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
A series of new quinazolinedione derivatives have been readily synthesized and evaluated for their in vitro antiplasmodial growth inhibition activity. Most of the compounds inhibited P. falciparum FcB1 strain in the low to medium micromolar concentration. The 2-ethoxy 8ag', 2-trifluoromethoxy 8ai' and 4-fluoro-2-methoxy 8ak' showed the best inhibitory activity with EC50 values around 5 µM and were non-toxic to the primary human fibroblast cell line AB943. However, these compounds were less potent than the original hit MMV665916, which showed remarkable growth inhibition with EC50 value of 0.4 µM and presented the highest selectivity index (SI > 250). In addition, a novel approach for determining the docking poses of these quinazolinedione derivatives with their potential protein target, the P. falciparum farnesyltransferase PfFT, was investigated.
Assuntos
Antimaláricos/farmacologia , Inibidores Enzimáticos/farmacologia , Farnesiltranstransferase/antagonistas & inibidores , Plasmodium falciparum/efeitos dos fármacos , Antimaláricos/síntese química , Antimaláricos/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Farnesiltranstransferase/metabolismo , Modelos Moleculares , Estrutura Molecular , Testes de Sensibilidade Parasitária , Plasmodium falciparum/enzimologia , Relação Estrutura-AtividadeRESUMO
Aminobenzosuberone-based PfA-M1 inhibitors were explored as novel antimalarial agents against two different Plasmodium falciparum strains. The 4-phenyl derivative 7c exhibited the most encouraging growth inhibitory activity with IC50 values of 6.5-11.2 µM. X-ray crystal structures and early assessment of DMPK/ADME-Tox parameters allowed us to initiate structure-based drug design approach and understand the liabilities (such as potential metabolic and aqueous solubility issues) as well as identify the opportunities for improvement of this aminobenzosuberone series. It also suggested that compound 7c should be regarded as an attractive chemical tool to investigate the different biological roles of this multifunctional PfA-M1 protein.
Assuntos
Aminopeptidases/antagonistas & inibidores , Anisóis/farmacologia , Antimaláricos/farmacologia , Cicloeptanos/farmacologia , Inibidores Enzimáticos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Aminopeptidases/metabolismo , Anisóis/síntese química , Anisóis/química , Antimaláricos/síntese química , Antimaláricos/química , Cicloeptanos/síntese química , Cicloeptanos/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Estrutura Molecular , Testes de Sensibilidade Parasitária , Plasmodium falciparum/enzimologia , Relação Estrutura-AtividadeRESUMO
The synthesis of racemic substituted 7-amino-5,7,8,9-tetrahydrobenzocyclohepten-6-one hydrochlorides was optimized to enhance reproducibility and increase the overall yield. In order to investigate their specificity, series of enzyme inhibition assays were carried out against a diversity of proteases, covering representative members of aspartic, cysteine, metallo and serine endopeptidases and including eight members of the monometallic M1 family of aminopeptidases as well as two members of the bimetallic M17 and M28 aminopeptidase families. This aminobenzosuberone scaffold indeed demonstrated selective inhibition of M1 aminopeptidases to the exclusion of other tested protease families; it was particularly potent against mammalian APN and its bacterial/parasitic orthologues EcPepN and PfAM1.
Assuntos
Aminopeptidases/antagonistas & inibidores , Aminopeptidases/química , Cumarínicos/química , Cumarínicos/farmacologia , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Animais , Ativação Enzimática/efeitos dos fármacos , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Ligação Proteica , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
BACKGROUND: Plasmodium falciparum M1 family aminopeptidase is currently considered as a promising target for anti-malarial chemotherapy. Several series of inhibitors developed by various research groups display IC50/Ki values down to nM range on native PfA-M1 or recombinant forms and block the parasite development in culture at µM to sub-µM concentrations. A handful of these inhibitors has been tested on murine models of malaria and has shown anti plasmodial in vivo activity. However, most of these inhibitors do also target the other neutral malarial aminopeptidase, PfA-M17, often with lower Ki values, which questions the relative involvement and importance of each enzyme in the parasite biology. RESULTS: An amino-benzosuberone derivative from a previously published collection of chemicals targeting specifically the M1-aminopeptidases has been identified; it is highly potent on PfA-M1 (Ki = 50 nM) and devoid of inhibitory activity on PfA-M17 (no inhibition up to 100 µM). This amino-benzosuberone derivative (T5) inhibits, in the µM range, the in vitro growth of two P. falciparum strains, 3D7 and FcB1, respectively chloroquino-sensitive and resistant. Evaluated in vivo, on the murine non-lethal model of malaria Plasmodium chabaudi chabaudi, this amino-benzosuberone derivative was able to reduce the parasite burden by 44 and 40% in a typical 4-day Peters assay at a daily dose of 12 and 24 mg/kg by intraperitoneal route of administration. CONCLUSIONS: The evaluation of a highly selective inhibitor of PfA-M1, over PfA-M17, active on Plasmodium parasites in vitro and in vivo, highlights the relevance of PfA-M1 in the biological development of the parasite as well as in the list of promising anti-malarial targets to be considered in combination with current or future anti-malarial drugs.
