Different Molecular Mechanisms of Inhibition of Bovine Viral Diarrhea Virus and Hepatitis C Virus RNA-Dependent RNA Polymerases by a Novel Benzimidazole.

Biochemistry. 2013 April 29; 52 (21): 3752–3764

Asthana S1, Shukla S2, Vargiu AV1, Ceccarelli M1, Ruggerone P1, Paglietti G3, Marongiu ME2, Blois S2, Giliberti G2, La Colla P2.

 

1. Dipartimento di Fisica, Università degli Studi di Cagliari, Cittadella Universitaria, 09042 Monserrato (CA), Italy

2. Dipartimento di Scienze Biomediche, Università degli Studi di Cagliari, Cittadella Universitaria, 09042 Monserrato (CA), Italy

3. Dipartimento di Scienze del Farmaco, Università degli Studi di Sassari, Via Muroni 23/a, 07100 Sassari, Italy

 

Abstract

The virus-encoded RNA-dependent RNA polymerase (RdRp) has emerged as a primary target in the search for selective inhibitors of Flaviviridae. Recently, we reported on the selective inhibition, in cell-based assays, of both BVDV (EC50 = 0.80 ± 0.06 μM) and HCV (EC50 = 1.11 ± 0.15 μM) by 2-{1-[2-(2,4-dimethoxyphenyl)-1H-benzimidazol-5-yl]ethylidene}hydrazinecarbothioamide (227G). Here we show that, in enzyme assays with recombinant enzymes, 227G inhibits, in a dose-dependent manner, the RdRp of both BVDV (IC50 = 0.0020 ± 0.0004 μM) and HCV (IC50 = 0.40 ± 0.04 μM). In addition, we report on the selection and molecular analysis of a BVDV-resistant mutant, characterized by the presence of the I261M mutation. By applying a multilevel computational approach, we identified different 227G binding sites on the two RdRps. They were further validated by the good agreement between the calculated affinities and those extrapolated from IC50 values. Our findings suggest different molecular mechanisms of inhibition of the HCV and BVDV RdRps by 227G and indicate the importance of understanding ligand-enzyme interactions at the molecular level for the rational design of new and more potent leads.

 

Supplement

This study reports about the first benzimidazole Non-Nucleoside Inhibitor (NNI), 227G, active at low micromolar concentration against the two viruses HCV and BVDV, representative of two different Pesti- and Hepaci- genera of the Flaviviridae family. In both cases, the target of 227G appears to be the RdRp, as shown by enzyme assays for these polymerases, where 227G inhibits (although with a different potency) the activity of BVDV and HCV recombinant enzymes.

Asthana et. al. underscored the different molecular mechanisms by which 227G inhibits the RdRps of the two viruses (Figure 1). They performed a thorough computational study to identify the most likely binding region of 227G on the two polymerases, and the effect this binding has on the structure and the functional dynamics of the proteins. In HCV 227G is found to bind with a good affinity within the λ1 loop cavity of the Thumb domain (Figure 1B). As a result, 277G displaces the λ1 loop from its functional site (Figure 1C), thus impairing the coordination of the movement of the Thumb and Finger domains provided by the Fingertip subdomain [1] and essential for RNA template translocation (Figure 1F). In other words, 227G switch the enzyme from a closed to an open and inactive conformation.

In BVDV a novel high-affinity site was found for 227G in the Finger domain (Figure 1B), localized at the entrance of the RNA template tunnel, a region clearly crucial for proper enzyme functioning. 227G stacks onto motif I, playing an essential role in binding the incoming NTPs [2, 3]. Moreover, upon binding to this site the inhibitor is “embraced” by the four loops lining the RNA template entrance. In particular, 227G is the first benzimidazole compound found to interact with the Linker (Figure 1D) loop, which is thought to escort the template into the catalytic site [2]. This interaction occludes the RNA gate, and (interestingly) also causes the closure of the NTP entrance gate and dsRNA exit gates (Figure 1G). Finally, the interaction of 227G with the Linker loop (part of N-terminal) might hinder dimerization of RdRp, thus impairing activity.

