-L. infantum promastigotes treated with HMK
6 and the complex II-inhibitor malonate. ADP addition prevents its potential role as a limiting substrate
for ATP synthesis by complex V. Panel A: Variation of the respiration rate by addition of TMPD-Asc on
promastigotes treated with 50 μM 6. Panel B: Variation of respiration after addition of α-GP under
inhibition of complex II. Panel C: Inability of α-glycerophosphate to restore the inhibition of the
respiration rate induced by compound 6. Panels A’, B’ and C’: Graphical interpretation of the effects of
HMK 6, malonate and α-glycerophosphate on respiration from the respective panels on their left (A, B
and C). Final concentration for substrates and inhibitors: 60 μM digitonin (Dig), 100 μM ADP, 10 mM
malonate, 6.7 mM α-glycerophosphate (α-GP), TMPD-ascorbate (TMPD-Asc) 0.1 mM and 1.7 mM,
respectively.
132
As the addition of quinones 72, 75 and 76 increases respiration rates on intact parasites
(Figure 23), a different approach was taken. Under inhibition of complex V by oligomycin,
the addition of any of these three quinones increased the respiration rate (Figure 26,
panels A, B and C), as seen for the protonophore FCCP in Figure 24. These results confirmed
a mild uncoupling effect of these compounds. Nevertheless, a dual action of these quinones
was anticipated. In fact, these three quinones reduced the oxygen consumption rate of
FCCP-treated mitochondria, demonstrating inhibitory activity on the electron transport chain
of the parasite (Figure 26 D, E and F). Inhibition of respiration by the quinones was restored
after addition of TMPD-Asc, therefore the target of inhibition of compounds 72, 75 and 76
is upstream complex IV (Figure 26 G, H and I). Nevertheless, the acceleration of respiration
in living parasites induced by these quinones (Figure 23) points towards a higher uncoupling
than inhibitory effect in vivo.
Results and discussion
133
Figure 26. Oxygen consumption rates of digitonin-permeabilized L. infantum parasites. Panels A, B
and C: Uncoupling effect of compounds 72, 75 and 76 (respectively) in oligomycin-treated
mitochondria. Panels D, E and F: Inhibition of respiration by compounds 72, 75 and 76 (respectively) in
FCCP-uncoupled mitochondria. Panels G, H and I: Restoration of the respiration inhibited by compounds
72, 75 and 76 (respectively) after addition of TMPD-ascorbate. Final concentrations of substrates and
inhibitors: 60 μM digitonin (Dig), 100 μM ADP, 10 μM FCCP, 12.6 μM oligomycin (Omy),
TMPD-ascorbate (TMPD-Asc) 0.1 mM and 1.7 mM respectively.
134
4.4.1.5. Induction of programmed cell death in L. infantum promastigotes
Most of the morphological features of apoptosis in mammalian cells, such a chromatin
condensation, shrinkage of the cytoplasm, or blebbing of the plasma membrane are
reproduced in Leishmania, but the molecular executioners are poorly defined in the
parasite.222 In trypanosomatids, the only feasible apoptotic pathway is the intrinsic one, as
the external apoptotic pathway is absent.223 The role of the mitochondrion in the intrinsic
pathway of Leishmania is mandatory; its depolarization is an early apoptotic stage, followed
by chromatin degradation at later stages with increase of the subG0/G1 population in cell
cycle studies.141 Hence, we evaluated the induction of programmed cell death in Leishmania
promastigotes with the mitochondrial depolarizing compounds 6, 72, 75 and 76. Other
leishmanicidal drugs with known apoptotic-inducing effects linked to inhibition of the
respiratory chain of Leishmania include miltefosine, tafenoquine and sitamaquine, targeting
complex IV, III and II of the respiratory chain, respectively.215,216,224 Pentamidine and
paromomycin also induce programmed cell death by targeting the mitochondrion but their
target remains unveiled.127,131
The individual content of DNA in a population of L. infantum promastigotes was
analysed by flow cytometry with propidium iodide (PI). PI is a fluorescent probe that
stoichiometrically binds to DNA. Since DNA fragmentation reduces the capacity of DNA to
bind PI, apoptotic cells are found in the SubG0/G1 population, with a much lower PI
fluorescence with respect to other cell populations in different phases of the cell cycle
(G0/G1, S, G2/M).141 For this study, compounds 6, 72, 75 and 76 were assayed at their
respective IC80. Miltefosine at 15 μM was used as a control to obtain an apoptotic
histogram.225
Both the HMK and the quinones tested increased the SubG0/G1 population, with lower
levels of PI accumulation in apoptotic parasites due to chromatin degradation (Figure 27).
