RESUMO
African sleeping sickness or human African trypanosomiasis, caused by Trypanosoma brucei spp., is responsible for approximately 30,000 deaths each year. Available treatments for this disease are poor, with unacceptable efficacy and safety profiles, particularly in the late stage of the disease when the parasite has infected the central nervous system. Here we report the validation of a molecular target and the discovery of associated lead compounds with the potential to address this lack of suitable treatments. Inhibition of this target-T. brucei N-myristoyltransferase-leads to rapid killing of trypanosomes both in vitro and in vivo and cures trypanosomiasis in mice. These high-affinity inhibitors bind into the peptide substrate pocket of the enzyme and inhibit protein N-myristoylation in trypanosomes. The compounds identified have promising pharmaceutical properties and represent an opportunity to develop oral drugs to treat this devastating disease. Our studies validate T. brucei N-myristoyltransferase as a promising therapeutic target for human African trypanosomiasis.
Assuntos
Aciltransferases/antagonistas & inibidores , Antiparasitários/farmacologia , Antiparasitários/uso terapêutico , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/enzimologia , Tripanossomíase Africana/tratamento farmacológico , Tripanossomíase Africana/parasitologia , Aciltransferases/metabolismo , Aminopiridinas/química , Aminopiridinas/metabolismo , Aminopiridinas/farmacologia , Aminopiridinas/uso terapêutico , Animais , Antiparasitários/química , Antiparasitários/metabolismo , Ensaios Enzimáticos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Feminino , Humanos , Camundongos , Estrutura Molecular , Pirazóis/química , Pirazóis/metabolismo , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Ratos , Sulfonamidas/química , Sulfonamidas/metabolismo , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Fatores de Tempo , Trypanosoma brucei brucei/crescimento & desenvolvimentoRESUMO
We report here a series of five chemically diverse scaffolds that have in vitro activities on replicating and hypoxic nonreplicating bacilli by targeting the respiratory bc1 complex in Mycobacterium tuberculosis in a strain-dependent manner. Deletion of the cytochrome bd oxidase generated a hypersusceptible mutant in which resistance was acquired by a mutation in qcrB. These results highlight the promiscuity of the bc1 complex and the risk of targeting energy metabolism with new drugs.
Assuntos
Antituberculosos/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Mycobacterium tuberculosis/efeitos dos fármacos , Tuberculose/tratamento farmacológico , Sítios de Ligação , Transporte de Elétrons/efeitos dos fármacos , Complexo IV da Cadeia de Transporte de Elétrons/genética , Metabolismo Energético/efeitos dos fármacos , Técnicas de Inativação de Genes , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Oxazinas/química , Estrutura Terciária de Proteína , Piridinas/farmacologia , Xantenos/químicaRESUMO
Enzymes involved in rescuing stalled ribosomes and recycling translation machinery are ubiquitous in bacteria and required for growth. Peptidyl tRNA drop-off is a type of abortive translation that results in the release of a truncated peptide that is still bound to tRNA (peptidyl tRNA) into the cytoplasm. Peptidyl tRNA hydrolase (Pth) recycles the released tRNA by cleaving off the unfinished peptide and is essential in most bacteria. We developed a sequencing-based strategy called copper sulfate-based tRNA sequencing (Cu-tRNAseq) to study the physiological role of Pth in Mycobacterium tuberculosis (Mtb). While most peptidyl tRNA species accumulated in a strain with impaired Pth expression, peptidyl prolyl-tRNA was particularly enriched, suggesting that Pth is required for robust peptidyl prolyl-tRNA turnover. Reducing Pth levels increased Mtb's susceptibility to tRNA synthetase inhibitors that are in development to treat tuberculosis (TB) and rendered this pathogen highly susceptible to macrolides, drugs that are ordinarily ineffective against Mtb. Collectively, our findings reveal the potency of Cu-tRNAseq for profiling peptidyl tRNAs and suggest that targeting Pth would open new therapeutic approaches for TB. IMPORTANCE Peptidyl tRNA hydrolase (Pth) is an enzyme that cuts unfinished peptides off tRNA that has been prematurely released from a stalled ribosome. Pth is essential in nearly all bacteria, including the pathogen Mycobacterium tuberculosis (Mtb), but it has not been clear why. We have used genetic and novel biochemical approaches to show that when Pth levels decline in Mtb, peptidyl tRNA accumulates to such an extent that usable tRNA pools drop. Thus, Pth is needed to maintain normal tRNA levels, most strikingly for prolyl-tRNAs. Many antibiotics act on protein synthesis and could be affected by altering the availability of tRNA. This is certainly true for tRNA synthetase inhibitors, several of which are drug candidates for tuberculosis. We find that their action is potentiated by Pth depletion. Furthermore, Pth depletion results in hypersensitivity to macrolides, drugs that are not active enough under ordinary circumstances to be useful for tuberculosis.
