Preclinical Characterization and In Vivo Efficacy of GSK8853, a Small-Molecule Inhibitor of the Hepatitis C Virus NS4B Protein

Abstract

dThe hepatitis C virus (HCV) NS4B protein is an antiviral therapeutic target for which small-molecule inhibitors have not been shown to exhibit in vivo efficacy. We describe here the in vitro and in vivo antiviral activity of GSK8853, an imidazo[1,2-a]pyrimidine inhibitor that binds NS4B protein. GSK8853 was active against multiple HCV genotypes and developed in vitro resistance mutations in both genotype 1a and genotype 1b replicons localized to the region of NS4B encoding amino acids 94 to 105. A 20-day in vitro treatment of replicons with GSK8853 resulted in a 2-log drop in replicon RNA levels, with no resistance mutation breakthrough. Chimeric replicons containing NS4B sequences matching known virus isolates showed similar responses to a compound with genotype 1a sequences but altered efficacy with genotype 1b sequences, likely corresponding to the presence of known resistance polymorphs in those isolates. In vivo efficacy was tested in a humanized-mouse model of HCV infection, and the results showed a 3-log drop in viral RNA loads over a 7-day period. Analysis of the virus remaining at the end of in vivo treatment revealed resistance mutations encoding amino acid changes that had not been identified by in vitro studies, including NS4B N56I and N99H. Our findings provide an in vivo proof of concept for HCV inhibitors targeting NS4B and demonstrate both the promise and potential pitfalls of developing NS4B inhibitors. Hepatitis C virus (HCV) is a significant public health threat, with up to 3% of the world population estimated to be harboring the infection. Although the virus can be naturally cleared, it more often becomes established as a chronic infection with an increased risk of poor long-term outcomes, including liver cirrhosis and cancer (1). Treatment of the disease historically used a combination of ribavirin and pegylated interferon, a cocktail with a 50% cure rate against genotype 1 but with a high likelihood of undesirable side effects (2, 3). The regulatory approval of directacting small-molecule antivirals represents a major advance in therapeutics by increasing the chance of efficacious treatment (4, 5), but there is still a need for novel antiviral therapies with reduced side effects and complementary resistance profiles. HCV inhibitors of the NS3 protease, of the multifunctional protein NS5A, and of the NS5B RNA-dependent RNA polymerase have been shown to be efficacious in the clinic, but inhibitors of NS4B have not been validated in vivo. We (6-8) and others (9-12) have described compounds targeting NS4B in vitro that, if developed, would offer a complementary mechanism that may improve the standard of care for hepatitis C virus infection treatment.HCV RNA replication occurs in vesicle-induced intracellular membranes derived from the cellular endoplasmic reticulum (ER), catalyzed by a complex of proteins encoded by the virus and host factors. NS4B is an integral membrane protein (13) that induces the rearrangement of intracellular membranes into a "membranous web," a collection of vesicles th...

Preclinical Evaluation of Amphihevir, a First-in-Class Clinical Hepatitis C Virus NS4B Inhibitor

“…A total of 11 mutations of the NS4B gene were found in drug resistance screening of HCV-1b, 3 of which occurred more than three times ( Table 3). The mutation with the highest frequency was H94R, followed by V105M and F98L ( Table 3).The cross-resistance and drug resistance mutation profiles of amphihevir were highly similar to those of other recently reported NS4B inhibitors, including anguizole, GSK8853, PTC725, and our previously reported imidazo[2,1b]thiazole NS4B inhibitors (11,14,17,18). This finding suggests that amphihevir mainly acts on the NS4B protein of HCV.…”

Deciphering the Role of Bovine Viral Diarrhea Virus Non-Structural NS4B Protein in Viral Pathogenesis

“…In these viruses, the immunogenic role of NS4B and other non-structural proteins has been well-established using the same methodology as adopted in this study [ 9 , 10 , 15 ]. The role of NS4B as a potential antiviral therapeutic target and vaccine candidate has already been well established in other members of the Flavivirida family [ 22 , 23 , 24 , 25 , 26 , 27 ] and could be actively pursued to control viral infections belonging to BVDV. Future exploration of anti-NS4B antibodies in acute and chronic infections and epitope mapping could contribute further to our current understanding of NS4B’s immunogenicity during BVDV pathogenesis and its assessment as a vaccine candidate.…”

PTC725, an NS4B-Targeting Compound, Inhibits a Hepatitis C Virus Genotype 3 Replicon, as Predicted by Genome Sequence Analysis and Determined Experimentally

“…Recently, an NS4B-targeting compound (GSK8853) structurally unrelated to PTC725 was reported to inhibit HCV gt1a replication in a humanized mouse model and elicited resistance mutations in vivo which were distinct from those identified in resistant replicons in vitro , i.e., those encoding substitutions at positions 94, 98, and 105 (42). RAVs were identified in vivo at N56I and N99H and were confirmed to confer resistance in genetically engineered replicons.…”