Hepatitis C virus (HCV) NS3/4A protease is an important and attractive target 
for anti-HCV drug development and discovery. Vaniprevir (phase III clinical 
trials) and MK-5172 (phase II clinical trials) are two potent antiviral 
compounds that target NS3/4A protease. However, the emergence of resistance to 
these two inhibitors reduced the effectiveness of vaniprevir and MK-5172 against 
viral replication. Among the drug resistance mutations, three single-site 
mutations at residues Arg155, Ala156, and Asp168 in NS3/4A protease are 
especially important due to their resistance to nearly all inhibitors in 
clinical development. A detailed understanding of drug resistance mechanism to 
vaniprevir and MK-5172 is therefore very crucial for the design of novel potent 
agents targeting viral variants. In this work, molecular dynamics (MD) 
simulation, binding free energy calculation, free energy decomposition, residue 
interaction network (RIN), and substrate envelope analysis were used to study 
the detailed drug resistance mechanism of the three mutants R155K, A156T, and 
D168A to vaniprevir and MK-5172. MD simulation was used to investigate the 
binding mode for these two inhibitors to wild-type and resistant mutants of HCV 
NS3/4A protease. Binding free energy calculation and free energy decomposition 
analysis reveal that drug resistance mutations reduced the interactions between 
the active site residues and substituent in the P2 to P4 linker of vaniprevir 
and MK-5172. Furthermore, RIN and substrate envelope analysis indicate that the 
studied mutations of the residues are located outside the substrate (4B5A) 
binding site and selectively decrease the affinity of inhibitors but not the 
activity of the enzyme and consequently help NS3/4A protease escape from the 
effect of the inhibitors without influencing the affinity of substrate binding. 
These findings can provide useful information for understanding the drug 
resistance mechanism against vaniprevir and MK-5172. The results can also 
provide some potential clues for further design of novel inhibitors that are 
less susceptible to drug resistance.