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1.
PLoS Pathog ; 20(7): e1012382, 2024 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-38991025

RESUMO

Liposomal amphotericin B is an important frontline drug for the treatment of visceral leishmaniasis, a neglected disease of poverty. The mechanism of action of amphotericin B (AmB) is thought to involve interaction with ergosterol and other ergostane sterols, resulting in disruption of the integrity and key functions of the plasma membrane. Emergence of clinically refractory isolates of L. donovani and L. infantum is an ongoing issue and knowledge of potential resistance mechanisms can help to alleviate this problem. Here we report the characterisation of four independently selected L. donovani clones that are resistant to AmB. Whole genome sequencing revealed that in three of the moderately resistant clones, resistance was due solely to the deletion of a gene encoding C24-sterol methyltransferase (SMT1). The fourth, hyper-resistant resistant clone (>60-fold) was found to have a 24 bp deletion in both alleles of a gene encoding a putative cytochrome P450 reductase (P450R1). Metabolic profiling indicated these parasites were virtually devoid of ergosterol (0.2% versus 18% of total sterols in wild-type) and had a marked accumulation of 14-methylfecosterol (75% versus 0.1% of total sterols in wild-type) and other 14-alpha methylcholestanes. These are substrates for sterol 14-alpha demethylase (CYP51) suggesting that this enzyme may be a bona fide P450R specifically involved in electron transfer from NADPH to CYP51 during catalysis. Deletion of P450R1 in wild-type cells phenocopied the metabolic changes observed in our AmB hyper-resistant clone as well as in CYP51 nulls. Likewise, addition of a wild type P450R1 gene restored sterol profiles to wild type. Our studies indicate that P450R1 is essential for L. donovani amastigote viability, thus loss of this gene is unlikely to be a driver of clinical resistance. Nevertheless, investigating the mechanisms underpinning AmB resistance in these cells provided insights that refine our understanding of the L. donovani sterol biosynthetic pathway.

2.
J Physiol ; 600(10): 2499-2513, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35413129

RESUMO

The human TE671 cell line was originally used as a model of medulloblastoma but has since been reassigned as rhabdomyosarcoma. Despite the characterised endogenous expression of voltage-sensitive sodium currents in these cells, the specific voltage-gated sodium channel (VGSC) subtype underlying these currents remains unknown. To profile the VGSC subtype in undifferentiated TE671 cells, endpoint and quantitative reverse transcription-PCR (qRT-PCR), western blot and whole-cell patch clamp electrophysiology were performed. qRT-PCR profiling revealed that expression of the SCN9A gene was ∼215-fold greater than the SCN4A gene and over 400-fold greater than any of the other VGSC genes, while western blot confirmed that the dominant SCN9A RNA was translated to a protein with a molecular mass of ∼250 kDa. Elicited sodium currents had a mean amplitude of 2.6 ± 0.7 nA with activation and fast inactivation V50 values of -31.9 ± 1.1 and -69.6 ± 1.0 mV, respectively. The currents were completely and reversibly blocked by tetrodotoxin at concentrations greater than 100 nm (IC50  = 22.3 nm). They were also very susceptible to the NaV 1.7 specific blockers Huwentoxin-IV and Protoxin-II with IC50 values of 14.6 nm and 0.8 nm, respectively, characteristic of those previously determined for NaV 1.7. Combined, the results revealed the non-canonical and highly dominant expression of NaV 1.7 in the human TE671 rhabdomyosarcoma cell line. We show that the TE671 cell line is an easy to maintain and cost-effective model for the study of NaV 1.7, a major target for the development of analgesic drugs and more generally for the study of pain. KEY POINTS: Undifferentiated TE671 cells produce a voltage-sensitive sodium current when depolarised. The voltage-gated sodium channel isoform expressed in undifferentiated TE671 cells was previously unknown. Through qRT-PCR, western blot and toxin pharmacology, it is shown that undifferentiated TE671 cells dominantly (>99.5%) express the NaV 1.7 isoform that is strongly associated with pain. The TE671 cell line is, therefore, a very easy to maintain and cost-effective model to study NaV 1.7-targeting drugs.