This study implies that, albeit an inhibitor is active against two closely related viral targets, the equivalence of the underlying mechanisms of action cannot be emphasized. As a consequence, although BVDV remains a useful surrogate model for identifying new HCV RdRp NIs [4], caution should be applied as far as NNIs are concerned. In fact, the concordance, in terms of the molecular mechanism of action, between drugs capable of inhibiting the replication of both BVDV and HCV is still largely unknown. Thus, as in the case of 227G, inhibition of both BVDV and HCV could be due only to serendipity and, the possibility of starting a successful rational design from data obtained in the surrogate model has to be proven.

 

shailendra asthana-1

Figure 1

A) Chemical structure of 2-(1-(2-(2,4-dimethoxyphenyl)-1H-benzimidazol-5-yl)ethylidene) hydrazinecarbothioamide (227G).

B) Structural overlay between representative complex structures of HCV (yellow) and BVDV (blue) as extracted from MD simulations. Proteins are shown with transparent tubes with Fingertip (HCV) and extended Fingertip (including Linker and Hood; BVDV [2, 3]) subdomains in solid cartoon representation. 227G is shown with vdW spheres with the same color as for the proteins. The regions common to both RdRps (Thumb, Palm, Finger domains and the Fingertip) are indicated by red labels, while those belonging to HCV and BVDV only are indicated by yellow and blue labels respectively. The different binding sites of 227G on the two enzymes are shown, i.e. l1-site in Thumb domain of HCV and template entrance-site (TE-site) in Finger domain of BVDV.

C) Superimposition between the conformation of the l1-loop in the Apo-enzyme (PDB-ID 1CSJ, red) and a representative structure of the complex extracted from the MD trajectory (227G·RdRp, yellow). The arrow indicates highlights the displacement of the l1-loop from its binding site on the Thumb domain (closed/active form of Apo-RdRp). The open conformation (227G·RdRp) corresponds to an inactive conformation of the enzyme..

D) Superimposition between the conformation of the Linker loop in the Apo-enzyme (PDB-ID 1S48, red) and a representative structure of the complex extracted from the MD trajectory (227G·RdRp, blue). The entrance gate for the RNA template (TE-site) is open in the active form of the enzyme (Apo). Upon binding to the enzyme 227G gets embraced by the four loops lining the TE-site. In particular, the Linker loop undergoes the most significant shift towards 227G (indicated by an arrow). As a result, the TE-site is occluded.

E) Thermodynamics of 227G binding to HCV and BVDV RdRps. The trend seen for the experimental EC50 and IC50 values (µM) is well reproduced by that of the calculated free energies of binding ∆G (kcal/mol).

F) Inhibition of HCV-RdRp by 227G. The Thumb (T), Finger (F) and Palm (P) domains are shown by transparent peach, yellow and orange transparent spheres, respectively, while 227G is represented by yellow vdW spheres. The functional region of enzyme involved in inhibition by 227G is shown in cartoon (red and yellow in the apo- and holo-enzymes respectively). Binding of the l1-loop (highlighted by an orange spot) to its site on the Thumb domain in the active form of RdRp  is supposed to coordinate (indicated by the orange double arrowed dashed line) the movement of that domain to that of the Finger domain, and thus is essential for polymerization [1]. In the holo-enzyme the loop is displaced by the binding of 227G, and the enzyme is forced into an open and inactive conformation, where coordinated motions are reduced or inhibited.

G) Inhibition of BVDV-RdRp by 227G. The representation is similar to panel F, with different colors. Binding of 227G closes the entrance of the RNA template channel.

 

References

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(3) Choi KH, Gallei A, Becher P, Rossmann MG 2006 The structure of bovine viral diarrhea virus RNA-dependent RNA polymerase and its amino-terminal domain. Structure 14, 1107−1113.

(4) Finkielsztein LM, Moltrasio GY, Caputto ME, Castro EF, Cavallaro LV, Moglioni AG 2010 What is Known About the Antiviral Agents Active Against Bovine Viral Diarrhea Virus (BVDV)? Curr. Med. Chem. 23, 2933−2955.

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