Encoded Library Technology Screening of Hepatitis C Virus NS4B Yields a Small-Molecule Compound Series with In Vitro Replicon Activity

Abstract

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 M 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. Significant progress has been made in recent years in the discovery and development of novel direct-acting antivirals (DAAs) to treat hepatitis C virus (HCV) infection. However, the ability of HCV to rapidly evolve and develop resistance is such that there is a continued need to discover DAAs that act through novel mechanisms. The standard of care for chronic HCV infection was a combination of pegylated alpha interferon and ribavirin up until the approval of the NS3 protease inhibitors telaprevir and boceprevir in 2011 (1, 2). Other classes of DAAs showing clinical efficacy target the NS5A protein (3, 4) or viral polymerase (NS5B; reviewed in reference 5). Of the other viral proteins, several smallmolecule DAAs to nonstructural protein NS4B have been identified (6-14), but to date, there are no examples of clinically active NS4B-targeting compounds.NS4B is a small (27-kDa), hydrophobic, membrane-associated protein that is derived through processing of the HCV polyprotein by the viral protease NS3/4A (reviewed in references 15, 16, and 17). It complexes with other HCV nonstructural and possibly a host cell factor(s) to form the viral replicase (18-22), which serves to replicate the viral RNA and is likely coupled to the assembly process (20, 23). Besides its structural role in the replicase, NS4B is thought to be involved in the induction of membranous vesicles that provide a platform for HCV RNA replication (24, 25), and it may also participate in virion assembly and release (26,27). NS4B is also reported to hydr...

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

“…The lack of activity against gt2 virus was consistent with the high frequency of L98 in reported gt2 sequences, including the JFH-1 virus used in many in vitro studies. Notably, other reported NS4B-targeting compounds have shown similar resistance and activity profiles (19–23). …”

“…Many of these have selected for resistance mutations in the coding region of NS4B residues 94 to 109 in gt1 HCV, a region predicted as the first of four transmembrane α-helices (19–23). Furthermore, the most prominent substitutions (at residues 94, 98, 105, and 109) lie on the same face of the α-helix according to the standard model based on 3.5 amino acids per turn.…”

“…In HCV gt3 replicons, minor substitutions were also identified N terminal to TM1 after de novo selection, including L64I, S85A, and S88C. The magnitude of resistance to PTC725 and the effect on HCV fitness are yet to be determined for these substitutions; however, an HCV NS4B-targeting compound discovered through encoded library technology was found to exhibit 3-fold-less potency against replicons containing S85A (23). Notably, S85 and S88 are located in the region postulated to be a fifth membrane-spanning domain (TMX), suggesting that NS4B-targeting compounds such as PTC725 may affect the interaction of these two transmembrane regions (33, 34).…”

A Journey around the Medicinal Chemistry of Hepatitis C Virus Inhibitors Targeting NS4B: From Target to Preclinical Drug Candidates

“…As expected, in vitro resistance passaging experiments employing wild-type HCV replicons revealed that single-point mutations within the NS4B protein (H94N or V105M) rendered the virus partially resistant to compound 18 (Figure 10). 104 Finally very recently, Thompson et al at GlaxoSmithKline have reported the anti-HCV activity of compound 18 against a panel of different gts 85. In particular, this compound showed EC 50 in the nM range against gts 3a (42.6 nM), 4a (6.98 nM), and 5a (2.53 nM), whilst it was only a weak inhibitor of gts 2b (2.4 µM) and 6a (4.47 µM) and resulted inactive against gts 2a and 6o.…”

“…85 ELT approach is a technology platform that bridges the fields of combinatorial chemistry and molecular biology. 89,90 Different combinatorial libraries are built by conjugating drug-like building blocks with coding short double strand DNA tags, used as markers of each chemical library.…”