Assuntos
Canal de Sódio Disparado por Voltagem NAV1.7 , Rabdomiossarcoma , Linhagem Celular , Humanos , Canal de Sódio Disparado por Voltagem NAV1.4 , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Dor , Rabdomiossarcoma/genética , Bloqueadores dos Canais de Sódio/farmacologia , Tetrodotoxina/farmacologia
3.
Antimicrob Agents Chemother ; 66(1): e0153521, 2022 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-34606338

RESUMO

Phenotypic screening identified an arylsulfonamide compound with activity against Trypanosoma cruzi, the causative agent of Chagas' disease. Comprehensive mode of action studies revealed that this compound primarily targets the T. cruzi proteasome, binding at the interface between ß4 and ß5 subunits that catalyze chymotrypsin-like activity. A mutation in the ß5 subunit of the proteasome was associated with resistance to compound 1, while overexpression of this mutated subunit also reduced susceptibility to compound 1. Further genetically engineered and in vitro-selected clones resistant to proteasome inhibitors known to bind at the ß4/ß5 interface were cross-resistant to compound 1. Ubiquitinated proteins were additionally found to accumulate in compound 1-treated epimastigotes. Finally, thermal proteome profiling identified malic enzyme as a secondary target of compound 1, although malic enzyme inhibition was not found to drive potency. These studies identify a novel pharmacophore capable of inhibiting the T. cruzi proteasome that may be exploitable for anti-chagasic drug discovery.


Assuntos
Doença de Chagas , Trypanosoma cruzi , Doença de Chagas/tratamento farmacológico , Descoberta de Drogas , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Trypanosoma cruzi/química
4.
Proc Natl Acad Sci U S A ; 116(19): 9318-9323, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-30962368

RESUMO

Visceral leishmaniasis (VL), caused by the protozoan parasites Leishmania donovani and Leishmania infantum, is one of the major parasitic diseases worldwide. There is an urgent need for new drugs to treat VL, because current therapies are unfit for purpose in a resource-poor setting. Here, we describe the development of a preclinical drug candidate, GSK3494245/DDD01305143/compound 8, with potential to treat this neglected tropical disease. The compound series was discovered by repurposing hits from a screen against the related parasite Trypanosoma cruzi Subsequent optimization of the chemical series resulted in the development of a potent cidal compound with activity against a range of clinically relevant L. donovani and L. infantum isolates. Compound 8 demonstrates promising pharmacokinetic properties and impressive in vivo efficacy in our mouse model of infection comparable with those of the current oral antileishmanial miltefosine. Detailed mode of action studies confirm that this compound acts principally by inhibition of the chymotrypsin-like activity catalyzed by the ß5 subunit of the L. donovani proteasome. High-resolution cryo-EM structures of apo and compound 8-bound Leishmania tarentolae 20S proteasome reveal a previously undiscovered inhibitor site that lies between the ß4 and ß5 proteasome subunits. This induced pocket exploits ß4 residues that are divergent between humans and kinetoplastid parasites and is consistent with all of our experimental and mutagenesis data. As a result of these comprehensive studies and due to a favorable developability and safety profile, compound 8 is being advanced toward human clinical trials.


Assuntos
Antiprotozoários/administração & dosagem , Leishmania donovani/efeitos dos fármacos , Leishmania infantum/efeitos dos fármacos , Leishmaniose Visceral/diagnóstico por imagem , Inibidores de Proteassoma/administração & dosagem , Proteínas de Protozoários/antagonistas & inibidores , Animais , Antiprotozoários/química , Sítios de Ligação , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Humanos , Leishmania donovani/química , Leishmania donovani/enzimologia , Leishmania infantum/química , Leishmania infantum/enzimologia , Leishmaniose Visceral/parasitologia , Masculino , Camundongos , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/química , Conformação Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo
5.
Proc Natl Acad Sci U S A ; 115(38): 9616-9621, 2018 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-30185555

