RESUMO
Non-nucleoside inhibitors of HCV NS5b RNA polymerase were discovered by a fragment-based lead discovery approach, beginning with crystallographic fragment screening. The NS5b binding affinity and biochemical activity of fragment hits and inhibitors was determined by surface plasmon resonance (Biacore) and an enzyme inhibition assay, respectively. Crystallographic fragment screening hits with approximately 1-10mM binding affinity (K(D)) were iteratively optimized to give leads with approximately 200nM biochemical activity and low microM cellular activity in a Replicon assay.
Assuntos
Antivirais/uso terapêutico , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Hepacivirus/química , Hepatite C/enzimologia , Proteínas não Estruturais Virais/farmacologia , Antivirais/síntese química , Sítios de Ligação , Cristalografia por Raios X , Ativação Enzimática , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície , Proteínas não Estruturais Virais/química , Replicação Viral/fisiologiaRESUMO
Lipid A modification with 4-amino-4-deoxy-L-arabinose confers on certain pathogenic bacteria, such as Salmonella, resistance to cationic antimicrobial peptides, including those derived from the innate immune system. ArnB catalysis of amino group transfer from glutamic acid to the 4"-position of a UDP-linked ketopyranose molecule to form UDP-4-amino-4-deoxy-L-arabinose represents a key step in the lipid A modification pathway. Structural and functional studies of the ArnB aminotransferase were undertaken by combining X-ray crystallography with biochemical analyses. High-resolution crystal structures were solved for two native forms and one covalently inhibited form of S. typhimurium ArnB. These structures permitted identification of key residues involved in substrate binding and catalysis, including a rarely observed nonprolyl cis peptide bond in the active site.