Identifying and Assessing Putative Allosteric Sites and Modulators for CXCR4 Predicted through Network Modeling and Site Identification by Ligand Competitive Saturation

The chemokine receptor CXCR4 is a critical target for the treatment of several cancer types and HIV-1 infections. While orthosteric and allosteric modulators have been developed targeting its extracellular or transmembrane regions, the intramembrane region of CXCR4 may also include allosteric binding sites suitable for the development of allosteric drugs. To investigate this, we apply the Gaussian Network Model (GNM) to the monomeric and dimeric forms of CXCR4 to identify residues essential for its local and global motions located in the hinge regions of the protein. Residue interaction network (RIN) analysis suggests hub residues that participate in allosteric communication throughout the receptor. Mutual residues from the network models reside in regions with a high capacity to alter receptor dynamics upon ligand binding. We then investigate the druggability of these potential allosteric regions using the site identification by ligand competitive saturation (SILCS) approach, revealing two putative allosteric sites on the monomer and three on the homodimer. Two screening campaigns with Glide and SILCS-Monte Carlo docking using FDA-approved drugs suggest 20 putative hit compounds including antifungal drugs, anticancer agents, HIV protease inhibitors, and antimalarial drugs. In vitro assays considering mAB 12G5 and CXCL12 demonstrate both positive and negative allosteric activities of these compounds, supporting our computational approach. However, in vivo functional assays based on the recruitment of β-arrestin to CXCR4 do not show significant agonism and antagonism at a single compound concentration. The present computational pipeline brings a new perspective to computer-aided drug design by combining conformational dynamics based on network analysis and cosolvent analysis based on the SILCS technology to identify putative allosteric binding sites using CXCR4 as a showcase.


Figure S28 .
Figure S28.RMSD, ligand interaction diagrams and interaction fractions of Itraconazole docked in DA3.

Figure S32 .
Figure S32.RMSD, ligand interaction diagrams and interaction fractions of Montelukast docked in DA3.

Figure S34 .
Figure S34.RMSD, ligand interaction diagrams and interaction fractions of Ritonavir docked in DA3.

Figure S42 .
Figure S42.(a) Potential allosteric binding site DA3 of monomer CXCR4 with the hit compound Itraconazole.Hotspots (b) occupied pockets of interest with selected fragments, and FragMaps (c) are shown with the protein.(d) Selected fragments are shown with the docked ligand.

Table S4 .
Binding site residues of each pocket.

Table S5 :
Docking scores, MM/GBSA energies and medical uses of the hit compounds for monomer CXCR4.

Table S6 :
Docking scores, MM/GBSA energies and medical uses of the hit compounds for homodimer CXCR4.

Table S7 :
SILCS Docking results and medical uses of the top 20 compounds on each allosteric site for monomer CXCR4.

Table S8 :
SILCS Docking results and medical uses of the top 20 compounds for homodimer CXCR4.

Table S9 :
SILCS Docking results and medical uses of the top 20 compounds on orthosteric site of CXCR4.

Table S10 :
ANOVA and Multiple Comparisons of 12G5 and CXCL12 Binding Data