RESUMO

African trypanosomes cause lethal and neglected tropical diseases, known as sleeping sickness in humans and nagana in animals. Current therapies are limited, but fortunately, promising therapies are in advanced clinical and veterinary development, including acoziborole (AN5568 or SCYX-7158) and AN11736, respectively. These benzoxaboroles will likely be key to the World Health Organization's target of disease control by 2030. Their mode of action was previously unknown. We have developed a high-coverage overexpression library and use it here to explore drug mode of action in Trypanosoma brucei Initially, an inhibitor with a known target was used to select for drug resistance and to test massive parallel library screening and genome-wide mapping; this effectively identified the known target and validated the approach. Subsequently, the overexpression screening approach was used to identify the target of the benzoxaboroles, Cleavage and Polyadenylation Specificity Factor 3 (CPSF3, Tb927.4.1340). We validated the CPSF3 endonuclease as the target, using independent overexpression strains. Knockdown provided genetic validation of CPSF3 as essential, and GFP tagging confirmed the expected nuclear localization. Molecular docking and CRISPR-Cas9-based editing demonstrated how acoziborole can specifically block the active site and mRNA processing by parasite, but not host CPSF3. Thus, our findings provide both genetic and chemical validation for CPSF3 as an important drug target in trypanosomes and reveal inhibition of mRNA maturation as the mode of action of the trypanocidal benzoxaboroles. Understanding the mechanism of action of benzoxaborole-based therapies can assist development of improved therapies, as well as the prediction and monitoring of resistance, if or when it arises.


Assuntos
Fator de Especificidade de Clivagem e Poliadenilação/antagonistas & inibidores , Proteínas de Protozoários/antagonistas & inibidores , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/fisiologia , Tripanossomíase Africana/prevenção & controle , Animais , Benzamidas/farmacologia , Benzamidas/uso terapêutico , Compostos de Boro/farmacologia , Compostos de Boro/uso terapêutico , Sistemas CRISPR-Cas , Núcleo Celular/genética , Núcleo Celular/metabolismo , Fator de Especificidade de Clivagem e Poliadenilação/genética , Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Resistência a Medicamentos/efeitos dos fármacos , Resistência a Medicamentos/genética , Técnicas de Silenciamento de Genes , Biblioteca Gênica , Ensaios de Triagem em Larga Escala/métodos , Humanos , Simulação de Acoplamento Molecular , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , RNA Mensageiro/metabolismo , RNA de Protozoário/metabolismo , Tripanossomicidas/uso terapêutico , Trypanosoma brucei brucei/efeitos dos fármacos , Tripanossomíase Africana/transmissão , Tripanossomíase Africana/veterinária , Valina/análogos & derivados , Valina/farmacologia , Valina/uso terapêutico
6.
Cell Microbiol ; 21(10): e13082, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31283102

RESUMO

The myosin superfamily comprises of actin-dependent eukaryotic molecular motors important in a variety of cellular functions. Although well studied in many systems, knowledge of their functions in Plasmodium, the causative agent of malaria, is restricted. Previously, six myosins were identified in this genus, including three Class XIV myosins found only in Apicomplexa and some Ciliates. The well characterized MyoA is a Class XIV myosin essential for gliding motility and invasion. Here, we characterize all other Plasmodium myosins throughout the parasite life cycle and show that they have very diverse patterns of expression and cellular location. MyoB and MyoE, the other two Class XIV myosins, are expressed in all invasive stages, with apical and basal locations, respectively. Gene deletion revealed that MyoE is involved in sporozoite traversal, MyoF and MyoK are likely essential in the asexual blood stages, and MyoJ and MyoB are not essential. Both MyoB and its essential light chain (MCL-B) are localised at the apical end of ookinetes but expressed at completely different time points. This work provides a better understanding of the role of actomyosin motors in Apicomplexan parasites, particularly in the motile and invasive stages of Plasmodium during sexual and asexual development within the mosquito.


Assuntos
Miosinas/metabolismo , Plasmodium/crescimento & desenvolvimento , Plasmodium/metabolismo , Proteínas de Protozoários/metabolismo , Esporozoítos/metabolismo , Animais , Feminino , Estágios do Ciclo de Vida , Espectrometria de Massas , Camundongos , Miosinas/química , Miosinas/genética , Fenótipo , Plasmodium/genética , Domínios Proteicos/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Esporozoítos/crescimento & desenvolvimento
7.
J Biol Chem ; 292(43): 17857-17875, 2017 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-28893907

RESUMO

Myosin A (MyoA) is a Class XIV myosin implicated in gliding motility and host cell and tissue invasion by malaria parasites. MyoA is part of a membrane-associated protein complex called the glideosome, which is essential for parasite motility and includes the MyoA light chain myosin tail domain-interacting protein (MTIP) and several glideosome-associated proteins (GAPs). However, most studies of MyoA have focused on single stages of the parasite life cycle. We examined MyoA expression throughout the Plasmodium berghei life cycle in both mammalian and insect hosts. In extracellular ookinetes, sporozoites, and merozoites, MyoA was located at the parasite periphery. In the sexual stages, zygote formation and initial ookinete differentiation precede MyoA synthesis and deposition, which occurred only in the developing protuberance. In developing intracellular asexual blood stages, MyoA was synthesized in mature schizonts and was located at the periphery of segmenting merozoites, where it remained throughout maturation, merozoite egress, and host cell invasion. Besides the known GAPs in the malaria parasite, the complex included GAP40, an additional myosin light chain designated essential light chain (ELC), and several other candidate components. This ELC bound the MyoA neck region adjacent to the MTIP-binding site, and both myosin light chains co-located to the glideosome. Co-expression of MyoA with its two light chains revealed that the presence of both light chains enhances MyoA-dependent actin motility. In conclusion, we have established a system to study the interplay and function of the three glideosome components, enabling the assessment of inhibitors that target this motor complex to block host cell invasion.


Assuntos
Estágios do Ciclo de Vida/fisiologia , Proteínas de Membrana , Miosinas , Plasmodium berghei , Plasmodium falciparum , Proteínas de Protozoários , Animais , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Miosinas/genética , Miosinas/metabolismo , Plasmodium berghei/genética , Plasmodium berghei/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo
8.
Artigo em Inglês | MEDLINE | ID: mdl-29844044

RESUMO

The lack of information regarding the mechanisms of action (MoA) or specific molecular targets of phenotypically active compounds can prove a barrier to their development as chemotherapeutic agents. Here, we report the results of our orthogonal genetic, molecular, and biochemical studies to determine the MoA of a novel 7-substituted 8-hydroxy-1,6-naphthyridine (8-HNT) series that displays promising activity against Trypanosoma brucei and Leishmania donovani High-throughput loss-of-function genetic screens in T. brucei highlighted two probable zinc transporters associated with resistance to these compounds. These transporters localized to the parasite Golgi apparatus. Directed by these findings, the role of zinc and other divalent cations in the MoA of these compounds was investigated. 8-HNT compounds were found to directly deplete intracellular levels of Zn2+, while the addition of exogenous Zn2+ and Fe2+ reduced the potency of compounds from this series. Detailed biochemical analyses confirmed that 8-HNT compounds bind directly to a number of divalent cations, predominantly Zn2+, Fe2+, and Cu2+, forming 2:1 complexes with one of these cations. Collectively, our studies demonstrate transition metal depletion, due to chelation, as the MoA of the 8-HNT series of compounds. Strategies to improve the selectivity of 8-HNT compounds are discussed.


Assuntos
Antiprotozoários/farmacologia , Proteínas de Transporte de Cátions/genética , Quelantes/farmacologia , Naftiridinas/farmacologia , Proteínas de Protozoários/genética , Zinco/metabolismo , Antiprotozoários/síntese química , Proteínas de Transporte de Cátions/metabolismo , Cátions Bivalentes , Quelantes/síntese química , Cobre/metabolismo , Expressão Gênica , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Ferro/metabolismo , Leishmania donovani/efeitos dos fármacos , Leishmania donovani/genética , Leishmania donovani/crescimento & desenvolvimento , Leishmania donovani/metabolismo , Mutação , Naftiridinas/síntese química , Testes de Sensibilidade Parasitária , Proteínas de Protozoários/metabolismo , Relação Estrutura-Atividade , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/crescimento & desenvolvimento , Trypanosoma brucei brucei/metabolismo
9.
J Biol Chem ; 291(47): 24768-24778, 2016 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-27703008

RESUMO

The aim of this study was to identify and characterize mechanisms of resistance to antifolate drugs in African trypanosomes. Genome-wide RNAi library screens were undertaken in bloodstream form Trypanosoma brucei exposed to the antifolates methotrexate and raltitrexed. In conjunction with drug susceptibility and folate transport studies, RNAi knockdown was used to validate the functions of the putative folate transporters. The transport kinetics of folate and methotrexate were further characterized in whole cells. RNA interference target sequencing experiments identified a tandem array of genes encoding a folate transporter family, TbFT1-3, as major contributors to antifolate drug uptake. RNAi knockdown of TbFT1-3 substantially reduced folate transport into trypanosomes and reduced the parasite's susceptibly to the classical antifolates methotrexate and raltitrexed. In contrast, knockdown of TbFT1-3 increased susceptibly to the non-classical antifolates pyrimethamine and nolatrexed. Both folate and methotrexate transport were inhibited by classical antifolates but not by non-classical antifolates or biopterin. Thus, TbFT1-3 mediates the uptake of folate and classical antifolates in trypanosomes, and TbFT1-3 loss-of-function is a mechanism of antifolate drug resistance.


