Affinity selection of a camelized V(H) domain antibody inhibitor of hepatitis C virus NS3 protease.
The HCV genome encodes, within the NS3 gene, a serine protease whose activity specifically cleaves the viral polyprotein precursor. Proteolytic processing of HCV polyprotein precursor by the viral NS3 proteinase is essential for virion maturation and designing specific inhibitors of this protease as possible anti-viral agents is a desirable and practical objective. With a view to studying both the function of HCV NS3 protease and to designing inhibitors of this enzyme, we directed our interest towards engineering macromolecular inhibitors of the viral protease catalytic activity. We describe here the affinity-selection and biochemical characterization of one inhibitor, cV(H)E2, a 'camelized' variable domain antibody fragment, isolated from a phage displayed synthetic repertoire, which is a potent and selective inhibitor of proteolysis by the NS3 enzyme. In addition to being useful as a biological probe to study the function of HCV protease, this inhibitor can serve as a potential pharmacophore model to design antivirals. Moreover, the results suggest a way of engineering improved human-derived small recognition units tailored for enzyme inhibition.
Martin
F
F
Istituto di Ricerche di Biologia Molecolare (IRBM) P. Angeletti, Pomezia (Rome), Italy.
Volpari
C
C
Steinkuhler
C
C
Dimasi
N
N
Brunetti
M
M
Biasiol
G
G
Altamura
S
S
Cortese
R
R
De Francesco
R
R
Sollazzo
M
M
D000818:Animals; D000956:Antigens, Viral / Q000276:immunology / Q000378:metabolism; D002162:Camelus / Q000276:immunology; D002384:Catalysis; D018360:Crystallography, X-Ray; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D015202:Protein Engineering; D020365:RNA Helicases; D012697:Serine Endopeptidases / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000138:chemical synthesis*; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D000956:Antigens, Viral; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D020365:RNA Helicases; D012697:Serine Endopeptidases; D015842:Serine Proteinase Inhibitors; D017361:Viral Nonstructural Proteins; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases