Resistance profiling of hepatitis C virus protease inhibitors using full-length NS3.
BACKGROUND
The NS3 protease of hepatitis C virus (HCV) is a prime target for anti-HCV drugs but resistance towards inhibitors of the enzyme is likely to emerge because of mutations in the viral genome that modify the structure of the protein. Enzyme inhibition data supporting this is limited to studies with few compounds and analysis performed with truncated NS3.


METHODS
The potential of HCV acquiring resistance towards NS3 protease inhibitors and the structural features associated with resistance has been explored with a series of inhibitors and by using full-length NS3 protease/helicase variants with amino acid substitutions (A156T, D168V and R155Q) in the protease domain.


RESULTS
The A156T and D168V substitutions did not influence the kinetic properties of the protease, whereas the R155Q substitution reduced the catalytic efficiency 20 times, as compared with the wild type. Inhibition studies revealed that these substitutions primarily affected the potency of compounds which effectively inhibit the wild-type enzyme, and had little effect on weak or moderate inhibitors. As a consequence, all compounds had similar inhibitory potencies to the substituted enzyme variants. An exception was VX-950, which inhibited the D168V enzyme more efficiently than the wild type. For this inhibitor, the present data correlated better with replicon data than data from assays with truncated enzyme.


CONCLUSIONS
These results have provided a structural basis for designing inhibitors that may be less susceptible to resistance by three known mutations, and suggest that the present variants of full-length NS3 constitute effective models for resistance profiling of NS3 protease inhibitors.
Dahl
Göran
G
Department of Biochemistry and Organic Chemistry, Uppsala University, BMC, Box 576, SE-751 23 Uppsala, Sweden.
Sandström
Anja
A
Akerblom
Eva
E
Danielson
U Helena
UH
D000818:Animals; D000998:Antiviral Agents / Q000737:chemistry / Q000494:pharmacology*; D020134:Catalytic Domain; D002460:Cell Line; D003001:Cloning, Molecular; D015195:Drug Design; D024921:Drug Resistance, Multiple, Viral / Q000235:genetics; D016174:Hepacivirus / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D006801:Humans; D007700:Kinetics; D015394:Molecular Structure; D009154:Mutation; D011480:Protease Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D000998:Antiviral Agents; 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