Assuntos
Transportadores de Ácido Fólico/metabolismo , Ácido Fólico/metabolismo , Metotrexato/farmacocinética , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/metabolismo , Transportadores de Ácido Fólico/genética , Estudo de Associação Genômica Ampla , Metotrexato/farmacologia , Proteínas de Protozoários/genética , Trypanosoma brucei brucei/genética
10.
PLoS Pathog ; 11(11): e1005273, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26565797

RESUMO

Cell-cycle progression and cell division in eukaryotes are governed in part by the cyclin family and their regulation of cyclin-dependent kinases (CDKs). Cyclins are very well characterised in model systems such as yeast and human cells, but surprisingly little is known about their number and role in Plasmodium, the unicellular protozoan parasite that causes malaria. Malaria parasite cell division and proliferation differs from that of many eukaryotes. During its life cycle it undergoes two types of mitosis: endomitosis in asexual stages and an extremely rapid mitotic process during male gametogenesis. Both schizogony (producing merozoites) in host liver and red blood cells, and sporogony (producing sporozoites) in the mosquito vector, are endomitotic with repeated nuclear replication, without chromosome condensation, before cell division. The role of specific cyclins during Plasmodium cell proliferation was unknown. We show here that the Plasmodium genome contains only three cyclin genes, representing an unusual repertoire of cyclin classes. Expression and reverse genetic analyses of the single Plant (P)-type cyclin, CYC3, in the rodent malaria parasite, Plasmodium berghei, revealed a cytoplasmic and nuclear location of the GFP-tagged protein throughout the lifecycle. Deletion of cyc3 resulted in defects in size, number and growth of oocysts, with abnormalities in budding and sporozoite formation. Furthermore, global transcript analysis of the cyc3-deleted and wild type parasites at gametocyte and ookinete stages identified differentially expressed genes required for signalling, invasion and oocyst development. Collectively these data suggest that cyc3 modulates oocyst endomitotic development in Plasmodium berghei.


Assuntos
Divisão Celular/fisiologia , Ciclinas/metabolismo , Malária/parasitologia , Plasmodium berghei/metabolismo , Proteínas de Protozoários/metabolismo , Animais , Culicidae , Ciclinas/genética , Feminino , Humanos , Camundongos , Oocistos , Proteínas de Protozoários/genética , Esporozoítos/crescimento & desenvolvimento
11.
Geophys Res Lett ; 44(12): 6092-6100, 2017 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-30166740

RESUMO

We combine geophysical and experimental observations to interpret preeruptive unrest at Volcán de Colima in 1998. 17,893 volcanic earthquakes were detected between 1 October and 31 December 1998, including 504 clusters. Using seismic ambient noise interferometry, we observe a drop in velocity prior to the eruption linked to damage accumulation during magma ascent. This is supported by experimental observations where static stress causes a velocity decrease prior to failure. Furthermore, we observe acoustic emission clusters during the experiments, with lower porosity samples producing higher numbers of repeaters. This behavior introduces tensile failure as an additional viable mechanism for clusters during magma ascent. The findings suggest that preeruptive magma ascent may be monitored to variable degrees of accuracy via descriptions of damage accumulation and associated seismic velocity changes.

12.
J Biol Chem ; 290(19): 12147-64, 2015 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-25802338

RESUMO

Myosin B (MyoB) is one of the two short class XIV myosins encoded in the Plasmodium genome. Class XIV myosins are characterized by a catalytic "head," a modified "neck," and the absence of a "tail" region. Myosin A (MyoA), the other class XIV myosin in Plasmodium, has been established as a component of the glideosome complex important in motility and cell invasion, but MyoB is not well characterized. We analyzed the properties of MyoB using three parasite species as follows: Plasmodium falciparum, Plasmodium berghei, and Plasmodium knowlesi. MyoB is expressed in all invasive stages (merozoites, ookinetes, and sporozoites) of the life cycle, and the protein is found in a discrete apical location in these polarized cells. In P. falciparum, MyoB is synthesized very late in schizogony/merogony, and its location in merozoites is distinct from, and anterior to, that of a range of known proteins present in the rhoptries, rhoptry neck or micronemes. Unlike MyoA, MyoB is not associated with glideosome complex proteins, including the MyoA light chain, myosin A tail domain-interacting protein (MTIP). A unique MyoB light chain (MLC-B) was identified that contains a calmodulin-like domain at the C terminus and an extended N-terminal region. MLC-B localizes to the same extreme apical pole in the cell as MyoB, and the two proteins form a complex. We propose that MLC-B is a MyoB-specific light chain, and for the short class XIV myosins that lack a tail region, the atypical myosin light chains may fulfill that role.


