title: Screening and rank ordering of reversible mechanism-based inhibitors of hepatitis C virus NS3 protease using electrospray ionization mass spectrometry.
abstract: An affinity-selection study using size exclusion chromatography (SEC) combined with off-line electrospray ionization mass spectrometry (ESI-MS) was performed on libraries of peptidic α-ketoamide inhibitors directed against the hepatitis C virus (HCV) NS3 protease. A limiting amount of HCV NS3 protease (25 µM) was incubated with equimolar amounts (100 µM) of 49 reversible mechanism-based ketoamide inhibitors, previously grouped into seven sets to ensure clearly distinguishable mass differences of the enzyme-inhibitor complexes (>10 Da). The unbound compounds were separated rapidly from the protease and the protease-inhibitor complexes by SEC spin columns. The eluate of the SEC was immediately analyzed by direct-infusion ESI-MS. An enzyme-inhibitor complex, with a molecular mass corresponding to the NS3 protease binding to the preferred inhibitor, SCH212986, was the only molecular species detected. By increasing the molar ratio of HCV NS3 protease to inhibitors to 1:2 while keeping the inhibitors' concentration constant, the complex of the second most tightly bound inhibitor, SCH215426, was also identified. Although the potencies of these inhibitors were virtually un-measurable by kinetic assays, a rank order of CVS4441 > SCH212986 > SCH215426 was deduced for their inhibition potencies by direct competition experiment with CVS4441 (K(i)*>80 µM). As discussed in the article, through judicious application of this strategy, even large libraries of fairly weak, reversible and slow-binding inhibitors could be rapidly screened and rank ordered to provide critical initial structure-activity insights.
authors.affiliation: Molecular Biomarkers, Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA. yan-hui.liu@merck.com
authors.forename: Yan-Hui
authors.initials: YH
authors.lastname: Liu
authors.forename: Lata
authors.initials: L
authors.lastname: Ramanathan
authors.forename: Bruce
authors.initials: B
authors.lastname: Malcolm
authors.forename: George
authors.initials: G
authors.lastname: Njoroge
authors.forename: Tin-Yau
authors.initials: TY
authors.lastname: Chan
authors.forename: Birendra N
authors.initials: BN
authors.lastname: Pramanik
mesh_terms: D000577:Amides / Q000737:chemistry / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism; D002850:Chromatography, Gel; D055808:Drug Discovery / Q000379:methods; D004795:Enzyme Stability; D020035:Holoenzymes / Q000737:chemistry / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D011480:Protease Inhibitors / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000378:metabolism; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D000577:Amides; D002352:Carrier Proteins; D020035:Holoenzymes; D047908:Intracellular Signaling Peptides and Proteins; D011480:Protease Inhibitors; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C455041:NS4A cofactor peptide, Hepatitis C virus