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The 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.

To identify novel antivirals to the hepatitis C virus (HCV) NS4B protein, we utilized encoded library technology (ELT), which enables purified proteins not amenable to standard biochemical screening methods to be tested against large combinatorial libraries in a short period of time. We tested NS4B against several DNA-encoded combinatorial libraries (DEL) and identified a single DEL feature that was subsequently progressed to off-DNA synthesis. The most active of the initial synthesized compounds had 50% inhibitory concentrations (IC 50 s) of 50 to 130 nM in a NS4B radioligand binding assay and 300 to 500 nM in an HCV replicon assay. Chemical optimization yielded compounds with potencies as low as 20 nM in an HCV genotype 1b replicon assay, 500 nM against genotype 1a, and 5 μM against genotype 2a. Through testing against other genotypes and genotype 2a-1b chimeric replicons and from resistance passage using the genotype 1b replicon, we confirmed that these compounds were acting on the proposed first transmembrane region of NS4B. A single sequence change (F98L) was identified as responsible for resistance, and it was thought to largely explain the relative lack of potency of this series against genotype 2a. Unlike other published series that appear to interact with this region, we did not observe sensitivity to amino acid substitutions at positions 94 and 105. The discovery of this novel compound series highlights ELT as a valuable approach for identifying direct-acting antivirals to nonenzymatic targets.