Physical methods to determine the binding mode of putative ligands for hepatitis C virus NS3 helicase.
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.
Sarver
Ronald W
RW
Structural, Analytical and Medicinal Chemistry, 301 Henrietta Street, Kalamazoo, MI 49001, USA.
Rogers
Joseph M
JM
Stockman
Brian J
BJ
Epps
Dennis E
DE
DeZwaan
Jack
J
Harris
Melissa S
MS
Baldwin
Eric T
ET
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D002151:Calorimetry / Q000379:methods; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D004247:DNA / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000032:analysis* / Q000378:metabolism / Q000494:pharmacology; D016174:Hepacivirus / Q000201:enzymology*; D007700:Kinetics; D008024:Ligands; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D000255:Adenosine Triphosphate; D004247:DNA; D004791:Enzyme Inhibitors; D008024:Ligands; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases