RESUMO
The RAS-MAPK pathway controls many cellular programs, including cell proliferation, differentiation, and apoptosis. In colorectal cancers, recurrent mutations in this pathway often lead to increased cell signaling that may contribute to the development of neoplasms, thereby making this pathway attractive for therapeutic intervention. To this end, we developed a 26-member gene signature of RAS-MAPK pathway activity utilizing the Affymetrix QuantiGene Plex 2.0 reagent system and performed both primary and confirmatory gene expression-based high-throughput screens (GE-HTSs) using KRAS mutant colon cancer cells (SW837) and leveraging a highly annotated chemical library. The screen achieved a hit rate of 1.4% and was able to enrich for hit compounds that target RAS-MAPK pathway members such as MEK and EGFR. Sensitivity and selectivity performance measurements were 0.84 and 1.00, respectively, indicating high true-positive and true-negative rates. Active compounds from the primary screen were confirmed in a dose-response GE-HTS assay, a GE-HTS assay using 14 additional cancer cell lines, and an in vitro colony formation assay. Altogether, our data suggest that this GE-HTS assay will be useful for larger unbiased chemical screens to identify novel compounds and mechanisms that may modulate the RAS-MAPK pathway.
Assuntos
Perfilação da Expressão Gênica/métodos , Ensaios de Triagem em Larga Escala/métodos , Neoplasias/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/isolamento & purificação , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Mutação , Neoplasias/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
ß-Lactamase inhibitors with a bicyclic urea core and a variety of heterocyclic side chains were prepared and evaluated as potential partners for combination with imipenem to overcome class A and C ß-lactamase mediated antibiotic resistance. The piperidine analog 3 (MK-7655) inhibited both class A and C ß-lactamases in vitro. It effectively restored imipenem's activity against imipenem-resistant Pseudomonas and Klebsiella strains at clinically achievable concentrations. A combination of MK-7655 and Primaxin® is currently in phase II clinical trials for the treatment of Gram-negative bacterial infections.
Assuntos
Compostos Azabicíclicos/química , Compostos Azabicíclicos/farmacologia , Cilastatina/química , Descoberta de Drogas , Inibidores Enzimáticos/química , Imipenem/química , Inibidores de beta-Lactamases , Cilastatina/farmacologia , Combinação Imipenem e Cilastatina , Cristalografia por Raios X , Combinação de Medicamentos , Farmacorresistência Bacteriana/efeitos dos fármacos , Imipenem/farmacologia , Concentração Inibidora 50 , Klebsiella/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Modelos Biológicos , Pseudomonas/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
The bridged monobactam ß-lactamase inhibitor MK-8712 (1) effectively inhibits class C ß-lactamases. Side chain N-alkylated and ring-opened analogs of 1 were prepared and evaluated for combination with imipenem to overcome class C ß-lactamase mediated resistance. Although some analogs were more potent inhibitors of AmpC, none exhibited better synergy with imipenem than 1.
Assuntos
Antibacterianos/química , Inibidores Enzimáticos/química , Monobactamas/síntese química , Inibidores de beta-Lactamases , Antibacterianos/síntese química , Antibacterianos/farmacologia , Sítios de Ligação , Simulação por Computador , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Imipenem/farmacologia , Testes de Sensibilidade Microbiana , Monobactamas/farmacologia , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , beta-Lactamases/metabolismoRESUMO
Bridged monobactam beta-lactamase inhibitors were prepared and evaluated as potential partners for combination with imipenem to overcome class C beta-lactamase mediated resistance. The (S)-azepine analog 2 was found to be effective in both in vitro and in vivo assays and was selected for preclinical development.