Hence, it was concluded that HMK 6 and quinones 72, 75 and 75 induced programmed cell
death in L. infantum promastigotes.
Results and discussion
135
Figure 27. Cell cycle of L. infantum promastigotes treated with quinones 72, 75 and 76 and the
halomethylketone 6 at their IC80. The values represented are the percentage of SubG0/G1 population
(delimited inside green bars) as the typical population for apoptotic-like process (degraded chromatin).
Miltefosine (HePc) at 15 μM was added as a control for induction of apoptosis.
Altogether, one halomethylketone and three naphthoquinones out of the 14
leishmanicidal LdGSK-3s inhibitors from our in-house chemical library induced a
bioenergetic collapse in the promastigote of Leishmania by targeting the respiratory chain
of the parasite, leading to apoptosis and revealing the multi-target nature of these protein
kinase inhibitors.
CONCLUSIONS
Conclusions
139
5. Conclusions
In this PhD thesis, new leishmanicidal agents belonging to three different chemical
families acting on Leishmania GSK-3s at micromolar concentrations have been identified
and characterized. The energy metabolism of Leishmania has been identified as an additional
off-target mechanism for members of two different chemical scaffolds.
From the results obtained, the following conclusions were drawn:
1. The usefulness of collections of small-size organic compounds as a source for the
identification of new leishmanicidal inhibitors for LdGSK-3 has been endorsed.
2. The combination of a target-based approach with an unbiased phenotypic assay is
highly relevant for a full interrogation of a chemical library in search of effective
compounds with a known mechanism of action.
3. The GSK-3s of Leishmania was confirmed as a druggable target by the identification
of 16 active compounds from four different chemical families; thiadiazolidindiones,
iminothiadiazoles, halomethylketones and quinones, broadening the range of
chemical scaffolds for inhibition of this enzyme.
4. A selectivity index over 10, a major requirement to endorse further hit optimization,
was obtained for TDZD 1, HMK 28 and quinones 75, 76 and 79.
5. The halomethylketones 6 and 32, and the quinones 72, 76 and 79, decreased the
parasite load in infected murine peritoneal macrophages.
6. Among the 24 1,4-naphthoquinones tested, simultaneous inhibition of both
Leishmania proliferation and LdGSK3-s activity took place in scaffolds with a
140
carbamate substituent at the nitrogen of the 2-amino group and a Cl atom at
position 3. Moreover, quinones with this pattern of substitution are the first selective
LdGSK-3s inhibitors with no activity against HsGSK-3β described so far. Hence,
this chemical pattern is the most appealing for further optimization.
7. The energy metabolism of the promastigote is a useful parameter to gauge its
intracellular homeostasis, and find feasible off-targets for GSK-3 inhibitors. Besides
inhibition of LdGSK-3s, the HMK 6 and the quinones 72, 75 and 76 caused a
bioenergetic collapse in Leishmania promastigotes, with mitochondrial
depolarization and inhibition of both respiration and ATP synthesis, driving the
parasite into a programmed cell death.
8. Inside the respiratory chain of Leishmania promastigotes, complex III was identified
as a target for HMK 6, whereas a double effect was observed for quinones 72, 75 and
76, with uncoupling and inhibitory activities on the respiration of the parasites.
In conclusion, the combination of a target-based screening focused on the GSK-3s of
Leishmania with a phenotypic screening in the parasite has led to the identification of new
compounds with appealing activities for their further development as new leishmanicidal
drugs. Moreover, some of these compounds have an additional target in the respiratory chain
of the parasite. The multi-target nature of these compounds is an advantageous trait for a
jeopardized raise of resistance caused by mutation in a single target.
