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

Abstract

Hepatitis C virus (HCV) infection is a global health burden with over 170 million people infected worldwide. In a significant portion of patients chronic hepatitis C infection leads to serious liver diseases, including fibrosis, cirrhosis, and hepatocellular carcinoma. The HCV NS3 protein is essential for viral polyprotein processing and RNA replication and hence viral replication. It is composed of an N-terminal serine protease domain and a C-terminal helicase/NTPase domain. For full activity, the protease requires the NS4A protein as a cofactor. HCV NS3/4A protease is a prime target for developing direct-acting antiviral agents. First-generation NS3/4A protease inhibitors have recently been introduced into clinical practice, markedly changing HCV treatment options. To date, crystal structures of HCV NS3/4A protease inhibitors have only been reported in complex with the protease domain alone. Here, we present a unique structure of an inhibitor bound to the full-length, bifunctional protease-helicase NS3/4A and show that parts of the P4 capping and P2 moieties of the inhibitor interact with both protease and helicase residues. The structure sheds light on inhibitor binding to the more physiologically relevant form of the enzyme and supports exploring inhibitor-helicase interactions in the design of the next generation of HCV NS3/4A protease inhibitors. In addition, small angle X-ray scattering confirmed the observed proteasehelicase domain assembly in solution.structure-based drug design | X-ray structure | solution scattering | medicinal chemistry C hronic hepatitis C virus (HCV) infection affects more than 3% of the world's population and is a leading cause of chronic liver diseases (1). Therapeutic options have been suboptimal, especially for HCV genotype 1, the most prevalent genotype in developed countries. Recently, the addition of direct-acting antiviral agents (DAAs) to the previous standard of care (combination therapy with pegylated interferon and ribavirin) have demonstrated considerable improvement in sustained virological response rates in patients infected with HCV genotype 1 (2). With the first DAAs now having been introduced into clinical practice, it is to be expected that in the near future standard therapy will change to a triple therapy including an HCV NS3/4A protease inhibitor in combination with pegylated interferon and ribavirin.The positive-strand RNA genome of HCV encodes a polyprotein precursor, which is proteolytically processed by host and viral proteases into 10 individual structural and nonstructural (NS) proteins. The viral NS3 protease in complex with the cofactor NS4A cleaves the polyprotein at four junctions releasing the NS proteins 4A, 4B, 5A, and 5B, and therefore is essential for viral replication (3, 4). A central, hydrophobic 14-mer peptide of the 54-residue NS4A, comprising residues 21-32, is necessary and sufficient for maximal activation in vitro (5). NS3/4A has also been shown to cleave cellular proteins leading to inhibition of interferon production, thereby impairin...

RNA helicases in infection and disease

“…The structure of full-length HCV-NS3 and of the isolated helicase has been determined in the absence of DNA, 22,35 in complex with ssDNA, 36 in complex with DNA and different nucleotides, 37 as well as in complex with inhibitors. 38 The HCV-NS3 helicase consists of two RecA domains (domains H1 and H2) and a third α-helical domain (domain 3) (ref. 22, Fig.…”

“…211). A recently reported structure of a protease-targeted inhibitor has revealed that this inhibitor also contacts the helicase domain, 38 underlining the possibility of targeting the helicase directly. Screening of chemical libraries has identified limited numbers of weak helicase inhibitors.…”

Infection of Common Marmosets with GB Virus B Chimeric Virus Encoding the Major Nonstructural Proteins NS2 to NS4A of Hepatitis C Virus

“…The NS4A polypeptide acts as a cofactor of NS3 serine protease for efficient polyprotein processing (30). The HCV NS3-NS4A complex is a heterodimeric serine protease that has been considered a prime target for developing direct-acting antivirals (31,32), such as novel protease inhibitors of noncovalent macrocyclic simeprevir/TMC435 (33,34), covalent linear boceprevir (BOC) (35)(36)(37), and telaprevir analog (TVR) (38,39). From an immunologic point of view, the HCV NS2 to -4A proteins are major antigens in HCV vaccines, a few of which are currently in clinical trials for induction of a protective T-cell response against HCV infection (40)(41)(42).…”

Vinylated linear P2 pyrimidinyloxyphenylglycine based inhibitors of the HCV NS3/4A protease and corresponding macrocycles

“…P3-P1 0 macrocycles have previously been investigated in P2 proline based inhibitors in a patent application. 49,50 The ring closure of 17 (Scheme 6) proved to be complicated. Full conversion of starting material was not obtained and large amounts of dimer, trimer, and ring-closed variants of the dimer and trimer were formed according to LC-MS. An interesting observation was made when a small fraction of 17 was purified on preparative HPLC with acetonitrile/water containing 0.05% formic acid as eluent, for biochemical evaluation.…”