A high-throughput radiometric assay for hepatitis C virus NS3 protease.
A novel radiometric in vitro assay for discovery of inhibitors of hepatitis C viral protease activity, suitable for high-throughput screening, was developed. The NS3 protein of hepatitis C virus (HCV) contains a serine protease, whose function is to process the majority of the nonstructural proteins of the viral polyprotein. The viral NS4A protein is a cofactor of NS3 protease activity in the cleavage of NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B junctions. To establish an in vitro assay system we used NS3 proteases from different HCV strains, purified from Escherichia coli and a synthetic radiolabeled peptide substrate that mimics the NS4A-NS4B junction. Upon incubation with the enzyme the substrate was separated from the radiolabeled cleavage product by addition of an ion exchange resin. The assay was performed in a microtiter plate format and offered the potential for assaying numerous samples using a laboratory robot. Taking advantage of these features, we used the assay to optimize reaction conditions by simultaneously varying different buffer components. We showed that physicochemical conditions affect NS3 protease activity in a strain-specific way. Furthermore, the sensitivity of the assay makes it suitable for detection and detailed mechanistic characterization of inhibitors with low-nanomolar affinities for the HCV serine protease.
Cerretani
M
M
Istituto di Ricerche di Biologia Molecolare (IRBM) "P. Angeletti,", Pomezia, Rome, 00040, Italy.
Di Renzo
L
L
Serafini
S
S
Vitelli
A
A
Gennari
N
N
Bianchi
E
E
Pessi
A
A
Urbani
A
A
Colloca
S
S
De Francesco
R
R
Steinkühler
C
C
Altamura
S
S
D002021:Buffers; D010450:Endopeptidases / Q000378:metabolism; D007700:Kinetics; D010455:Peptides / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D011874:Radiometry / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors; D017361:Viral Nonstructural Proteins / Q000032:analysis* / Q000037:antagonists & inhibitors; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D002021:Buffers; D010450:Endopeptidases; D010455:Peptides; 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