Structure-Based Discovery of Allosteric Inhibitors Targeting a New Druggable Site in the Respiratory Syncytial Virus Polymerase

Abstract

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory infections for which effective treatment options remain limited. Herein, we employed a computational structure-based design strategy aimed at identifying potential targets for a new class of allosteric inhibitors. Our investigation led to the discovery of a previously undisclosed allosteric binding site within the RSV polymerase, the large (L) protein. This discovery was achieved through a combination of virtual screening and molecular dynamics simulations. Subsequently, we identified two inhibitors, 6a and 10b, which both exhibited promising antiviral activity in the low micromolar range. Resistance profiling revealed a distinctive pattern in how RSV evaded treatment with this class of inhibitors. This pattern strongly suggested that this class of small molecules was targeting a new binding site in the RSV L protein, aligning with the computational predictions made in our study. This study paves the way for the development of more potent inhibitors for combating RSV infections by targeting a new druggable pocket within the RdRp which does not overlap with previously known resistance sites.

Figure 1

RSV L protein domains (domains are color-coded). The P protein tetramer is shown in red color, the RdRp domain in blue, and the capping domain in green (PDB ID 6PZK). The figure was generated using PyMOL. Reproduced with permission from ref (20). Copyright 2024 Elsevier.

Figure 2

(A) Structure of compound 1. (B) Flowchart of the virtual screening and computational investigations performed in this work.

Figure 3

Allosteric Pocket on the RSV L protein in complex with the P protein. (A) Druggable pockets identified in the L protein. (B) Surface representation of pocket 1 highlighting key pocket residues in red.

Figure 4

Putative docking pose of compound 1 (left) and 2D representation of the ligand–protein interactions (right). Predicted hydrogen bonds are shown as red dotted lines.

Figure 5

Summary of new derivatives design for SAR investigation as RSV RdRp inhibitors.

Figure 6

MD analysis of compound 1. (A) RMSD trends of compound 1 in complex with the L protein. (B) MD snapshot suggesting the putative binding pose of compound 1.

Figure 7

Binding poses of Group A compounds. (A) Chemical structures of compounds 6a and 6b from Group A. (B) RMSD of the backbone atoms of compound 6a in complex with L protein. (C) MD snapshot of compound 6a binding pose. (D) MD snapshot of compound 6b binding pose (hydrogen bonds are shown as red dashed lines).

Figure 8

Chemical Structure of compound 10b (left) and the lowest energy MD snapshot of the ligand–protein complex (right). Hydrogen bonds are depicted as red dashed lines.

Scheme 1. Synthesis of Group A Compounds

Reagents and conditions: (a) NaNO₂, aq. HCl, NaN₃, H₂O, 0 °C; (b) (i) trichloroacetyl chloride, methyl acetate, NaH, DCM, 0 °C (ii) N-methylimidazole, −45 °C, (iii) TiCl₄, Et₃N, −45 °C; (c) TsN₃, Et₃N, CH₃CN, rt; (d) 10 mol % Cu(OTf)₂, 5-hydroxyindole, DCM, rt.

Scheme 2. Synthesis of Intermediate Compound 9

Reagents and conditions: (i) glacial acetic acid, EtOH, reflux. (ii) Polyphosphoric acid, 120 °C.

Scheme 3. Synthesis of Group B Bis-indole Derivatives 10a–e

Reagents and conditions: 10 mol % Cu(OTf)₂, DCM, rt.

Figure 9

Effect of bis-indoles on RSV progeny production. (A) RSV viral progeny inhibition assay in the presence of the test compounds showing substantial inhibition of viral progeny in the presence of compound 6a (**** P < 0.0001). Viral titers were performed in triplicate and expressed as focus-forming units (FFU/mL) and compounds were tested at 10 μM concentration. (B) Dose response curve of compound 6a in RSV-GFP reporter assay. (C) Effect of group B compounds on viral infectivity. Viral expression is measured by GFP fluorescence 72 h postinfection. (D) Dose response curve of compound 10b in RSV-GFP reporter assay measured 48 h postinfection. EC₅₀ calculated through four-parameter variable slope regression modeling. Significance was determined by one-way ANOVA with Dunnett’s test of significance vs DMSO controls, **P = 0.0018, ****P < 0.0001.

Figure 10

Resistance mutants in the RSV L protein associated with Bis-indole treatment. (A) Schematic representation of the RSV L domains with resistance sites for the bis-indole compounds mapped on the RdRp domain (domains are color coded). (B) 3D model of the RSV L protein showing sites of the resistant mutants (red spheres) and the allosteric binding sites. Mutations are clustered around the computationally identified allosteric binding site in the RSV L protein.

Figure 11

Effect of the bis-indole chemotype induced mutations on the RSV L allosteric site. (A) Schematic diagram of the cryo-EM structure of the RSV L bound to the RNA template (PDB ID: 8SNX). The figure was generated using PyMOL. Reproduced with permission from ref (30). Copyright 2023 Springer Nature. Residues mutated in response to compound 1 treatment are shown as spheres and the RNA is shown as yellow sticks with hydrogen bond interactions shown as black dotted lines. (B) MDpocket analysis of pocket 1 pocket volume change from the wild-type RSV L (blue) and the mutated RSV L (red) throughout the 100 ns MD trajectory (representative 1000 frames from the whole trajectory (10000 frames) were used for the analysis). (C) RMSD analysis of the trajectory showing the stability of the mutated protein (orange) and the ligand (blue) showing a drastic change in the ligand’s RMSD from the starting coordinates.