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 HematoencefálicaRESUMO
Herein we describe the optimization of a phenotypic hit against Plasmodium falciparum based on an aminoacetamide scaffold. This led to N-(3-chloro-4-fluorophenyl)-2-methyl-2-{[4-methyl-3-(morpholinosulfonyl)phenyl]amino}propanamide (compound 28) with low-nanomolar activity against the intraerythrocytic stages of the malaria parasite, and which was found to be inactive in a mammalian cell counter-screen up to 25â µm. Inhibition of gametes in the dual gamete activation assay suggests that this family of compounds may also have transmission blocking capabilities. Whilst we were unable to optimize the aqueous solubility and microsomal stability to a point at which the aminoacetamides would be suitable for inâ vivo pharmacokinetic and efficacy studies, compound 28 displayed excellent antimalarial potency and selectivity; it could therefore serve as a suitable chemical tool for drug target identification.
Assuntos
Acetamidas/farmacologia , Antimaláricos/farmacologia , Acetamidas/síntese química , Acetamidas/farmacocinética , Animais , Antimaláricos/síntese química , Antimaláricos/farmacocinética , Humanos , Camundongos , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Testes de Sensibilidade Parasitária , Plasmodium berghei/efeitos dos fármacos , Plasmodium cynomolgi/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
Crystallography has guided the hybridization of two series of Trypanosoma brucei N-myristoyltransferase (NMT) inhibitors, leading to a novel highly selective series. The effect of combining the selectivity enhancing elements from two pharmacophores is shown to be additive and has led to compounds that have greater than 1000-fold selectivity for TbNMT vs HsNMT. Further optimization of the hybrid series has identified compounds with significant trypanocidal activity capable of crossing the blood-brain barrier. By using CF-1 mdr1a deficient mice, we were able to demonstrate full cures in vivo in a mouse model of stage 2 African sleeping sickness. This and previous work provides very strong validation for NMT as a drug target for human African trypanosomiasis in both the peripheral and central nervous system stages of disease.
Assuntos
Aciltransferases/antagonistas & inibidores , Barreira Hematoencefálica/efeitos dos fármacos , Desenho de Fármacos , Tripanossomicidas/química , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Tripanossomíase Africana/tratamento farmacológico , Animais , Sobrevivência Celular , Feminino , Humanos , Camundongos , Camundongos Endogâmicos , Modelos Moleculares , Estrutura Molecular , Conformação Proteica , Relação Estrutura-Atividade , Tripanossomíase Africana/microbiologiaRESUMO
N-Myristoyltransferase (NMT) represents a promising drug target within the parasitic protozoa Trypanosoma brucei (T. brucei), the causative agent for human African trypanosomiasis (HAT) or sleeping sickness. We have previously validated T. brucei NMT as a promising druggable target for the treatment of HAT in both stages 1 and 2 of the disease. We report on the use of the previously reported DDD85646 (1) as a starting point for the design of a class of potent, brain penetrant inhibitors of T. brucei NMT.
Assuntos
Aciltransferases/antagonistas & inibidores , Aminopiridinas/química , Aminopiridinas/farmacologia , Sulfonamidas/química , Sulfonamidas/farmacologia , Tripanossomicidas/química , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/enzimologia , Tripanossomíase Africana/tratamento farmacológico , Aciltransferases/metabolismo , Aminopiridinas/síntese química , Aminopiridinas/farmacocinética , Animais , Encéfalo/metabolismo , Cristalografia por Raios X , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/farmacologia , Humanos , Camundongos , Sulfonamidas/síntese química , Sulfonamidas/farmacocinética , Tripanossomicidas/síntese química , Tripanossomicidas/farmacocinética , Tripanossomíase Africana/metabolismoRESUMO
BACKGROUND: Protein kinases have been shown to be key drug targets, especially in the area of oncology. It is of interest to explore the possibilities of protein kinases as a potential target class in Plasmodium spp., the causative agents of malaria. However, protein kinase biology in malaria is still being investigated. Therefore, rather than assaying against individual protein kinases, a library of 4731 compounds with protein kinase inhibitor-like scaffolds was screened against the causative parasite, Plasmodium falciparum. This approach is more holistic and considers the whole kinome, making it possible to identify compounds that inhibit more than one P. falciparum protein kinase, or indeed other malaria targets. RESULTS: As a result of this screen, 9 active compound series were identified; further validation was carried out on 4 of these series, with 3 being progressed into hits to lead chemistry. The detailed evaluation of one of these series is described. DISCUSSION: This screening approach proved to be an effective way to identify series for further optimisation against malaria. Compound optimisation was carried out in the absence of knowledge of the molecular target. Some of the series had to be halted for various reasons. Mode of action studies to find the molecular target may be useful when problems prevent further chemical optimisation. CONCLUSIONS: Progressible series were identified through phenotypic screening of a relatively small focused kinase scaffold chemical library.
