RESUMO
For several decades, molecular recognition has been considered one of the most fundamental processes in biochemistry. For enzymes, substrate binding is often coupled to conformational changes that alter the local environment of the active site to align the reactive groups for efficient catalysis and to reach the transition state. Adaptive substrate recognition is a well-known concept; however, it has been poorly characterized at a structural level because of its dynamic nature. Here, we provide a detailed mechanism for an induced-fit process at atomic resolution. We take advantage of a slow, tight binding inhibitor-enzyme system, actinonin-peptide deformylase. Crystal structures of the initial open state and final closed state were solved, as well as those of several intermediate mimics captured during the process. Ligand-induced reshaping of a hydrophobic pocket drives closure of the active site, which is finally "zipped up" by additional binding interactions. Together with biochemical analyses, these data allow a coherent reconstruction of the sequence of events leading from the encounter complex to the key-lock binding state of the enzyme. A "movie" that reconstructs this entire process can be further extrapolated to catalysis.
Assuntos
Amidoidrolases/química , Proteínas de Arabidopsis/química , Arabidopsis/enzimologia , Inibidores Enzimáticos/química , Amidoidrolases/antagonistas & inibidores , Amidoidrolases/genética , Motivos de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/antagonistas & inibidores , Proteínas de Arabidopsis/genética , Domínio Catalítico , Cristalografia por Raios X , Ligação de Hidrogênio , Ácidos Hidroxâmicos/química , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Ligação Proteica/genética , TermodinâmicaRESUMO
New classes of antibiotics are urgently needed to counter increasing levels of pathogen resistance. Peptide deformylase (PDF) was originally selected as a specific bacterial target, but a human homologue, the inhibition of which causes cell death, was recently discovered. We developed a dual-screening strategy for selecting highly effective compounds with low inhibition effect against human PDF. We selected a new scaffold in vitro that discriminated between human and bacterial PDFs. Analyses of structure-activity relationships identified potent antibiotics such as 2-(5-bromo-1H-indol-3-yl)-N-hydroxyacetamide (6b) with the same mode of action in vivo as previously identified PDF inhibitors but without the apoptotic effects of these inhibitors in human cells.
Assuntos
Amidoidrolases/antagonistas & inibidores , Antibacterianos/síntese química , Ácidos Hidroxâmicos/síntese química , Indóis/síntese química , Amidoidrolases/química , Antibacterianos/química , Antibacterianos/farmacologia , Apoptose/efeitos dos fármacos , Bacillus subtilis/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Geobacillus stearothermophilus/enzimologia , Humanos , Ácidos Hidroxâmicos/química , Ácidos Hidroxâmicos/farmacologia , Indóis/química , Indóis/farmacologia , Células KB , Espectroscopia de Ressonância Magnética , Testes de Sensibilidade Microbiana , Modelos Moleculares , Relação Estrutura-AtividadeRESUMO
Peptide deformylase inhibitors (PDFIs) appear to be one of the most exciting classes of antibacterial agents discovered to date. Rapid progress in the development of PDFIs has been possible because peptide deformylase is a metalloprotease, and this class of enzymes shows a high degree of structure-function conservation, and because the most potent PDFIs are hydroxamate derivatives, a well known category of pharmacophores. The current challenge in structure-activity relationship analysis is obtaining molecules with potent in vivo antibacterial activity against a range of drug-resistant pathogens. The PDFIs currently in clinical trials target community-based bacterial infections, with a potential major pharmaceutical market.