Assuntos
Miosina não Muscular Tipo IIB/química , Plasmodium berghei/metabolismo , Plasmodium falciparum/metabolismo , Plasmodium knowlesi/metabolismo , Proteínas de Protozoários/química , Sequência de Aminoácidos , Calmodulina/química , Dicroísmo Circular , Técnica Indireta de Fluorescência para Anticorpo , Proteínas de Fluorescência Verde/química , Dados de Sequência Molecular , Cadeias Leves de Miosina/química , Miosina não Muscular Tipo IIA/química , Peptídeos/química , Ligação Proteica , Desnaturação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos
13.
mBio ; : e0180323, 2023 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-37929970

RESUMO

Visceral leishmaniasis (VL) is a parasitic disease endemic across multiple regions of the world and is fatal if untreated. New therapeutic options with diverse mechanisms of actions (MoAs) are required to consolidate progress toward control of this disease and combat drug resistance. Here, we describe the development of a scalable resistance library screen (RES-Seq) as a tool to facilitate the identification and prioritization of anti-leishmanial compounds acting via novel MoA. We have amassed a large collection of Leishmania donovani cell lines resistant to frontline drugs and compounds in the VL pipeline, with resistance-conferring mutations fully characterized. New phenotypic hits screened against this highly curated panel of resistant lines can determine cross-resistance and potentially shared MoA. The ability to efficiently identify compounds acting via previously established MoA is vital to maintain diversity within drug development portfolios. To expedite screening, short identifier DNA barcodes were introduced into resistant clones enabling pooling and simultaneous screening of multiple cell lines. Illumina sequencing of barcodes enables the growth kinetics and relative fitness of multiple cell lines under compound selection to be tracked. Optimal conditions allowing discrimination of resistant and sensitive clones were established (3× and 10× EC50 for 3 days) and applied to screening of a complex library with VL preclinical and clinical drug candidates. RES-Seq is set to play an important role in ensuring that anti-leishmanial compounds exploiting diverse mechanisms of action are developed, ultimately providing options for future drug combination strategies.IMPORTANCEVisceral leishmaniasis (VL) remains the third largest parasitic killer worldwide, responsible for 20,000-30,000 deaths each year. Control and ultimate elimination of VL will require a range of therapeutic options with diverse mechanisms of action to combat drug resistance. One approach to ensure that compounds in development exploit diverse mechanisms of action is to screen them against highly curated cell lines resistant to drugs already in the VL pipeline. The identification of cross-resistant cell lines indicates that test compounds are likely acting via previously established mechanisms. Current cross-resistance screens are limited by the requirement to profile individual resistant cell lines one at a time. Here, we introduce unique DNA barcodes into multiple resistant cell lines to facilitate parallel profiling. Utilizing the power of Illumina sequencing, growth kinetics and relative fitness under compound selection can be monitored revolutionizing our ability to identify and prioritize compounds acting via novel mechanisms.

14.
J Med Chem ; 66(13): 8896-8916, 2023 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-37343180

RESUMO

While treatment options for human African trypanosomiasis (HAT) have improved significantly, there is still a need for new drugs with eradication now a realistic possibility. Here, we report the development of 2,4-diaminothiazoles that demonstrate significant potency against Trypanosoma brucei, the causative agent of HAT. Using phenotypic screening to guide structure-activity relationships, potent drug-like inhibitors were developed. Proof of concept was established in an animal model of the hemolymphatic stage of HAT. To treat the meningoencephalitic stage of infection, compounds were optimized for pharmacokinetic properties, including blood-brain barrier penetration. However, in vivo efficacy was not achieved, in part due to compounds evolving from a cytocidal to a cytostatic mechanism of action. Subsequent studies identified a nonessential kinase involved in the inositol biosynthesis pathway as the molecular target of these cytostatic compounds. These studies highlight the need for cytocidal drugs for the treatment of HAT and the importance of static-cidal screening of analogues.


