title: Longer wavelength fluorescence resonance energy transfer depsipeptide substrates for hepatitis C virus NS3 protease.
abstract: Maturation of the hepatitis C virus (HCV) polyprotein occurs by a series of proteolytic processes catalyzed by host cell proteases and the virally encoded proteases NS2 and NS3. Although several peptidomimetic inhibitors of NS3 protease have been published, only a few small molecule inhibitors have been reported. In an effort to improve screening efficiency by minimizing the spectral interference of various test compounds, a substrate that contains the longer wavelength fluorescence resonance energy transfer (FRET) pair, TAMRA/QSY-7, was devised. For the optimized substrate T-Abu-Q, with sequence Ac-Asp-Glu-Lys(TAMRA)-Glu-Glu-Abu-Psi(COO)Ala-Ser-Lys(QSY-7)-amide, the kinetic parameters with HCV NS3 protease are K(m)=30 microM, k(cat)=0.6s(-1), and k(cat)/K(m)=20,100s(-1)M(-1). We show that this substrate is suitable for inhibitor analysis and mechanistic studies so long as the substrate concentration is low enough (0.5 microM) to avoid complications from high inner filter effects. The substrate is especially useful with ultra-high-density screening formats, such as microarrayed compound screening technology, because there is less spectral interference from the compounds being tested than with more traditional (EDANS/DABCYL) FRET protease substrates. The merits of the new substrate, as well as potential applications of this FRET pair to other protease substrates, are discussed.
authors.affiliation: Infectious Diseases Research, Abbott Laboratories, Abbott Park, IL 60064, USA.
authors.forename: Alex K
authors.initials: AK
authors.lastname: Konstantinidis
authors.forename: Paul L
authors.initials: PL
authors.lastname: Richardson
authors.forename: Kevin A
authors.initials: KA
authors.lastname: Kurtz
authors.forename: Rakesh
authors.initials: R
authors.lastname: Tripathi
authors.forename: Chih-Ming
authors.initials: CM
authors.lastname: Chen
authors.forename: Peggy
authors.initials: P
authors.lastname: Huang
authors.forename: John
authors.initials: J
authors.lastname: Randolph
authors.forename: Danli
authors.initials: D
authors.lastname: Towne
authors.forename: Jennifer
authors.initials: J
authors.lastname: Donnelly
authors.forename: Usha
authors.initials: U
authors.lastname: Warrior
authors.forename: Tim
authors.initials: T
authors.lastname: Middleton
authors.forename: Warren M
authors.initials: WM
authors.lastname: Kati
mesh_terms: D004305:Dose-Response Relationship, Drug; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D006868:Hydrolysis; D007700:Kinetics; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry*; D011480:Protease Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D012235:Rhodamines / Q000737:chemistry; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D005456:Fluorescent Dyes; D009842:Oligopeptides; D011480:Protease Inhibitors; D012235:Rhodamines; D017361:Viral Nonstructural Proteins; C114557:5-carboxytetramethylrhodamine succinimidyl ester; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C524379:Q-Abu-T peptide; C524378:T-Abu-Q peptide