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 (IC50s) 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.

FIG 1

Schematic of ELT technology. (A) DNA-encoded chemical libraries are synthesized. (B) Affinity selection is performed to separate DEL molecules bound to a protein target from unbound DEL molecules. (C) DNA tags of bound DEL molecules are amplified by PCR. (D) Amplified DNA tags are sequenced by a high-throughput sequencing method. (E) Sequence information is processed and translated into chemical structures. (F) Chemotypes are identified through analysis of structural information. (G) Exemplar compounds from identified chemotypes are synthesized. (H) Assays are performed on synthesized compounds to confirm activity. C1, C2, and C3 indicate cycles 1, 2, and 3, respectively.

FIG 2

Identification of chemotypes bound to HCV NS4B. (A) Graph of enrichment ratio (relative to no-target control) versus DEL library showing the generic structure of DEL 54. (B) Spotfire visualization of putative hit families from DEL 54, with chemistry cycle 2 depicted by color. Structures corresponding to the selected cycle 2–cycle 3–cycle 4 chemotypes are shown below along with minimized derivatives.

FIG 3

Structures of compounds derived from DEL 54. Et, ethyl group; tBu, tert-butyl group; Ph, phenyl group.

FIG 4

Alignment of NS4B amino acid sequences used in the construction of chimeric replicons. Chimeras of genotypes 1a (H77) and 4a were constructed using the genotype 1b (Con1 strain) backbone; hence, their sequences are aligned beneath that of genotype 1b. The remaining chimeras were constructed using the JFH1 (genotype 2a) backbone, and their sequences are shown beneath that of JFH1. Shading of amino acids in the JFH1 sequence indicates differences with the genotype 1b sequence. Lettering above the sequences denotes the 21-amino-acid blocks that were switched to create the genotype 1b-2a domain chimeras.

FIG 5

Recently published NS4B inhibitors. (A) Chemical structures. The structure shown for Shotwell et al. (7) is GSK8853. (B) Overlay of all structures except Gu et al. (9) using Molecular Operating Environment (Chemical Computing Group, Inc., Montreal, Canada). GSK0109 is highlighted in orange.