Assuntos
Aminopeptidases/antagonistas & inibidores , Plasmodium falciparum/crescimento & desenvolvimento , Inibidores de Proteases/farmacologia , Proteínas de Protozoários/antagonistas & inibidores , Anisóis/farmacologia , Antimaláricos/farmacologia , Cicloeptanos/farmacologia , Plasmodium falciparum/efeitos dos fármacosRESUMO
Cell surface aminopeptidase N (APN) is a membrane-bound ectoenzyme that hydrolyzes proteins and peptides and regulates numerous cell functions. APN participates in tumor cell expansion and motility, and is a target for cancer therapies. Small drugs that bind to the APN active site inhibit catalysis and suppress tumor growth. APN is also a major cell entry receptor for coronavirus, which binds to a region distant from the active site. Three crystal structures that we determined of human and pig APN ectodomains defined the dynamic conformation of the protein. These structures offered snapshots of closed, intermediate and open APN, which represent distinct functional states. Coronavirus envelope proteins specifically recognized the open APN form, prevented ectodomain progression to the closed form and substrate hydrolysis. In addition, drugs that bind the active site inhibited both coronavirus binding to cell surface APN and infection; the drugs probably hindered APN transition to the virus-specific open form. We conclude that allosteric inhibition of APN functions occurs by ligand suppression of ectodomain motions necessary for catalysis and virus cell entry, as validated by locking APN with disulfides. Blocking APN dynamics can thus be a valuable approach to development of drugs that target this ectoenzyme.
Assuntos
Antígenos CD13/antagonistas & inibidores , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Coronavirus/fisiologia , Neoplasias/patologia , Regulação Alostérica/efeitos dos fármacos , Animais , Antineoplásicos/farmacologia , Biocatálise/efeitos dos fármacos , Antígenos CD13/química , Antígenos CD13/metabolismo , Células CHO , Domínio Catalítico , Proliferação de Células/efeitos dos fármacos , Coronavirus/efeitos dos fármacos , Cricetinae , Cricetulus , Dissulfetos/metabolismo , Humanos , Modelos Moleculares , Domínios Proteicos , Sus scrofaRESUMO
Aminopeptidases are ubiquitous hydrolases that cleave the N-terminal residues of proteins and oligopeptides. They are broadly distributed throughout all kingdoms of life and have been implicated in a wide variety of physiological processes, including viral infection, parasite metabolism, protein processing, regulation of peptide hormones, and cancer cell proliferation. Members of the M1 family, also termed gluzincins, are defined by two highly conserved motifs in the catalytic domain: a zinc-binding motif, HEXXH-(X18)-E; and an exopeptidase motif, GXMEN. We report the high-resolution X-ray structures of E. coli aminopeptidase N (PepN) in complex with three aminobenzosuberone scaffolds that display various Ki values (50, 0.33, and 0.034 µM) and provide a compelling view of the outstanding selectivity of these chemical entities for the M1 aminopeptidases. This series of inhibitors interacts as transition state mimics with highly conserved residues of the catalytic machinery and substrate recognition sites. Structural comparisons and model-building studies allowed a deep interpretation of the SAR observed for bacterial, as well as mammalian enzymes. Proteins 2017; 85:1413-1421. © 2017 Wiley Periodicals, Inc.
Assuntos
Aminopeptidases/antagonistas & inibidores , Anisóis/química , Proteínas de Bactérias/antagonistas & inibidores , Cicloeptanos/química , Escherichia coli/química , Inibidores de Proteases/química , Motivos de Aminoácidos , Aminopeptidases/química , Aminopeptidases/genética , Aminopeptidases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Epóxido Hidrolases/química , Epóxido Hidrolases/genética , Epóxido Hidrolases/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Expressão Gênica , Humanos , Cinética , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia Estrutural de Proteína , Especificidade por Substrato , TermodinâmicaRESUMO
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
In order to probe the S1 and S1' mammalian aminopeptidase N subsites, racemic 1- or 4-substituted 7-aminobenzocyclohepten-6-one derivatives were synthesized and evaluated for their ability to inhibit mammalian aminopeptidase N. We focused on improving the physicochemical and ADME properties of this series by targeting lipophilicity and LELP score. Some 4-heteroaryl substituted analogues displayed reduced lipophilicity and enhanced inhibition potency with Ki values in the nanomolar range.
Assuntos
Aminobenzoatos/síntese química , Benzocicloeptenos/síntese química , Antígenos CD13/antagonistas & inibidores , Inibidores de Proteases/síntese química , Aminobenzoatos/química , Animais , Benzocicloeptenos/química , Antígenos CD13/química , Antígenos CD13/isolamento & purificação , Rim/química , Rim/enzimologia , Simulação de Acoplamento Molecular , Estrutura Molecular , Inibidores de Proteases/química , Estereoisomerismo , Relação Estrutura-Atividade , Suínos , TermodinâmicaRESUMO
Racemic trisubstituted benzocycloheptanes were synthesized and evaluated for their ability to inhibit metalloaminopeptidase activities. A highly selective nanomolar inhibitor of a prototypical 'two zinc' aminopeptidase from the M28 family was observed with these tridentate species, while bidentate analogs proved to be highly selective for the 'one zinc' M1 family of enzymes. The selectivity profile of these new, low molecular weight structures may guide the design of specific, non-peptidic inhibitors of binuclear aminopeptidases.