Assuntos
Antimaláricos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Avaliação Pré-Clínica de MedicamentosRESUMO
In late mitosis and G1, origins of DNA replication must be "licensed" for use in the upcoming S phase by being encircled by double hexamers of the minichromosome maintenance proteins MCM2-7. A "licensing checkpoint" delays cells in G1 until sufficient origins have been licensed, but this checkpoint is lost in cancer cells. Inhibition of licensing can therefore kill cancer cells while only delaying normal cells in G1. In a high-throughput cell-based screen for licensing inhibitors we identified a family of 2-arylquinolin-4-amines, the most potent of which we call RL5a. The binding of the origin recognition complex (ORC) to origin DNA is the first step of the licensing reaction. We show that RL5a prevents ORC forming a tight complex with DNA that is required for MCM2-7 loading. Formation of this ORC-DNA complex requires ATP, and we show that RL5a inhibits ORC allosterically to mimic a lack of ATP.
Assuntos
Aminas/farmacologia , Replicação do DNA/efeitos dos fármacos , DNA/metabolismo , Complexo de Reconhecimento de Origem/metabolismo , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Aminas/química , Aminas/metabolismo , Animais , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Cromatina/química , Cromatina/metabolismo , Humanos , Proteínas de Manutenção de Minicromossomo/química , Proteínas de Manutenção de Minicromossomo/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Complexo de Reconhecimento de Origem/antagonistas & inibidores , Quinolinas/farmacologia , Origem de Replicação/genética , Tiazóis/farmacologia , Xenopus , Proteínas de Xenopus/metabolismoRESUMO
The antiplasmodial activity, DMPK properties, and efficacy of a series of quinoline-4-carboxamides are described. This series was identified from a phenotypic screen against the blood stage of Plasmodium falciparum (3D7) and displayed moderate potency but with suboptimal physicochemical properties and poor microsomal stability. The screening hit (1, EC50 = 120 nM) was optimized to lead molecules with low nanomolar in vitro potency. Improvement of the pharmacokinetic profile led to several compounds showing excellent oral efficacy in the P. berghei malaria mouse model with ED90 values below 1 mg/kg when dosed orally for 4 days. The favorable potency, selectivity, DMPK properties, and efficacy coupled with a novel mechanism of action, inhibition of translation elongation factor 2 (PfEF2), led to progression of 2 (DDD107498) to preclinical development.
Assuntos
Antimaláricos/farmacologia , Descoberta de Drogas , Malária/tratamento farmacológico , Plasmodium falciparum/efeitos dos fármacos , Quinolinas/farmacologia , Animais , Antimaláricos/síntese química , Antimaláricos/química , Modelos Animais de Doenças , Camundongos , Estrutura Molecular , Quinolinas/síntese química , Quinolinas/química , Relação Estrutura-AtividadeRESUMO
In this paper we describe the optimization of a phenotypic hit against Plasmodium falciparum, based on a trisubstituted pyrimidine scaffold. This led to compounds with good pharmacokinetics and oral activity in a P. berghei mouse model of malaria. The most promising compound (13) showed a reduction in parasitemia of 96% when dosed at 30 mg/kg orally once a day for 4 days in the P. berghei mouse model of malaria. It also demonstrated a rapid rate of clearance of the erythrocytic stage of P. falciparum in the SCID mouse model with an ED90 of 11.7 mg/kg when dosed orally. Unfortunately, the compound is a potent inhibitor of cytochrome P450 enzymes, probably due to a 4-pyridyl substituent. Nevertheless, this is a lead molecule with a potentially useful antimalarial profile, which could either be further optimized or be used for target hunting.
