Importance of Ligand Bioactive Conformation in the Discovery of Potent Indole-Diamide Inhibitors of the Hepatitis C Virus NS5B

Abstract

Significant advances have led to receptor induced-fit and conformational selection models for describing bimolecular recognition, but a more comprehensive view must evolve to also include ligand shape and conformational changes. Here, we describe an example where a ligand's "structural hinge" influences potency by inducing an "L-shape" bioactive conformation, and due to its solvent exposure in the complex, reasonable conformation-activity-relationships can be qualitatively attributed. From a ligand design perspective, this feature was exploited by successful linker hopping to an alternate "structural hinge" that led to a new and promising chemical series which matched the ligand bioactive conformation and the pocket bioactive space. Using a combination of X-ray crystallography, NMR and modeling with support from binding-site resistance mutant studies and photoaffinity labeling experiments, we were able to derive inhibitor-polymerase complexes for various chemical series.

Dynamics of Hepatitis C Virus (HCV) RNA-dependent RNA Polymerase NS5B in Complex with RNA

“…1). Upon binding, the finger loop is likely to be displaced and adopts a disengaged flexible conformation (41). These inhibitors block RNA synthesis specifically at the level of initiation; presumably by affecting formation of a competent binary NS5B-RNA complex.…”

“…Here, we used a prototype inhibitor, i.e. compound C (11,41), to study whether the mechanism of action involves changes in the dynamic interaction between NS5B and its ssRNA template.…”

Mechanism of Inhibition for BMS-791325, a Novel Non-nucleoside Inhibitor of Hepatitis C Virus NS5B Polymerase

“…In a more recent Phase 2 study, 63 of 66 HCV-infected GT-1 patients treated for 12 or 24 weeks with a combination of BMS-791325 (75 or 150 mg twice daily), daclatasvir (60 mg once daily), and asunaprevir (200 mg twice daily) achieved sustained virologic response (9). The preclinical profile of BMS-791325, including potent activity in GT-1a and -1b enzyme and replicon assays (IC 50 and EC 50 values of 0.7-4 nM), selection of significant resistance at a single substitution site, and a robust pharmacokinetic profile in animal models, anticipated the strong antiviral effect observed in patients (10,11).…”

“…IC 50 nM Poly(C)⅐GTP, de novo 3.5 Ϯ 0.5 Poly(C)⅐pGpG, primer-dependent 2.9 Ϯ 0.9 Poly(A)⅐dT 12 , primer-dependent 4.5 Ϯ 2.3 HCV RNA⅐NTP, copy-back 3.9 Ϯ 0.4 the formation of new replication complexes (39). The progress curve for the poly(C) template⅐pGpG dinucleotide reaction with BMS-791325 added (10 M) parallels the progress curve with heparin added (10 g/ml).…”

“…Time-dependent Inhibition of HCV NS5B Polymerase Activity-To determine whether the inhibition of NS5B by BMS-791325 is time-dependent, the IC 50 was measured with and without preincubation of inhibitor with NS5B before replication was initiated by adding NTP. The potency of BMS-791325 was enhanced by increasing the time of preincubation ( Table 2).…”