Assuntos
Citostáticos , Tripanossomicidas , Trypanosoma brucei brucei , Tripanossomíase Africana , Animais , Humanos , Tripanossomíase Africana/tratamento farmacológico , Tripanossomicidas/uso terapêutico , Tripanossomicidas/farmacocinética , Citostáticos/uso terapêutico , Barreira Hematoencefálica
15.
Sci Transl Med ; 15(726): eadg8105, 2023 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-38091410

RESUMO

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, affects millions of people in the Americas and across the world, leading to considerable morbidity and mortality. Current treatment options, benznidazole (BNZ) and nifurtimox, offer limited efficacy and often lead to adverse side effects because of long treatment durations. Better treatment options are therefore urgently required. Here, we describe a pyrrolopyrimidine series, identified through phenotypic screening, that offers an opportunity to improve on current treatments. In vitro cell-based washout assays demonstrate that compounds in the series are incapable of killing all parasites; however, combining these pyrrolopyrimidines with a subefficacious dose of BNZ can clear all parasites in vitro after 5 days. These findings were replicated in a clinically predictive in vivo model of chronic Chagas disease, where 5 days of treatment with the combination was sufficient to prevent parasite relapse. Comprehensive mechanism of action studies, supported by ligand-structure modeling, show that compounds from this pyrrolopyrimidine series inhibit the Qi active site of T. cruzi cytochrome b, part of the cytochrome bc1 complex of the electron transport chain. Knowledge of the molecular target enabled a cascade of assays to be assembled to evaluate selectivity over the human cytochrome b homolog. As a result, a highly selective and efficacious lead compound was identified. The combination of our lead compound with BNZ rapidly clears T. cruzi parasites, both in vitro and in vivo, and shows great potential to overcome key issues associated with currently available treatments.


Assuntos
Doença de Chagas , Parasitos , Tripanossomicidas , Trypanosoma cruzi , Animais , Humanos , Citocromos b , Tripanossomicidas/efeitos adversos , Doença de Chagas/tratamento farmacológico , Doença de Chagas/induzido quimicamente , Doença de Chagas/parasitologia
16.
Sci Transl Med ; 15(726): eadh9902, 2023 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-38091406

RESUMO

New drugs for visceral leishmaniasis that are safe, low cost, and adapted to the field are urgently required. Despite concerted efforts over the last several years, the number of new chemical entities that are suitable for clinical development for the treatment of Leishmania remains low. Here, we describe the discovery and preclinical development of DNDI-6174, an inhibitor of Leishmania cytochrome bc1 complex activity that originated from a phenotypically identified pyrrolopyrimidine series. This compound fulfills all target candidate profile criteria required for progression into preclinical development. In addition to good metabolic stability and pharmacokinetic properties, DNDI-6174 demonstrates potent in vitro activity against a variety of Leishmania species and can reduce parasite burden in animal models of infection, with the potential to approach sterile cure. No major flags were identified in preliminary safety studies, including an exploratory 14-day toxicology study in the rat. DNDI-6174 is a cytochrome bc1 complex inhibitor with acceptable development properties to enter preclinical development for visceral leishmaniasis.


Assuntos
Leishmaniose Visceral , Leishmaniose , Ratos , Animais , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/parasitologia , Modelos Animais de Doenças
17.
ACS Infect Dis ; 8(9): 1962-1974, 2022 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-36037410

RESUMO

There is a pressing need for new medicines to prevent and treat malaria. Most antimalarial drug discovery is reliant upon phenotypic screening. However, with the development of improved target validation strategies, target-focused approaches are now being utilized. Here, we describe the development of a toolkit to support the therapeutic exploitation of a promising target, lysyl tRNA synthetase (PfKRS). The toolkit includes resistant mutants to probe resistance mechanisms and on-target engagement for specific chemotypes; a hybrid KRS protein capable of producing crystals suitable for ligand soaking, thus providing high-resolution structural information to guide compound optimization; chemical probes to facilitate pulldown studies aimed at revealing the full range of specifically interacting proteins and thermal proteome profiling (TPP); as well as streamlined isothermal TPP methods to provide unbiased confirmation of on-target engagement within a biologically relevant milieu. This combination of tools and methodologies acts as a template for the development of future target-enabling packages.