Assuntos
Aminoacil-tRNA Sintetases , Mycobacterium tuberculosis , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , RNA de Transferência/genética , Peptídeos , Aminoacil-tRNA Sintetases/genética , Hidrolases , Hidrolases de Éster Carboxílico/metabolismoRESUMO
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
Tuberculosis is a major global cause of both mortality and financial burden mainly in low and middle-income countries. Given the significant and ongoing rise of drug-resistant strains of Mycobacterium tuberculosis within the clinical setting, there is an urgent need for the development of new, safe and effective treatments. Here the development of a drug-like series based on a fused dihydropyrrolidino-pyrimidine scaffold is described. The series has been developed against M. tuberculosis lysyl-tRNA synthetase (LysRS) and cellular studies support this mechanism of action. DDD02049209, the lead compound, is efficacious in mouse models of acute and chronic tuberculosis and has suitable physicochemical, pharmacokinetic properties and an in vitro safety profile that supports further development. Importantly, preliminary analysis using clinical resistant strains shows no pre-existing clinical resistance towards this scaffold.
Assuntos
Lisina-tRNA Ligase , Mycobacterium tuberculosis , Tuberculose , Animais , Lisina-tRNA Ligase/química , Lisina-tRNA Ligase/genética , Lisina-tRNA Ligase/farmacologia , Camundongos , Mycobacterium tuberculosis/genética , Tuberculose/tratamento farmacológicoRESUMO
With the emergence of multi-drug-resistant strains of Mycobacterium tuberculosis, there is a pressing need for new oral drugs with novel mechanisms of action. A number of scaffolds with potent anti-tubercular in vitro activity have been identified from phenotypic screening that appear to target MmpL3. However, the scaffolds are typically lipophilic, which facilitates partitioning into hydrophobic membranes, and several contain basic amine groups. Highly lipophilic basic amines are typically cytotoxic against mammalian cell lines and have associated off-target risks, such as inhibition of human ether-à-go-go related gene (hERG) and IKr potassium current modulation. The spirocycle compound 3 was reported to target MmpL3 and displayed promising efficacy in a murine model of acute tuberculosis (TB) infection. However, this highly lipophilic monobasic amine was cytotoxic and inhibited the hERG ion channel. Herein, the related spirocycles (1-2) are described, which were identified following phenotypic screening of the Eli Lilly corporate library against M. tuberculosis. The novel N-alkylated pyrazole portion offered improved physicochemical properties, and optimization led to identification of a zwitterion series, exemplified by lead 29, with decreased HepG2 cytotoxicity as well as limited hERG ion channel inhibition. Strains with mutations in MmpL3 were resistant to 29, and under replicating conditions, 29 demonstrated bactericidal activity against M. tuberculosis. Unfortunately, compound 29 had no efficacy in an acute model of TB infection; this was most likely due to the in vivo exposure remaining above the minimal inhibitory concentration for only a limited time.
RESUMO
The leishmaniases are diseases that affect millions of people across the world, in particular visceral leishmaniasis (VL) which is fatal unless treated. Current standard of care for VL suffers from multiple issues and there is a limited pipeline of new candidate drugs. As such, there is a clear unmet medical need to identify new treatments. This paper describes the optimization of a phenotypic hit against Leishmania donovani, the major causative organism of VL. The key challenges were to balance solubility and metabolic stability while maintaining potency. Herein, strategies to address these shortcomings and enhance efficacy are discussed, culminating in the discovery of preclinical development candidate GSK3186899/DDD853651 (1) for VL.
