RESUMO
Bacterial peptide deformylase (PDF) belongs to a subfamily of metalloproteases catalyzing the removal of the N-terminal formyl group from newly synthesized proteins. We report the synthesis and biological activity of highly potent inhibitors of Mycobacterium tuberculosis (Mtb) PDF enzyme as well as the first X-ray crystal structure of Mtb PDF. Structure-activity relationship and crystallographic data clarified the structural requirements for high enzyme potency and cell based potency. Activities against single and multi-drug-resistant Mtb strains are also reported.
Assuntos
Amidoidrolases/antagonistas & inibidores , Amidoidrolases/química , Antituberculosos/uso terapêutico , Mycobacterium tuberculosis/efeitos dos fármacos , Tuberculose/tratamento farmacológico , Antituberculosos/química , Química Farmacêutica/métodos , Cristalografia por Raios X/métodos , Desenho de Fármacos , Resistência a Múltiplos Medicamentos , Fluoroquinolonas/farmacologia , Gatifloxacina , Humanos , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana , Modelos Químicos , Conformação Molecular , Mycobacterium bovis/metabolismo , Mycobacterium tuberculosis/metabolismoRESUMO
A series of inhibitors related to the benzoyl-norleucine-lysine-arginine-arginine (Bz-nKRR) tetrapeptide aldehyde was synthesized. When evaluated against the West Nile virus (WNV) NS3 protease, the measured IC(50) ranges from approximately 1 to 200 microM. Concurrently, a modeling study using the recently published crystal structure of the West Nile NS3/NS2B protease complex (pdb code 2FP7) was conducted. We found that the crystal structure is relevant in explaining the observed SAR for this series of tetrapeptides, with the S1 and S2 pockets being the key peptide recognition sites. In general, a residue capable of both pi-stacking and hydrogen bonding is favored in the S1 pocket, while a positively charged residue is preferred in the S2 pocket. This study not only confirms the importance of the NS2B domain in substrate-based inhibitor binding of WNV, it also suggests that the crystal structure would provide useful guidance in the drug discovery process of related Flavivirus proteases, given the high degree of homology.
Assuntos
Aldeídos/antagonistas & inibidores , Oligopeptídeos/farmacologia , Inibidores de Proteases/farmacologia , Serina Endopeptidases/efeitos dos fármacos , Vírus do Nilo Ocidental/enzimologia , Modelos Moleculares , Conformação Molecular , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Peptide deformylase (PDF) catalyzes the hydrolytic removal of the N-terminal formyl group from nascent proteins. This is an essential step in bacterial protein synthesis, making PDF an attractive target for antibacterial drug development. Essentiality of the def gene, encoding PDF from Mycobacterium tuberculosis, was demonstrated through genetic knockout experiments with Mycobacterium bovis BCG. PDF from M. tuberculosis strain H37Rv was cloned, expressed, and purified as an N-terminal histidine-tagged recombinant protein in Escherichia coli. A novel class of PDF inhibitors (PDF-I), the N-alkyl urea hydroxamic acids, were synthesized and evaluated for their activities against the M. tuberculosis PDF enzyme as well as their antimycobacterial effects. Several compounds from the new class had 50% inhibitory concentration (IC50) values of <100 nM. Some of the PDF-I displayed antibacterial activity against M. tuberculosis, including MDR strains with MIC90 values of <1 microM. Pharmacokinetic studies of potential leads showed that the compounds were orally bioavailable. Spontaneous resistance towards these inhibitors arose at a frequency of < or =5 x 10(-7) in M. bovis BCG. DNA sequence analysis of several spontaneous PDF-I-resistant mutants revealed that half of the mutants had acquired point mutations in their formyl methyltransferase gene (fmt), which formylated Met-tRNA. The results from this study validate M. tuberculosis PDF as a drug target and suggest that this class of compounds have the potential to be developed as novel antimycobacterial agents.
Assuntos
Amidoidrolases/antagonistas & inibidores , Antibacterianos , Mycobacterium/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Administração Oral , Animais , Antibacterianos/farmacocinética , Southern Blotting , Soluções Tampão , Meios de Cultura , Análise Mutacional de DNA , DNA Bacteriano/genética , Bases de Dados Genéticas , Farmacorresistência Bacteriana , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Feminino , Injeções Intravenosas , Camundongos , Testes de Sensibilidade Microbiana , Mycobacterium/genética , Mycobacterium bovis/efeitos dos fármacos , Mycobacterium bovis/genética , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Plasmídeos/genética , Inibidores de Proteases/farmacocinéticaRESUMO
Substrate-based tetrapeptide inhibitors with various warheads were designed, synthesized, and evaluated against the Dengue virus NS3 protease. Effective inhibition was achieved by peptide inhibitors with electrophilic warheads such as aldehyde, trifluoromethyl ketone, and boronic acid. A boronic acid has the highest affinity, exhibiting a K(i) of 43 nM.
Assuntos
Química Farmacêutica/métodos , Vírus da Dengue/enzimologia , Desenho de Fármacos , Peptídeos/farmacologia , Inibidores de Proteases/química , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/química , Ácidos Borônicos/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Cetonas/química , Cinética , Modelos Químicos , Peptídeos/química , Inibidores de Proteases/farmacologia , RNA Helicases/antagonistas & inibidores , RNA Helicases/química , Serina Endopeptidases/química , Especificidade por Substrato , Fatores de TempoRESUMO
With the aim of discovering potent and selective dengue NS3 protease inhibitors, we systematically synthesized and evaluated a series of tetrapeptide aldehydes based on lead aldehyde 1 (Bz-Nle-Lys-Arg-Arg-H, K(i)=5.8 microM). In general, we observe that interactions of P(2) side chain are more important than P(1) followed by P(3) and P(4). Tripeptide and dipeptide aldehyde inhibitors also show low micromolar activity. Additionally, an effective non-basic, uncharged replacement of P(1) Arg is identified.