Serpin Mechanism of Hepatitis C Virus Nonstructural 3 (NS3) Protease Inhibition

Abstract

Hepatitis C virus (HCV) nonstructural 3 (NS3) serine protease disrupts important cellular antiviral signaling pathways and plays a pivotal role in the proteolytic maturation of the HCV polyprotein precursor. This recent discovery has fostered the search for NS3 protease inhibitors. However, the enzyme's unusual induced fit behavior and peculiar molecular architecture have imposed considerable obstacles to the development of small molecule inhibitors. In this article, we demonstrate that such unique induced fit behavior and the chymotrypsin-like catalytic domain can provide the structural plasticity necessary to generate proteinbased inhibitors of the NS3 protease. We took advantage of the macromolecular scaffold of a Drosophila serpin, SP6, which intrinsically supports chymotrypsin-like enzyme inhibition, to design a novel class of potent and selective inhibitors. We show that altering the SP6 reactive site loop (RSL) resulted in the development of the first effective (K i of 34 nM) and selective serpin, SP6 EVC/S , directed at the NS3 protease. SP6 EVC/S operates as a suicide substrate inhibitor, and its partitioning between the complex-forming and proteolytic pathways for the NS3 protease is HCV NS4A cofactor-dependent and -specific. Once bound to the protease active site, SP6 EVC/S partitions with equal probability to undergo proteolysis by NS3 at the C-terminal site of the engineered RSL, (P 6 )GluIle-(P 4 )Val-Met-Thr-(P 1 )Cys-2-(P 1 )Ser, or to form a covalent acyl-enzyme complex characteristic of cognate protease-serpin pairs. Our results also reveal a novel cofactor-induced serpin mechanism of enzyme inhibition that could be explored for developing effective and selective inhibitors of other important induced fit viral proteases of the Flaviviridae family such as the West Nile virus NS3 endoprotease. Hepatitis C virus (HCV)1 infection has reached worldwide epidemic proportions, with Ͼ3% of the world population infected and 3-4 million people newly infected each year (1). The clinical significance of HCV and the need to rapidly identify new therapeutic approaches has resulted in intensive study of the molecular properties of this virus; yet, thus far, no efficient therapy or vaccine exists (1, 2). Moreover, although considerable progress has been made in understanding HCV biology (3, 4), the molecular mechanisms by which HCV induces liver cirrhosis, fibrosis, and hepatocellular carcinoma remain unclear (1). The discovery that HCV nonstructural 3 (NS3) protease may be involved in the modulation of important host cell functions (5), in addition to its role in the maturation of the viral polyprotein precursor (NS3-NS5) (6), has fostered the search for NS3 protease inhibitors (2, 7).The NS3 protease is an induced fit serine protease that adopts a chymotrypsin-like fold (8). The action of a virusencoded protein cofactor, NS4A, is required for some but not all of the NS3-dependent proteolytic cleavage events in vitro (9) and ex vivo (10,11). The commonly accepted model of action for the NS3 protease suggests t...

The Spn4 gene of Drosophila encodes a potent furin-directed secretory pathway serpin

“…3A) and dPC2 (Fig. 4A) by Spn4A obeyed slow-binding inhibition kinetics (16), as indicated by biphasic plots, where maximal inhibition was achieved more rapidly with increasing concentrations of serpin (19,22). The serpin-enzyme complexes were kinetically trapped because no PC activity was recovered for up to 3 h (Figs.…”

“…19 and 22 by using ENZFITTER (Version 2.0, Elsevier-Biosoft, Cambridge, U.K.). Measurements also were made by using the progress curve method (19,22). These fluorescence data were fitted by using least-squares regression in SCIENTIST (Version 2.01, Micromath Scientific Software, Salt Lake City) to an equation describing slow inhibitor binding (24).…”

“…The enzyme assay data were obtained by using a SpectraMax Gemini XS spectrofluorometer equipped with a temperature-controlled 96-well plate reader (Molecular Devices) at excitation and emission wavelengths of 370 and 460 nm to measure release of 7-amino-4-methylcoumarin (22). Furin and PC2 assays were performed by using pERTKR-MCA as a fluorogenic substrate (19).…”

“…Recombinant serpin variants were expressed in the cytosol of bacteria (Escherichia coli BL21 pLysS) and purified as previously reported by using an AKTApurifier FPLC system (Amersham Biosciences) (22). Protein purity and composition were demonstrated by Coomassie blue staining of SDS͞PAGE gels, RP-HPLC, amino acid analysis (Advanced Protein Technology Center, University of Toronto), electrospray MS (M-Scan, West Chester, PA), and Western blot.…”

“…The samples were then mixed immediately with reduced SDS-loading buffer at 95°C and heated for 10 min. SDS-gel electrophoresis was performed in a 10% gel (22), and the protein was visualized by Coomassie blue or analyzed by Western blot with anti-FLAG Ab (22) or anti-PC2 Ab (9) after transfer to nitrocellulose membranes (22). Western blots were visualized directly by using the VersaDoc imaging system (BioRad).…”