Assuntos
Leishmaniose Visceral/tratamento farmacológico , Morfolinas/uso terapêutico , Pirazóis/uso terapêutico , Pirimidinas/uso terapêutico , Tripanossomicidas/uso terapêutico , Animais , Feminino , Células Hep G2 , Humanos , Leishmania donovani/efeitos dos fármacos , Masculino , Camundongos Endogâmicos BALB C , Estrutura Molecular , Morfolinas/síntese química , Morfolinas/toxicidade , Testes de Sensibilidade Parasitária , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/uso terapêutico , Inibidores de Proteínas Quinases/toxicidade , Pirazóis/síntese química , Pirazóis/toxicidade , Pirimidinas/síntese química , Pirimidinas/toxicidade , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Tripanossomicidas/síntese química , Tripanossomicidas/toxicidadeRESUMO
With the emergence of multidrug-resistant strains of Mycobacterium tuberculosis there is a pressing need for new oral drugs with novel mechanisms of action. Herein, we describe the identification of a novel morpholino-thiophenes (MOT) series following phenotypic screening of the Eli Lilly corporate library against M. tuberculosis strain H37Rv. The design, synthesis, and structure-activity relationships of a range of analogues around the confirmed actives are described. Optimized leads with potent whole cell activity against H37Rv, no cytotoxicity flags, and in vivo efficacy in an acute murine model of infection are described. Mode-of-action studies suggest that the novel scaffold targets QcrB, a subunit of the menaquinol cytochrome c oxidoreductase, part of the bc1-aa3-type cytochrome c oxidase complex that is responsible for driving oxygen-dependent respiration.
Assuntos
Citocromos c/metabolismo , Morfolinas/química , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Oxirredutases/metabolismo , Tiofenos/química , Tiofenos/farmacologia , Animais , Antituberculosos/química , Antituberculosos/farmacocinética , Antituberculosos/farmacologia , Antituberculosos/toxicidade , Chlorocebus aethiops , Camundongos , Relação Estrutura-Atividade , Tiofenos/farmacocinética , Tiofenos/toxicidade , Células VeroRESUMO
A potent, noncytotoxic indazole sulfonamide was identified by high-throughput screening of >100,000 synthetic compounds for activity against Mycobacterium tuberculosis (Mtb). This noncytotoxic compound did not directly inhibit cell wall biogenesis but triggered a slow lysis of Mtb cells as measured by release of intracellular green fluorescent protein (GFP). Isolation of resistant mutants followed by whole-genome sequencing showed an unusual gene amplification of a 40 gene region spanning from Rv3371 to Rv3411c and in one case a potential promoter mutation upstream of guaB2 (Rv3411c) encoding inosine monophosphate dehydrogenase (IMPDH). Subsequent biochemical validation confirmed direct inhibition of IMPDH by an uncompetitive mode of inhibition, and growth inhibition could be rescued by supplementation with guanine, a bypass mechanism for the IMPDH pathway. Beads containing immobilized indazole sulfonamides specifically interacted with IMPDH in cell lysates. X-ray crystallography of the IMPDH-IMP-inhibitor complex revealed that the primary interactions of these compounds with IMPDH were direct pi-pi interactions with the IMP substrate. Advanced lead compounds in this series with acceptable pharmacokinetic properties failed to show efficacy in acute or chronic murine models of tuberculosis (TB). Time-kill experiments in vitro suggest that sustained exposure to drug concentrations above the minimum inhibitory concentration (MIC) for 24 h were required for a cidal effect, levels that have been difficult to achieve in vivo. Direct measurement of guanine levels in resected lung tissue from tuberculosis-infected animals and patients revealed 0.5-2 mM concentrations in caseum and normal lung tissue. The high lesional levels of guanine and the slow lytic, growth-rate-dependent effect of IMPDH inhibition pose challenges to developing drugs against this target for use in treating TB.
Assuntos
Antituberculosos/farmacologia , IMP Desidrogenase/antagonistas & inibidores , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Sulfonamidas/farmacologia , Animais , Desenho de Fármacos , Descoberta de Drogas , Farmacorresistência Bacteriana , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Mutação , Conformação Proteica , Coelhos , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/farmacocinética , Tuberculose/tratamento farmacológicoRESUMO
A novel short synthetic route to annelated benzazepines, using additive enhanced palladium-indium catalytic 3-component cascade methodology is described.
Assuntos
Benzazepinas/síntese química , Catálise , Índio , Métodos , PaládioRESUMO
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
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
N-Myristoyltransferase (NMT) represents a promising drug target for human African trypanosomiasis (HAT), which is caused by the parasitic protozoa Trypanosoma brucei. We report the optimization of a high throughput screening hit (1) to give a lead molecule DDD85646 (63), which has potent activity against the enzyme (IC(50) = 2 nM) and T. brucei (EC(50) = 2 nM) in culture. The compound has good oral pharmacokinetics and cures rodent models of peripheral HAT infection. This compound provides an excellent tool for validation of T. brucei NMT as a drug target for HAT as well as a valuable lead for further optimization.