Assuntos
Antimaláricos , Lisina-tRNA Ligase , Malária , Antimaláricos/química , Antimaláricos/farmacologia , Descoberta de Drogas , Humanos , Lisina-tRNA Ligase/química , Lisina-tRNA Ligase/genética , Lisina-tRNA Ligase/metabolismo , Plasmodium falciparum/metabolismo
18.
J Med Chem ; 65(7): 5606-5624, 2022 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-35303411

RESUMO

African animal trypanosomiasis or nagana, caused principally by infection of the protozoan parasites Trypanosoma congolense and Trypanosoma vivax, is a major problem in cattle and other livestocks in sub-Saharan Africa. Current treatments are threatened by the emergence of drug resistance and there is an urgent need for new, effective drugs. Here, we report the repositioning of a compound series initially developed for the treatment of human African trypanosomiasis. A medicinal chemistry program, focused on deriving more soluble analogues, led to development of a lead compound capable of curing cattle infected with both T. congolense and T. vivax via intravenous dosing. Further optimization has the potential to yield a single-dose intramuscular treatment for this disease. Comprehensive mode of action studies revealed that the molecular target of this promising compound and related analogues is the cyclin-dependent kinase CRK12.


Assuntos
Trypanosoma congolense , Tripanossomíase Africana , Animais , Bovinos , Quinases Ciclina-Dependentes , Reposicionamento de Medicamentos , Trypanosoma vivax , Tripanossomíase Africana/tratamento farmacológico , Tripanossomíase Africana/parasitologia , Tripanossomíase Africana/veterinária
19.
Microorganisms ; 9(7)2021 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-34210040

RESUMO

Current treatment options for visceral leishmaniasis have several drawbacks, and clinicians are confronted with an increasing number of treatment failures. To overcome this, the Drugs for Neglected Diseases initiative (DNDi) has invested in the development of novel antileishmanial leads, including a very promising class of oxaboroles. The mode of action/resistance of this series to Leishmania is still unknown and may be important for its further development and implementation. Repeated in vivo drug exposure and an in vitro selection procedure on both extracellular promastigote and intracellular amastigote stages were both unable to select for resistance. The use of specific inhibitors for ABC-transporters could not demonstrate the putative involvement of efflux pumps. Selection experiments and inhibitor studies, therefore, suggest that resistance to oxaboroles may not emerge readily in the field. The selection of a genome-wide cosmid library coupled to next-generation sequencing (Cos-seq) was used to identify resistance determinants and putative targets. This resulted in the identification of a highly enriched cosmid, harboring genes of chromosome 2 that confer a subtly increased resistance to the oxaboroles tested. Moderately enriched cosmids encompassing a region of chromosome 34 contained the cleavage and polyadenylation specificity factor (cpsf) gene, encoding the molecular target of several related benzoxaboroles in other organisms.

20.
Cell Chem Biol ; 28(5): 711-721.e8, 2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-33691122

RESUMO

Phenotypic screening identified a benzothiophene compound with activity against Leishmania donovani, the causative agent of visceral leishmaniasis. Using multiple orthogonal approaches, oxidosqualene cyclase (OSC), a key enzyme of sterol biosynthesis, was identified as the target of this racemic compound and its enantiomers. Whole genome sequencing and screening of a genome-wide overexpression library confirmed that OSC gene amplification is associated with resistance to compound 1. Introduction of an ectopic copy of the OSC gene into wild-type cells reduced susceptibility to these compounds confirming the role of this enzyme in resistance. Biochemical analyses demonstrated the accumulation of the substrate of OSC and depletion of its product in compound (S)-1-treated-promastigotes and cell-free membrane preparations, respectively. Thermal proteome profiling confirmed that compound (S)-1 binds directly to OSC. Finally, modeling and docking studies identified key interactions between compound (S)-1 and the LdOSC active site. Strategies to improve the potency for this promising anti-leishmanial are proposed.


Assuntos
Antiprotozoários/farmacologia , Inibidores Enzimáticos/farmacologia , Transferases Intramoleculares/antagonistas & inibidores , Leishmania donovani/efeitos dos fármacos , Piperidinas/farmacologia , Antiprotozoários/síntese química , Antiprotozoários/química , Cristalografia por Raios X , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Transferases Intramoleculares/metabolismo , Leishmania donovani/enzimologia , Modelos Moleculares , Estrutura Molecular , Testes de Sensibilidade Parasitária , Piperidinas/síntese química , Piperidinas/química
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