A scintillation proximity active site binding assay for the hepatitis C virus serine protease.
A binding assay suitable for the identification of active site-directed inhibitors of the hepatitis C virus serine protease NS3 was developed. A C-terminal extension of 13 residues that is specifically recognized by the Escherichia coli biotin holoenzyme synthetase (Bir A) was fused to a truncated NS3 protease domain, allowing the efficient production of in vivo biotinylated protease. This enzyme was purified and shown to have the same properties as its wild-type counterpart concerning substrate binding and turnover, interaction with a cofactor peptide, and inhibition by three different classes of inhibitors. Immobilization of the biotinylated protease, using streptavidin-coated scintillation proximity beads, allowed detection, by scintillation counting, of its interaction with a tritiated active site ligand spanning the whole substrate binding site of the protease from P6 to P4('). Immobilization did not measurably affect accessibility to either the active site or the cofactor binding site of the protease as judged by the unchanged affinities for a cofactor peptide and for two active site binders. Using the displacement of the radioligand as readout, we were able to set up a rapid, robust, and fully automated assay, suitable for the selective identification of novel active site ligands of the NS3 protease.
Department of Biochemistry, Istituto di Ricerche di Biologia Molecolare P.Angeletti (IRBM), Merck Research Laboratories, Rome, Italy.
Christian
C
Steinkühler
Gabriella
G
Biasiol
Mauro
M
Cerretani
Laura
L
Di Renzo
Mirko
M
Brunetti
Paolo
P
Ingallinella
Raffaele
R
De Francesco
Sergio
S
Altamura
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000502:physiology; D001667:Binding, Competitive; D019731:Carbon-Nitrogen Ligases / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D016174:Hepacivirus / Q000201:enzymology*; D006801:Humans; D008024:Ligands; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D012097:Repressor Proteins; D012588:Scintillation Counting / Q000379:methods*; D012697:Serine Endopeptidases / Q000378:metabolism*; D014157:Transcription Factors; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D001426:Bacterial Proteins; D019731:Carbon-Nitrogen Ligases; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D008024:Ligands; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D012697:Serine Endopeptidases; D014157:Transcription Factors; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C058125:birA protein, E coli