Abstract
The nonstructural (NS) protein NS3/4A protease is a critical factor for hepatitis C virus (HCV) maturation that requires activation by NS4A. Synthetic peptide mutants of NS4A were found to inhibit NS3 function. The bridging from peptide inhibitors to heterocyclic peptidomimetics of NS4A has not been considered in the literature and, therefore, we decided to explore this strategy for developing a new class of NS3 inhibitors. In this report, a structure-based design approach was used to convert the bound form of NS4A into 1H-imidazole-2,5-dicarboxamide derivatives as first generation peptidomimetics. This scaffold mimics the buried amino acid sequence Ile-25` to Arg-28` at the core of NS4A21`–33` needed to activate the NS3 protease. Some of the synthesized compounds (Coded MOC) were able to compete with and displace NS4A21`–33` for binding to NS3. For instance, N5-(4-guanidinobutyl)-N2-(n-hexyl)-1H-imidazole-2,5-dicarboxamide (MOC-24) inhibited the binding of NS4A21`–33` with a competition half maximal inhibitory concentration (IC50) of 1.9 ± 0.12 µM in a fluorescence anisotropy assay and stabilized the denaturation of NS3 by increasing the aggregation temperature (40% compared to NS4A21`–33`). MOC-24 also inhibited NS3 protease activity in a fluorometric assay. Molecular dynamics simulations were conducted to rationalize the differences in structure–activity relationship (SAR) between the active MOC-24 and the inactive MOC-26. Our data show that MOC compounds are possibly the first examples of NS4A peptidomimetics that have demonstrated promising activities against NS3 proteins.
Highlights
Hepatitis C is a life-threatening, widespread viral infection [1]
Awareness of hepatitis C health problems and forceful efforts to combat the spread of hepatitis C virus (HCV) in healthcare settings have led to a significant decline in new hepatitis C cases
Spanning through Val-26` to Arg-28` (Figure 1A, torsion table in Supplementary Material Table S1A). This cis bond largely depends on the presence of Gly-27` as it allows lessening the steric conflict with the Val-26`side chain. This glycine turn is conserved in all crystal structures of HCV NS3 protease in the Protein Data Bank (PDB) deposited to date, regardless of their genotype [33,34,35] or of the sequence Bcoionmsotlercuucletss2u02s0e, d10(, N47S94A fused with NS3 or non-fused) [14,23,35]
Summary
The virus initially infects liver cells though remains asymptomatic for extended periods. This invisibility constitutes epidemiological challenges as patients diagnosed with hepatitis C come from two pools: recently infected and late-stage asymptomatic. The initial symptoms, such as fever, fatigue, nausea, and liver tenderness, can be misleading, as they are tolerated by most patients. The NS3/4A protease was the first and foremost targeted viral protein with DAAs that bind to its substrate site [21]. We present compounds that were designed to compete with and replace NS4A on its NS3 binding site, leading to NS3 inhibition [22]
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