Assuntos
Antimaláricos/química , Antimaláricos/uso terapêutico , Malária/tratamento farmacológico , Plasmodium berghei/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Pirimidinas/química , Pirimidinas/uso terapêutico , Animais , Antimaláricos/farmacocinética , Antimaláricos/farmacologia , Humanos , Malária/parasitologia , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Camundongos SCID , Parasitemia/tratamento farmacológico , Parasitemia/parasitologia , Pirimidinas/farmacocinética , Pirimidinas/farmacologiaRESUMO
The enzyme N-myristoyltransferase (NMT) from Trypanosoma brucei has been validated both chemically and biologically as a potential drug target for human African trypanosomiasis. We previously reported the development of some very potent compounds based around a pyrazole sulfonamide series, derived from a high-throughput screen. Herein we describe work around thiazolidinone and benzomorpholine scaffolds that were also identified in the screen. An X-ray crystal structure of the thiazolidinone hit in Leishmania major NMT showed the compound bound in the previously reported active site, utilising a novel binding mode. This provides potential for further optimisation. The benzomorpholinone was also found to bind in a similar region. Using an X-ray crystallography/structure-based design approach, the benzomorpholinone series was further optimised, increasing activity against T.â brucei NMT by >1000-fold. A series of trypanocidal compounds were identified with suitable in vitro DMPK properties, including CNS exposure for further development. Further work is required to increase selectivity over the human NMT isoform and activity against T.â brucei.
Assuntos
Aciltransferases/antagonistas & inibidores , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Trypanosoma brucei brucei/enzimologia , Aciltransferases/metabolismo , Sítios de Ligação/efeitos dos fármacos , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Modelos Moleculares , Estrutura Molecular , Testes de Sensibilidade Parasitária , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Trypanosoma brucei brucei/efeitos dos fármacosRESUMO
A screen of a focused kinase inhibitor library against Trypanosoma brucei rhodesiense led to the identification of seven series, totaling 121 compounds, which showed >50 % inhibition at 5â µm. Screening of these hits in a T.â b.â brucei proliferation assay highlighted three compounds with a 1H-imidazo[4,5-b]pyrazin-2(3H)-one scaffold that showed sub-micromolar activity and excellent selectivity against the MRC5 cell line. Subsequent rounds of optimisation led to the identification of compounds that exhibited good in vitro drug metabolism and pharmacokinetics (DMPK) properties, although in general this series suffered from poor solubility. A scaffold-hopping exercise led to the identification of a 1H-pyrazolo[3,4-b]pyridine scaffold, which retained potency. A number of examples were assessed in a T.â b.â brucei growth assay, which could differentiate static and cidal action. Compounds from the 1H-imidazo[4,5-b]pyrazin-2(3H)-one series were found to be either static or growth-slowing and not cidal. Compounds with the 1H-pyrazolo[3,4-b]pyridine scaffold were found to be cidal and showed an unusual biphasic nature in this assay, suggesting they act by at least two mechanisms.
Assuntos
Descoberta de Drogas , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Trypanosoma brucei rhodesiense/efeitos dos fármacos , Linhagem Celular , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Testes de Sensibilidade Parasitária , Fenótipo , Inibidores de Proteínas Quinases/química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Trypanosoma brucei rhodesiense/crescimento & desenvolvimentoRESUMO
There is an urgent need for new, brain penetrant small molecules that target the central nervous system second stage of human African trypanosomiasis (HAT). We report that a series of novel indoline-2-carboxamides have been identified as inhibitors of Trypanosoma brucei from screening of a focused protease library against Trypanosoma brucei brucei in culture. We describe the optimization and characterization of this series. Potent antiproliferative activity was observed. The series demonstrated excellent pharmacokinetic properties, full cures in a stage 1 mouse model of HAT, and a partial cure in a stage 2 mouse model of HAT. Lack of tolerability prevented delivery of a fully curative regimen in the stage 2 mouse model and thus further progress of this series.
Assuntos
Encéfalo/efeitos dos fármacos , Tripanossomicidas/química , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Tripanossomíase Africana/tratamento farmacológico , Animais , Técnicas de Química Sintética , Modelos Animais de Doenças , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Indóis/química , Camundongos Endogâmicos , Estereoisomerismo , Relação Estrutura-Atividade , Tripanossomicidas/farmacocinética , Trypanosoma brucei brucei/crescimento & desenvolvimento , Tripanossomíase Africana/parasitologiaRESUMO
There is an urgent need for new drugs to treat malaria, with broad therapeutic potential and novel modes of action, to widen the scope of treatment and to overcome emerging drug resistance. Here we describe the discovery of DDD107498, a compound with a potent and novel spectrum of antimalarial activity against multiple life-cycle stages of the Plasmodium parasite, with good pharmacokinetic properties and an acceptable safety profile. DDD107498 demonstrates potential to address a variety of clinical needs, including single-dose treatment, transmission blocking and chemoprotection. DDD107498 was developed from a screening programme against blood-stage malaria parasites; its molecular target has been identified as translation elongation factor 2 (eEF2), which is responsible for the GTP-dependent translocation of the ribosome along messenger RNA, and is essential for protein synthesis. This discovery of eEF2 as a viable antimalarial drug target opens up new possibilities for drug discovery.