Assuntos
Aminopeptidases/antagonistas & inibidores , Benzocicloeptenos/química , Inibidores de Proteases/síntese química , Aeromonas/enzimologia , Aminopeptidases/metabolismo , Benzocicloeptenos/síntese química , Benzocicloeptenos/metabolismo , Sítios de Ligação , Domínio Catalítico , Escherichia coli/enzimologia , Simulação de Acoplamento Molecular , Inibidores de Proteases/química , Inibidores de Proteases/metabolismo , Ligação Proteica , Relação Estrutura-Atividade , Zinco/químicaRESUMO
Racemic 5-substituted 7-aminobenzocyclohepten-6-one were synthesized and evaluated for their ability to inhibit metalloaminopeptidase activities. Unexpectedly, 5-thio substituted compounds showed enhanced inhibition potency with K(i) values in the nanomolar range against the 'one zinc' aminopeptidases from the M1 family, while most of them were rather poor inhibitors of the 'two zincs' enzymes from the M17 family. This interesting selectivity profile may guide the design of new, specific inhibitors of target mammalian aminopeptidases with one active site zinc.
Assuntos
Aminopeptidases/antagonistas & inibidores , Anisóis/farmacologia , Cicloeptanos/farmacologia , Inibidores Enzimáticos/farmacologia , Aminopeptidases/metabolismo , Animais , Anisóis/síntese química , Anisóis/química , Cicloeptanos/síntese química , Cicloeptanos/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Rim/enzimologia , Estrutura Molecular , Estereoisomerismo , Relação Estrutura-Atividade , SuínosRESUMO
This paper describes the design and synthesis of compounds belonging to a novel class of highly selective mammalian CD13 inhibitors. Racemic homologues of 3-amino-2-tetralone 1 were synthesised and evaluated for their ability to selectively inhibit the membrane-bound, zinc-dependent aminopeptidase-N/CD13 (EC 3.4.11.2). Some of these novel non-peptidic compounds are potent, competitive inhibitors of the mammalian enzyme, with K(i) values in the low micromolar range in spite of their minimal size (MW <200 Da). Moreover, they show an interesting selectivity profile against representative members of the aminopeptidase family, that is leucine aminopeptidase (EC 3.4.11.1), Aeromonas proteolytica aminopeptidase (EC 3.4.11.10) and the aminopeptidase activity of leukotriene A4 hydrolase (EC 3.3.2.6). The amino-benzosuberone derivative 4 is the most promising compound in terms of potency, stability and selectivity. A hypothetical binding mode of 4 to the catalytic zinc and several conserved active site residues is proposed, based on the observed structure-activity relationships, structural insights from aminopeptidase-N homologues of known three-dimensional structure.
Assuntos
Anisóis/química , Antígenos CD13/antagonistas & inibidores , Cicloeptanos/química , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Anisóis/farmacologia , Cicloeptanos/farmacologia , Dimerização , Humanos , Modelos Moleculares , Estrutura Molecular , Solventes/química , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
The silver-catalysed Doyle-Kirmse reaction of propargyl and allyl sulfides with diazo compounds is disclosed. The carbon-carbon bond forming process proceeds with a range of substituents and functionality under mild conditions.
Assuntos
Alcinos/química , Compostos Alílicos/química , Compostos Organometálicos/química , Prata/química , Sulfetos/química , Sulfetos/síntese química , Compostos Azo/química , Catálise , Estrutura Molecular , EstereoisomerismoRESUMO
The in situ-generation of sulfur ylides by the gold-catalysed rearrangement of propargylic carboxylates in the presence of sulfides has resulted in highly efficient and novel transformations.
RESUMO
Racemic derivatives of 3-amino-2-tetralone were synthesised and evaluated for their ability to inhibit metallo-aminopeptidase activities. New compounds substituted in position 2 by methyl ketone, substituted oximes or hydroxamic acids as well as heterocyclic derivatives were evaluated against representative members of zinc-dependent aminopeptidases: leucine aminopeptidase (E.C. 3.4.11.1), aminopeptidase-N (E.C. 3.4.11.2), Aeromonas proteolytica aminopeptidase (E.C. 3.4.11.10), and the aminopeptidase activity of leukotriene A(4) hydrolase (E.C. 3.3.2.6). Several compounds showed K(i) values in the low micromolar range against the 'one-zinc' aminopeptidases, while most of them were rather poor inhibitors of the 'two-zinc' enzymes. This interesting selectivity profile may guide the design of new, specific inhibitors of target mammalian aminopeptidases with one active site zinc.