Use of the Fused NS4A Peptide−NS3 Protease Domain To Study the Importance of the Helicase Domain for Protease Inhibitor Binding to Hepatitis C Virus NS3-NS4A

Abstract

The NS3 protein of hepatitis C virus is unusual because it encodes two unrelated enzymatic activities in linked protease and helicase domains. It has also been intensively studied because inhibitors targeting its protease domain have potential to significantly improve treatment options for those infected with this virus. Many enzymological studies and inhibitor discovery programs have been carried out using the isolated protease domain in complex with a peptide derived from NS4A which stimulates activity. However, some recent publications have suggested that the NS3 helicase domain may influence inhibitor binding and thus suggest work should focus on the full-length NS3-NS4A protein. Here we present the characterization of a single-chain protease in which the NS4A peptide activator is linked to the N-terminus of the NS3 protease domain. This protein behaves well in solution, and its protease activity is very similar to that of full-length NS3-NS4A. We find that this fusion protein, as well as the noncovalent complex of the NS4A peptide with NS3, gives similar Ki values, spanning 3 orders of magnitude, for a set of 25 structurally diverse inhibitors. We also show that simultaneous mutation of three residues on the surface of the helicase domain which has been hypothesized to interact with the protease does not significantly affect enzymatic activity or inhibitor binding. Thus, the protease domain with the NS4A peptide, in a covalent or noncovalent complex, is a good model for the protease activity of native NS3-NS4A.

A macrocyclic HCV NS3/4A protease inhibitor interacts with protease and helicase residues in the complex with its full-length target

“…The SAXS data unambiguously prove the domain orientation as in the crystal structure. The experimental data cannot be reconciled with the scattering patterns computed from a full-length flaviviral NS2B/3 structure with an extended domain orientation, exemplified by the Dengue virus structure 2VBC (17) for which no satisfactory fit could be obtained (discrepancy χ ¼ 7.5; Fig. 2B).…”

“…Dahl et al observed a 10-fold decrease in binding affinity of BILN2061 to the full-length NS3/4A harboring an H528S mutation (16). Thibeault et al, on the other hand, report that the full-length NS3/4A triple mutant M485A, V524A, Q526A did not lead to a significant change in K I for eight inhibitors tested (17). An example where the structural information of helicase residues was explicitly considered in the design of P2-P4 macrocyclic protease inhibitors was reported by Liverton et al (18).…”

Combined X-ray, NMR, and Kinetic Analyses Reveal Uncommon Binding Characteristics of the Hepatitis C Virus NS3-NS4A Protease Inhibitor BI 201335

“…The HCV genotype 1b Con1 strain NS3-NS4A protein (FL-protease), the HCV genotype 1b J strain NS3 protease domain, and the same domain with an NS4A peptide fused to its N terminus (sc-protease) were expressed and purified as described previously (16). The NS4A peptide used for crystallography (KKGSVVIVGRIILSGRK) has also been described previously (16), as well as the depsipeptide fluorogenic substrate anthranilyl-DDIVP-Abu[C(O)-O]AMY(3-NO 2 )-TW-OH, which is derived from the NS5A-NS5B cleavage site. FL-protease and sc-protease recognize and process this substrate with a similar efficiency (16).…”

“…The NS4A peptide used for crystallography (KKGSVVIVGRIILSGRK) has also been described previously (16), as well as the depsipeptide fluorogenic substrate anthranilyl-DDIVP-Abu[C(O)-O]AMY(3-NO 2 )-TW-OH, which is derived from the NS5A-NS5B cleavage site. FL-protease and sc-protease recognize and process this substrate with a similar efficiency (16).…”

“…NMR Sample Preparation and Spectroscopy-Uniformly 15 N-labeled samples of the sc-protease were expressed and purified as described previously (16). The final buffer consisted of 25 mM NaPO 4 , 150 mM NaCl, 1 mM deuterated Tris (2-carboxyethyl) phosphine hydrochloride at pH 6.0 in 90% H 2 O, 10% D 2 O.…”