Assuntos
Antimaláricos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Malária/parasitologia , Plasmodium/efeitos dos fármacos , Plasmodium/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Quinolinas/farmacologia , Animais , Antimaláricos/administração & dosagem , Antimaláricos/efeitos adversos , Antimaláricos/farmacocinética , Descoberta de Drogas , Feminino , Estágios do Ciclo de Vida/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/parasitologia , Malária/tratamento farmacológico , Masculino , Modelos Moleculares , Fator 2 de Elongação de Peptídeos/antagonistas & inibidores , Fator 2 de Elongação de Peptídeos/metabolismo , Plasmodium/genética , Plasmodium/crescimento & desenvolvimento , Plasmodium berghei/efeitos dos fármacos , Plasmodium berghei/fisiologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/metabolismo , Plasmodium vivax/efeitos dos fármacos , Plasmodium vivax/metabolismo , Quinolinas/administração & dosagem , Quinolinas/química , Quinolinas/farmacocinéticaRESUMO
Trypanosoma brucei N-myristoyltransferase (TbNMT) is an attractive therapeutic target for the treatment of human African trypanosomiasis (HAT). From previous studies, we identified pyrazole sulfonamide, DDD85646 (1), a potent inhibitor of TbNMT. Although this compound represents an excellent lead, poor central nervous system (CNS) exposure restricts its use to the hemolymphatic form (stage 1) of the disease. With a clear clinical need for new drug treatments for HAT that address both the hemolymphatic and CNS stages of the disease, a chemistry campaign was initiated to address the shortfalls of this series. This paper describes modifications to the pyrazole sulfonamides which markedly improved blood-brain barrier permeability, achieved by reducing polar surface area and capping the sulfonamide. Moreover, replacing the core aromatic with a flexible linker significantly improved selectivity. This led to the discovery of DDD100097 (40) which demonstrated partial efficacy in a stage 2 (CNS) mouse model of HAT.
Assuntos
Aciltransferases/antagonistas & inibidores , Pirazóis/síntese química , Sulfonamidas/síntese química , Tripanossomicidas/síntese química , Tripanossomíase Africana/tratamento farmacológico , Aminopiridinas/química , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Sistema Nervoso Central/efeitos dos fármacos , Feminino , Humanos , Concentração Inibidora 50 , Camundongos , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacosRESUMO
Glycogen synthase kinase 3 (GSK3) is a genetically validated drug target for human African trypanosomiasis (HAT), also called African sleeping sickness. We report the synthesis and biological evaluation of aminopyrazole derivatives as Trypanosoma brucei GSK3 short inhibitors. Low nanomolar inhibitors, which had high selectivity over the off-target human CDK2 and good selectivity over human GSK3ß enzyme, have been prepared. These potent kinase inhibitors demonstrated low micromolar levels of inhibition of the Trypanosoma brucei brucei parasite grown in culture.
Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Pirazóis/síntese química , Pirazóis/farmacologia , Tripanossomíase Africana/tratamento farmacológico , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Técnicas de Química Sintética , Quinase 2 Dependente de Ciclina/química , Quinase 2 Dependente de Ciclina/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Humanos , Modelos Moleculares , Conformação Proteica , Pirazóis/química , Pirazóis/uso terapêutico , Especificidade por SubstratoAssuntos
Academias e Institutos/organização & administração , Serviços Contratados/organização & administração , Descoberta de Drogas/métodos , Tecnologia Farmacêutica/organização & administração , Academias e Institutos/economia , Academias e Institutos/normas , Fortalecimento Institucional/organização & administração , Fortalecimento Institucional/normas , Serviços Contratados/economia , Serviços Contratados/métodos , Serviços Contratados/normas , Descoberta de Drogas/economia , Descoberta de Drogas/organização & administração , Comunicação Interdisciplinar , Tecnologia Farmacêutica/economia , Tecnologia Farmacêutica/métodosRESUMO
Uridine diphosphate N-acetylglucosamine pyrophosphorylase (UAP) catalyzes the final reaction in the biosynthesis of UDP-GlcNAc, an essential metabolite in many organisms including Trypanosoma brucei, the etiological agent of Human African Trypanosomiasis. High-throughput screening of recombinant T. brucei UAP identified a UTP-competitive inhibitor with selectivity over the human counterpart despite the high level of conservation of active site residues. Biophysical characterization of the UAP enzyme kinetics revealed that the human and trypanosome enzymes both display a strictly ordered bi-bi mechanism, but with the order of substrate binding reversed. Structural characterization of the T. brucei UAP-inhibitor complex revealed that the inhibitor binds at an allosteric site absent in the human homologue that prevents the conformational rearrangement required to bind UTP. The identification of a selective inhibitory allosteric binding site in the parasite enzyme has therapeutic potential.
