Characterization of the Anti-Hepatitis C Virus Activity of New Nonpeptidic Small-Molecule Cyclophilin Inhibitors with the Potential for Broad Anti-Flaviviridae Activity

Abstract

Although members of the Flaviviridae display high incidence, morbidity, and mortality rates, the development of specific antiviral drugs for each virus is unlikely. Cyclophilins, a family of host peptidyl-prolyl cis-trans isomerases (PPIases), play a pivotal role in the life cycles of many viruses and therefore represent an attractive target for broad-spectrum antiviral development. We report here the pangenotypic anti-hepatitis C virus (HCV) activity of a small-molecule cyclophilin inhibitor (SMCypI). Mechanistic and modeling studies revealed that the SMCypI bound to cyclophilin A in competition with cyclosporine (CsA), inhibited its PPIase activity, and disrupted the CypA-nonstructural protein 5A (NS5A) interaction. Resistance selection showed that the lead SMCypI hardly selected amino acid substitutions conferring low-level or no resistance in vitro Interestingly, the SMCypI selected D320E and Y321H substitutions, located in domain II of the NS5A protein. These substitutions were previously associated with low-level resistance to cyclophilin inhibitors such as alisporivir. Finally, the SMCypI inhibited the replication of other members of the Flaviviridae family with higher 50% effective concentrations (EC₅₀s) than for HCV. Thus, because of its chemical plasticity and simplicity of synthesis, our new family of SMCypIs represents a promising new class of drugs with the potential for broad-spectrum anti-Flaviviridae activity as well as an invaluable tool to explore the role of cyclophilins in viral life cycles.

Keywords: Flaviviridae; antiviral agents; broad-spectrum antiviral activity; cyclophilin inhibitors; hepatitis C virus; resistance; small molecule.

FIG 1

Molecular modeling of the interaction of C31 and CsA with CypA, competition between C31 and CsA for CypA binding, and the relationship between the anti-PPIase activity of the SMCypIs in an enzyme assay and their anti-HCV activity against a genotype 1b HCV-SGR. (A and B) Surface representations of CypA in complex with CsA (A) and C31 (B) showing occupation of the catalytic site and the gatekeeper pocket. The side chain of Arg55 is represented in stick format and highlighted in purple. (C) Competition between C31 and CsA for CypA binding, assessed by a TR-FRET assay. The graphs represent the FRET emission ratios measured in the presence of increasing concentrations of C31. Unlabeled CsA and ALV were used as internal controls. The data are shown as means ± SD of results from three independent experiments. AU, arbitrary units. (D) Graph representing the relationship between the 50% inhibitory concentration (IC₅₀) in a CypA PPIase enzyme assay and the EC₅₀ in a genotype 1b HCV-SGR assay of 6 SMCypIs related to C31 (listed in Table S1 in the supplemental material). The Pearson correlation coefficient (R) and P value are shown on the graph.

FIG 2

Disruption of the CypA-NS5A interaction by the cyclophilin inhibitors. The interaction between NS5A-Rluc and CypA-6His was assessed by means of a protein-protein interaction assay with Ni-NTA magnetic beads. After loading of CypA-6His, the beads were incubated with a lysate of Huh7.5 cells expressing WT-NS5A-Rluc (A) or NS5A-D320E-Rluc (B) and two concentrations of C31 and ALV. After washing, CypA-6His-interacting proteins were eluted with imidazole. The NS5A-Rluc activity was measured in the eluate. Huh7.5 cells expressing Rluc were used as a negative interaction control. The data are shown as means ± SD of results from at least three independent experiments. NS, not significant; ***, P < 0.001.

FIG 3

Studies of the combination of C31 (2.5 μM) and LDV (10 pM) in a genotype 1a HCV-SGR in Huh7.5 cells. Huh7.5 cells stably expressing a genotype 1a HCV-SGR were cultured through 5 passages in the presence of C31 (2.5 μM), LDV (10 pM), or both drugs, in the presence (top) or in the absence (bottom) of G418. The cells were stained with crystal violet.

FIG 4

Amino acid changes selected by serial C31 passages at increasing concentrations in a genotype 1b HCV-SGR. Huh7.5 cells stably harboring a genotype 1b HCV-SGR were cultured in medium containing 1.5 mg/ml of G418 in the presence of increasing doses of C31 until resistant clones were selected. Total RNAs of two resistant clones were extracted, and the NS5A-coding region was sequenced. The amino acid changes observed in the sequences of the NS5A regions spanning domains I to III are shown. Neo, neomycin.

FIG 5

In vitro activity of C31 against three flaviviruses. Shown are dose-dependent curves of antiviral activity against DENV, ZIKV, and YFV. Viral replication was assessed by RT-qPCR at 48 h postinfection. Relative replication was expressed as a percentage of the value for the untreated control and plotted against the log₁₀ concentration of C31. The EC₅₀s are shown on the graph and are represented by a dashed line. Data shown are means ± SD of results from at least two independent experiments performed in triplicate.