Physical methods to determine the binding mode of putative ligands for hepatitis C virus NS3 helicase.
Anal Biochem
; 309(2): 186-95, 2002 Oct 15.
Article
em En
| MEDLINE
| ID: mdl-12413450
ABSTRACT
Several small molecules identified by high-throughput screening (HTS) were evaluated for their ability to bind to a nonstructural protein 3 (NS3) helicase from hepatitis C virus (HCV). Equilibrium dissociation constants (K(d)'s) of the compounds for this helicase were determined using several techniques including an assay measuring the kinetics of isothermal enzyme denaturation at several concentrations of the test molecule. Effects of two nonhydrolyzable ATP analogs on helicase denaturation were measured as controls using the isothermal denaturation (ITD) assay. Two compounds, 4-(2,4-dimethylphenyl)-2,7,8-trimethyl-4,5-quinolinediamine and 2-phenyl-N-(5-piperazin-1-ylpentyl)quinazolin-4-amine, were identified from screening that inhibited the enzyme and had low micromolar dissociation constants for NS3 helicase in the ITD assay. Low micromolar affinity of the quinolinediamine to helicase was also confirmed by nuclear magnetic resonance experiments. Unfortunately, isothermal titration calorimetry (ITC) experiments indicated that a more water-soluble analog bound to the 47/23-mer oligonucleotide helicase substrate with low micromolar affinity as did the substituted quinazolinamine. There was no further interest in these templates as helicase inhibitors due to the nonspecific binding to enzyme and substrate. A combination of physical methods was required to discern the mode of action of compounds identified by HTS and remove undesirable lead templates from further consideration.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Proteínas não Estruturais Virais
/
Hepacivirus
/
Inibidores Enzimáticos
Idioma:
En
Revista:
Anal Biochem
Ano de publicação:
2002
Tipo de documento:
Article
País de afiliação:
Estados Unidos