Assuntos
Nucleotidiltransferases/antagonistas & inibidores , Tripanossomicidas/química , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/enzimologia , Regulação Alostérica/efeitos dos fármacos , Domínio Catalítico , Humanos , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Conformação Proteica , Trypanosoma brucei brucei/crescimento & desenvolvimento , Tripanossomíase Africana/tratamento farmacológico , Tripanossomíase Africana/parasitologia , Uridina Difosfato N-Acetilglicosamina/metabolismoRESUMO
Human African trypanosomiasis (HAT) is a life-threatening disease with approximately 30 000-40 000 new cases each year. Trypanosoma brucei protein kinase GSK3 short (TbGSK3) is required for parasite growth and survival. Herein we report a screen of a focused kinase library against T.â brucei GSK3. From this we identified a series of several highly ligand-efficient TbGSK3 inhibitors. Following the hit validation process, we optimised a series of diaminothiazoles, identifying low-nanomolar inhibitors of TbGSK3 that are potent inâ vitro inhibitors of T.â brucei proliferation. We show that the TbGSK3 pharmacophore overlaps with that of one or more additional molecular targets.
Assuntos
Descoberta de Drogas , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/enzimologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Modelos Moleculares , Estrutura Molecular , Testes de Sensibilidade Parasitária , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Relação Estrutura-Atividade , Tripanossomicidas/síntese química , Tripanossomicidas/químicaRESUMO
Visceral leishmaniasis is a neglected tropical disease with significant health impact. The current treatments are poor, and there is an urgent need to develop new drugs. Primary screening assays used for drug discovery campaigns have typically used free-living forms of the Leishmania parasite to allow for high-throughput screening. Such screens do not necessarily reflect the physiological situation, as the disease-causing stage of the parasite resides inside human host cells. Assessing the drug sensitivity of intracellular parasites on scale has recently become feasible with the advent of high-content screening methods. We describe here a 384-well microscopy-based intramacrophage Leishmania donovani assay and compare it to an axenic amastigote system. A panel of eight reference compounds was tested in both systems, as well as a human counterscreen cell line, and our findings show that for most clinically used compounds both axenic and intramacrophage assays report very similar results. A set of 15,659 diverse compounds was also screened using both systems. This resulted in the identification of seven new antileishmanial compounds and revealed a high false-positive rate for the axenic assay. We conclude that the intramacrophage assay is more suited as a primary hit-discovery platform than the current form of axenic assay, and we discuss how modifications to the axenic assay may render it more suitable for hit-discovery.
Assuntos
Antiprotozoários/farmacologia , Leishmania donovani/efeitos dos fármacos , Leishmaniose Visceral/tratamento farmacológico , Testes de Sensibilidade Parasitária , Cultura Axênica , Linhagem Celular , Descoberta de Drogas , Ensaios de Triagem em Larga Escala , Humanos , Macrófagos/parasitologiaRESUMO
CDP-ME kinase (IspE) contributes to the non-mevalonate or deoxy-xylulose phosphate (DOXP) pathway for isoprenoid precursor biosynthesis found in many species of bacteria and apicomplexan parasites. IspE has been shown to be essential by genetic methods and since it is absent from humans it constitutes a promising target for antimicrobial drug development. Using in silico screening directed against the substrate binding site and in vitro high-throughput screening directed against both, the substrate and co-factor binding sites, non-substrate-like IspE inhibitors have been discovered and structure-activity relationships were derived. The best inhibitors in each series have high ligand efficiencies and favourable physico-chemical properties rendering them promising starting points for drug discovery. Putative binding modes of the ligands were suggested which are consistent with established structure-activity relationships. The applied screening methods were complementary in discovering hit compounds, and a comparison of both approaches highlights their strengths and weaknesses. It is noteworthy that compounds identified by virtual screening methods provided the controls for the biochemical screens.