Assuntos
Aciltransferases/antagonistas & inibidores , Aminopiridinas/síntese química , Sulfonamidas/síntese química , Tripanossomicidas/síntese química , Administração Oral , Aminopiridinas/farmacocinética , Aminopiridinas/farmacologia , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cristalografia por Raios X , Bases de Dados Factuais , Humanos , Modelos Moleculares , Conformação Molecular , Testes de Sensibilidade Parasitária , Relação Estrutura-Atividade , Sulfonamidas/farmacocinética , Sulfonamidas/farmacologia , Tripanossomicidas/farmacocinética , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Tripanossomíase Africana/tratamento farmacológicoRESUMO
Screening of the Sigma-Aldrich Library of Pharmacologically Active Compounds (LOPAC) against cultured Trypanosoma brucei, the causative agent of African sleeping sickness, resulted in the identification of a number of compounds with selective antiproliferative activity over mammalian cells. These included (+)-(1R,2R)-U50488, a weak opioid agonist with an EC(50) value of 59 nM as determined in our T. brucei in vitro assay reported previously. This paper describes the modification of key structural elements of U50488 to investigate structure-activity relationships (SAR) and to optimise the antiproliferative activity and pharmacokinetic properties of this compound.
Assuntos
(trans)-Isômero de 3,4-dicloro-N-metil-N-(2-(1-pirrolidinil)-ciclo-hexil)-benzenoacetamida/química , (trans)-Isômero de 3,4-dicloro-N-metil-N-(2-(1-pirrolidinil)-ciclo-hexil)-benzenoacetamida/farmacologia , Antiprotozoários/química , Antiprotozoários/farmacologia , Antagonistas de Entorpecentes , Trypanosoma brucei brucei/efeitos dos fármacos , (trans)-Isômero de 3,4-dicloro-N-metil-N-(2-(1-pirrolidinil)-ciclo-hexil)-benzenoacetamida/farmacocinética , Antiprotozoários/farmacocinética , Humanos , Modelos Moleculares , Receptores Opioides/metabolismo , Relação Estrutura-Atividade , Tripanossomíase Africana/tratamento farmacológicoRESUMO
New drugs are urgently needed for the treatment of tropical parasitic diseases such as leishmaniasis and human African trypanosomiasis (HAT). This work involved a high-throughput screen of a focussed kinase set of ~3400 compounds to identify potent and parasite-selective inhibitors of an enzymatic Leishmania CRK3-cyclin 6 complex. The aim of this study is to provide chemical validation that Leishmania CRK3-CYC6 is a drug target. Eight hit series were identified, of which four were followed up. The optimisation of these series using classical SAR studies afforded low-nanomolar CRK3 inhibitors with significant selectivity over the closely related human cyclin dependent kinase CDK2.
Assuntos
Proteína Quinase CDC2/antagonistas & inibidores , Leishmania/efeitos dos fármacos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas de Protozoários/antagonistas & inibidores , Sítios de Ligação , Proteína Quinase CDC2/metabolismo , Simulação por Computador , Avaliação Pré-Clínica de Medicamentos , Humanos , Leishmania/enzimologia , Leishmaniose/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Estrutura Terciária de Proteína , Proteínas de Protozoários/metabolismo , Pirazóis/química , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Pirimidinas/química , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Relação Estrutura-Atividade , Ureia/química , Ureia/farmacologia , Ureia/uso terapêuticoRESUMO
There is an urgent need for new drugs for the treatment of tropical parasitic diseases such as human African trypanosomiasis, which is caused by Trypanosoma brucei. The enzyme trypanothione reductase (TryR) is a potential drug target within these organisms. Herein we report the screening of a 62,000 compound library against T. brucei TryR. Further work was undertaken to optimise potency and selectivity of two novel-compound series arising from the enzymatic and whole parasite screens and mammalian cell counterscreens. Both of these series, containing either a quinoline or pyrimidinopyrazine scaffold, yielded low micromolar inhibitors of the enzyme and growth of the parasite. The challenges of inhibiting TryR with druglike molecules is discussed.