RESUMO
DNA gyrase is an essential enzyme in bacteria, and its inhibition results in the disruption of DNA synthesis and, subsequently, cell death. The pyrrolamides are a novel class of antibacterial agents targeting DNA gyrase. These compounds were identified by a fragment-based lead generation (FBLG) approach using nuclear magnetic resonance (NMR) screening to identify low-molecular-weight compounds that bind to the ATP pocket of DNA gyrase. A pyrrole hit with a binding constant of 1 mM formed the basis of the design and synthesis of a focused library of compounds that resulted in the rapid identification of a lead compound that inhibited DNA gyrase with a 50% inhibitory concentration (IC(50)) of 3 µM. The potency of the lead compound was further optimized by utilizing iterative X-ray crystallography to yield DNA gyrase inhibitors that also displayed antibacterial activity. Spontaneous mutants were isolated in Staphylococcus aureus by plating on agar plates containing pyrrolamide 4 at the MIC. The resistant variants displayed 4- to 8-fold-increased MIC values relative to the parent strain. DNA sequencing revealed two independent point mutations in the pyrrolamide binding region of the gyrB genes from these variants, supporting the hypothesis that the mode of action of these compounds was inhibition of DNA gyrase. Efficacy of a representative pyrrolamide was demonstrated against Streptococcus pneumoniae in a mouse lung infection model. These data demonstrate that the pyrrolamides are a novel class of DNA gyrase inhibitors with the potential to deliver future antibacterial agents targeting multiple clinical indications.
Assuntos
Amidas/farmacologia , Antibacterianos/farmacologia , Pirróis/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Streptococcus pneumoniae/efeitos dos fármacos , Inibidores da Topoisomerase II , Amidas/química , Animais , Antibacterianos/química , Sítios de Ligação , Cristalografia por Raios X , DNA Girase/química , DNA Girase/metabolismo , Farmacorresistência Bacteriana , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Concentração Inibidora 50 , Espectroscopia de Ressonância Magnética , Camundongos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Mutação , Ligação Proteica , Pirróis/química , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética , Staphylococcus aureus/crescimento & desenvolvimento , Streptococcus pneumoniae/crescimento & desenvolvimentoRESUMO
Synthetic lethal screening is a chemical biology approach to identify small molecules that selectively kill oncogene-expressing cell lines with the goal of identifying pathways that provide specific targets against cancer cells. We performed a high-throughput screen of 303,282 compounds from the National Institutes of Health-Molecular Libraries Small Molecule Repository (NIH-MLSMR) against immortalized BJ fibroblasts expressing HRAS(G12V) followed by a counterscreen of lethal compounds in a series of isogenic cells lacking the HRAS(G12V) oncogene. This effort led to the identification of two novel molecular probes (PubChem CID 3689413, ML162 and CID 49766530, ML210) with nanomolar potencies and 4-23-fold selectivities, which can potentially be used for identifying oncogene-specific pathways and targets in cancer cells.
Assuntos
Apoptose/efeitos dos fármacos , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Fibroblastos , Humanos , Concentração Inibidora 50 , Estrutura Molecular , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Ratos , Relação Estrutura-Atividade , Tiofenos/química , Tiofenos/farmacologiaRESUMO
The effectiveness of the potent antifungal drug fluconazole is being compromised by the rise of drug-resistant fungal pathogens. While inhibition of Hsp90 or calcineurin can reverse drug resistance in Candida, such inhibitors also impair the homologous human host protein and fungal-selective chemosensitizers remain rare. The MLPCN library was screened to identify compounds that selectively reverse fluconazole resistance in a Candida albicans clinical isolate, while having no antifungal activity when administered as a single agent. A piperazinyl quinoline was identified as a new small-molecule probe (ML189) satisfying these criteria.
Assuntos
Antifúngicos/farmacologia , Inibidores de Calcineurina , Candida albicans/efeitos dos fármacos , Fluconazol/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Quinolinas/farmacologia , Antifúngicos/química , Calcineurina/metabolismo , Candida albicans/crescimento & desenvolvimento , Candida albicans/isolamento & purificação , Relação Dose-Resposta a Droga , Fluconazol/química , Proteínas de Choque Térmico HSP90/metabolismo , Testes de Sensibilidade Microbiana , Estrutura Molecular , Quinolinas/síntese química , Quinolinas/química , Bibliotecas de Moléculas Pequenas , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
We report the outcome of a high-throughput small-molecule screen to identify novel, nontoxic, inhibitors of Trypansoma cruzi, as potential starting points for therapeutics to treat for both the acute and chronic stages of Chagas disease. Two compounds were identified that displayed nanomolar inhibition of T. cruzi and an absence of activity against host cells at the highest tested dose. These compounds have been registered with NIH Molecular Libraries Program (probes ML157 and ML158).
Assuntos
Bibliotecas de Moléculas Pequenas , Tripanossomicidas/síntese química , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Concentração Inibidora 50 , Estrutura Molecular , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade , Tripanossomicidas/químicaRESUMO
An SAR study of an HTS screening hit generated a series of pyridodiazepine amines as potent inhibitors of Helicobacter pylori glutamate racemase (MurI) showing highly selective anti-H. pylori activity, marked improved solubility, and reduced plasma protein binding. X-ray co-crystal E-I structures were obtained. These uncompetitive inhibitors bind at the MurI dimer interface.
Assuntos
Aminas/química , Isomerases de Aminoácido/química , Química Farmacêutica/métodos , Infecções por Helicobacter/tratamento farmacológico , Helicobacter pylori/enzimologia , Animais , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Ligação Competitiva , Dimerização , Desenho de Fármacos , Humanos , Concentração Inibidora 50 , Camundongos , Conformação Molecular , Relação Estrutura-AtividadeRESUMO
A high-throughput screen of the NIH-MLSMR compound collection, along with a series of secondary assays to identify potential targets of hit compounds, previously identified a 1,3-diaminobenzene scaffold that targets protease-activated receptor 1 (PAR1). We now report additional structure-activity relationship (SAR) studies that delineate the requirements for activity at PAR1 and identify plasma-stable analogues with nanomolar inhibition of PAR1-mediated platelet activation. Compound 4 was declared as a probe (ML161) with the NIH Molecular Libraries Program. This compound inhibited platelet aggregation induced by a PAR1 peptide agonist or by thrombin but not by several other platelet agonists. Initial studies suggest that ML161 is an allosteric inhibitor of PAR1. These findings may be important for the discovery of antithrombotics with an improved safety profile.
RESUMO
Macrocyclic Hedgehog (Hh) pathway inhibitors have been discovered with improved potency and maximal inhibition relative to the previously reported macrocycle robotnikinin. Analogues were prepared using a modular and efficient build-couple-pair (BCP) approach, with a ring-closing metathesis step to form the macrocyclic ring. Varying the position of the macrocycle nitrogen and oxygen atoms provided inhibitors with improved activity in cellular assays; the most potent analogue was 29 (BRD-6851), with an IC(50) of 0.4 µM against C3H10T1/2 cells undergoing Hh-induced activation, as measured by Gli1 transcription and alkaline phosphatase induction. Studies with Patched knockout (Ptch(-/-)) cells and competition studies with the Smoothened (Smo) agonists SAG and purmorphamine demonstrate that in contrast to robotnikinin, select analogues are Smo antagonists.