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RESULTS DISSEMINATION
Results dissemination
167
Results dissemination
Publications from the PhD thesis
1. Martínez de Iturrate, P.; Sebastián-Perez, V.; Nácher-Vázquez, M.; Tremper, C. S.;
Smirlis, D.; Martín, J.; Martínez, A.; Campillo, N. E.; Rivas, L.; Gil, C., Towards discovery
of new leishmanicidal scaffolds able to inhibit Leishmania GSK-3. J Enzyme Inhib Med
Chem, 2020, 35, 199-210.
Participation in Conferences, Symposiums and Congresses
1. Poster at XXIV International Symposium on Medicinal Chemistry. Manchester, UK,
2016. “Drug repurposing of human kinase inhibitors as new hits against Leishmania”
(Abstract book, P155). Authors: Gil, C.; Abengózar, M. A.; Martínez de Iturrate, P.;
Sebastián-Pérez, V.; Martínez, A.; Campillo, N. E.; Rivas, L.
2. Poster at 3rd COST Action CM1307 Conference. Madrid, Spain, 2016. “Drug
repurposing of human kinase inhibitors as new hits against Leishmania” (Abstract book,
P.10.). Authors: Gil, C.; Abengózar, M. A.; Martínez de Iturrate, P.; Sebastián-Pérez, V.;
Martínez, A.; Campillo, N. E.; Rivas, L.
3. Poster at WorldLeish6. Toledo, Spain, 2017. “New small heterocyclic scaffolds with
leishmanicidal activity” (Abstract book, C1313). Authors: Rivas, L.; Martínez de Iturrate,
P.; Abengózar, M. A.; Sebastián-Pérez, V.; Nácher, M.; Martínez, A.; Gil, C.
4. Poster at EUROPIN Summer School on Drug Design. Vienna, Austria, 2017.
“Targeting protein kinases in Leishmania as a novel approach to discover new drugs”
(Abstract and certification). Authors: Sebastián-Pérez, V.; Estañ, N.; Nácher-Vázquez, M.;
Martínez de Iturrate, P.; Rivas, L.; Campillo, N.E.; Gil, C.
168
5. Poster at I Jornada PhDay Facultad de Farmacia UCM. Madrid, Spain 2017. “Protein
kinase inhibitors as leads for new chemotherapeutics against Leishmania” (Abstract book,
P29). Authors: Martínez de Iturrate, P.; Sebastián-Pérez, V.; Nácher-Vázquez, M.;
Abengózar, M. A.; Martínez, A.; Campillo, N.E.; Rivas, L.; Gil, C.
6. Oral Presentation at I Jornada PhDay Facultad de Farmacia UCM. Madrid, Spain 2017.
“Protein kinase inhibitors as leads for new chemotherapeutics against Leishmania” (Abstract
book and certification).
7. Poster at 20th European Bioenergetics Conference. Budapest, Hungary, 2018. “The
energy metabolism of Leishmania as a drug target” (Abstract book, P10/P16). Authors: Rial,
E.; Lastra-Romero, A.; Martínez-de-Iturrate, P.; Nácher-Vázquez, M.; Rivas, L.
8. Poster at 41 Congreso de la Sociedad Española de Bioquímica y Biología Molecular.
Santander, Spain, 2018. “In house libraries of human protein kinase inhibitors as a source
for new leishmanicidal agents” (Abstract book, P0232). Authors: Rivas, L.; Martínez de
Iturrate, P.; Sebastián-Pérez, V.; Nácher-Vázquez, M.; Tremper, C.; Lastra, A.; Rial, E.;
Campillo, N.E.; Gil, C.
9. Poster at 6th Young Reasearchers Symposium. Madrid, Spain, 2019. “The quest for
new Leishmania GSK3 inhibitors from large drug libraries: Leishbox as a representative
example” (Abstract book, P14). Authors: Tremper, C.S.; Sebastián-Pérez, V.; Martínez de
Iturrate, P.; Nácher-Vázquez, M.; Campillo, N.E.; Gil, C.; Rivas, L.
10. Poster at 6th Young Researchers Symposium. Madrid, Spain, 2019. “In-house protein
kinase inhibitors against Leishmania. The energy metabolism of the parasite as an off-target”
(Abstract book, P15). Authors: Martínez de Iturrate, P.; Sebastián-Pérez, V.;
Nácher-Vázquez, M.; Abengózar, M.A.; Martínez, A.; Campillo, N.E.; Rivas, L.; Gil, C.
