The effect of prime-site occupancy on the hepatitis C virus NS3 protease structure

Abstract

We recently reported a new class of inhibitors of the chymotrypsin-like serine protease NS3 of the hepatitis C virus. These inhibitors exploit the binding potential of the S' site of the protease, which is not generally used by the natural substrates. The effect of prime-site occupancy was analyzed by circular dichroism spectroscopy and limited proteolysis-mass spectrometry. Generally, nonprime inhibitors cause a structural change in NS3. Binding in the S' site produces additional conformational changes with different binding modes, even in the case of the NS3/4A cofactor complex. Notably, inhibitor binding either in the S or S' site also has profound effects on the stabilization of the protease. In addition, the stabilization propagates to regions not in direct contact with the inhibitor. In particular, the N-terminal region, which according to structural studies is endowed with low structural stability and is not stabilized by nonprime inhibitors, was now fully protected from proteolytic degradation. From the perspective of drug design, P-P' inhibitors take advantage of binding pockets, which are not exploited by the natural HCV substrates; hence, they are an entry point for a novel class of NS3/4A inhibitors. Here we show that binding of each inhibitor is associated with a specific structural rearrangement. The development of a range of inhibitors belonging to different classes and an understanding of their interactions with the protease are required to address the issue of the most likely outcome of viral protease inhibitor therapy, that is, viral resistance.

Fig. 1.

Ribbon representation of the backbone of the crystal structure of the NS3 protease/Pep4A complex (Yan et al. 1998). A gray ribbon is used for the backbone of the NS3 protease and a magenta ribbon for Pep4A. The protease residues of the catalytic triad are represented in yellow sticks. The backbone of a decapeptide inhibitor was modeled by energy minimization and molecular dynamics (Ingallinella et al. 2000). The peptide is shown as a stick presentation, with the P region in green and the P` in blue. The red spheres indicate the chymotrypsin sensitive proteolytic sites in the NS3 structure.

Fig. 2.

Reverse-phase HPLC analysis of NS3/PepB (A) and NS3/PepC (B) complexes digested with chymotrypsin under controlled conditions using an enzyme/substrate ratio of 1:100 (w/w) in the presence of a 1:10 molar excess of each peptide. Individual fractions were collected and analyzed by ESMS. The CHAPS peak is marked with an asterisk; peaks corresponding to the inhibitor PepB and PepC are also indicated.

Fig. 3.

Reverse-phase HPLC analysis of NS3/4A/PepA (A) and NS3/4A/PepC (B) ternary complexes incubated with chymotrypsin under controlled conditions using an enzyme/substrate ratio of 1:100 (w/w). Individual fractions were collected and analyzed by ESMS. The CHAPS peak is marked with an asterisk and those accounting for Pep4A and its proteolytic fragments are marked with P; peaks corresponding to the inhibitor PepB and PepC are also indicated.

Fig. 4.

Inhibitor-induced conformational change of NS3 as detected by near-UV circular dichroism in 15% glycerol, 2% CHAPS, 3 mM DTT, and 50 mM phosphate buffer at pH 7.5. The spectrum of the uncomplexed protease at 60 μM is indicated as NS3; upon formation of complexes with the hexapeptide inhibitor PepA, +A curve, and with the decapeptide inhibitor PepC, +C curve.

Fig. 5.

Inhibitor-induced conformational change of NS3 as detected by near-UV circular dichroism. The spectrum of the uncomplexed protease at 60 μM is indicated as NS3 and upon formation of complexes with the decapeptide inhibitors: PepB, +B curve; PepC, +C curve; PepD, +D curve.

Fig. 6.

Inhibitor-induced conformational change of the NS3/4A complex as detected by near-UV circular dichroism. The spectrum of the NS3/4A protease at 60 μM is indicated as NS3/4A; upon formation of complexes with the hexapeptide inhibitor PepA, +A curve, and with the decapeptide inhibitor PepC, +C curve.

Fig. 7.

Inhibitor-induced conformational change of the NS3/4A complex as detected by near-UV circular dichroism. The spectrum of the NS3/4A protease is indicated as NS3/4A, and those of the complex with the inhibitors as PepB, +B curve; PepC, +C curve; and PepD, +D curve.