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Study of Structures of CCR5 and Its Interactions with CCR5 Inhibitors

Hiroaki Mitsuya

2 Collaborator(s)

Funding source

National Cancer Institute (NIH)
We hypothesized that small molecules that bind to amino acid residues in the orthosteric binding site of CXCR4 would likely inhibit the CXCR4-gp120 interactions. To determine these residues, fifty-six amino acid residues in the extracellular and transmembrane regions of CXCR4 were selected for introducing amino acid substitution(s) and the HIV-1-gp120-elicited cell fusion levels were determined and compared to that of wild type CXCR4. The significance of some of the residues selected for substitution are as follows: Asp133, Arg134, and Tyr135 in TM-3 are the conserved DRY motif in various GPCRs and are reportedly important in triggering ligand-induced conformational changes that lead to receptor activation. The second extracellular loop (ECL2) is known to be important for the structure and function of CXCR4, CCR5 and other GPCRs; therefore, several residues in ECL2 were selected. Cys109 located in the extracellular region of TM3 forms a disulfide bond with Cys186 in ECL2. This disulfide linkage is conserved for class-A GPCRs. When we determined the changes in the magnitude of cell-cell fusion levels with wild type or mutant CXCR4, 11 amino acid(s) substitutions (D97A, Y116A, F174A, A175F, D182A, D187A, R188A, Y190A, D262A, E288A, and F292A) resulted in a substantial reduction in the fusion level by more than 30%. It is noteworthy that there are a number of negatively charged acidic residues (D97A, D182A, D187A, D262A, and E288A) whose substitution significantly decreased gp120 fusion. These acidic residues may interact with the basic residues of gp120 to affect co-receptor selectivity. We analyzed the crystal structures of CXCR4 to understand the interactions and orientation of the residues whose substitution adversely impacted the interactions of CXCR4 with the HIV-1-envelope protein. Six amino acid substitutions (F174A, A175F, D182A, D187A, R188A, and Y190A) that impacted the fusion event were identified in or near ECL2, strongly suggesting that this region as a whole probably affects fusion more than any other loop or transmembrane region of CXCR4. Site-directed mutagenesis studies have suggested that Asp262 is important for the binding of AMD3100. Glu288 (TM7) has a hydrogen bond with the side chain of Tyr116 (TM3) and an intra-helix hydrogen bond with Phe292 (TM7), and these three residues form part of the binding pocket within the transmembrane domain. The side chain of Asp97 forms part of the binding pocket for small molecules whereas the carboxylate side chain of Asp182 is oriented towards the extracellular region and away from the binding pocket located inside the transmembrane domain. Substitution of residues in TM5 or ECL3 did not affect the fusion event in the assay. We determined the shape similarity of the molecules, from the ChemBridge library to IT1t. The highest shape similarity Tanimoto coefficient to IT1t from the database was 0.85. Seven hundred fifty three (753) unique molecules with 1005 configurations/conformations had shape Tanimoto coefficient of at least 0.70. The binding mode and interactions of these molecules with CXCR4 were determined by molecular docking using Glide (version 5.6, Schrodinger, LLC). To avoid any issues that may arise through using conformations generated by Omega with Glide docking, Ligprep was used to generate molecular configurations and conformations for docking, and it generated 14,226 of those. These configurations/conformations were docked to the crystal structure of CXCR4 to determine their possible binding modes. Since it had been determined that CXCR4 transmembrane residues Asp97, Tyr116, Asp262, Glu288, Phe292 and ECL2 residues Phe174, Ala175, Asp182, Asp187, Arg188 and Tyr190 appeared to be important for the cell fusion event, we hypothesized that molecules that bound around the active site determined in the crystal structure, and formed hydrogen bond interactions with at least two of these residues are likely to competitively inhibit the interactions of CXCR4 with gp120. Our candidate molecule selection was based on shape similarity to a known inhibitor (IT1t) and putative binding mode and interactions with residues that were determined to be important for the HIV-1-gp120-elicited cell fusion event with CXCR4. Of note, we did not use any energy-based or empirical scoring functions for estimating the relative affinity for the selection of compounds. Based on the hypothesis described above, we selected sixteen compounds (named CX1 to CX16) for biological assays from ChemBridge general screening library (the minimum purity of the selected compounds was 90% or greater). Three most interesting compounds were piperidinylethanamine derivatives. When we asked whether the selected compounds bind to CXCR4 by blocking the intracellular Ca2+ mobilization induced by SDF-1alpha, the data strongly suggest that the PEA derivatives bind to CXCR4 with specificity and are antagonists of CXCR4. Subsequently, we newly synthesized CX6, and more than twenty PEA derivatives in high purity. The sixteen compounds were selected through docking simulations suggesting that they bound to the a potential orthosteric binding site of CXCR4, formed hydrogen bonds to at least two amino acid residues most likely to be important for the fusion event, and thereby have the potential to competitively inhibit the fusion event. We thus determined if the compounds were actually able to inhibit the interactions of the HIV-1NL4-3 envelope protein with CXCR4, and to block the fusion event in the HIV-1-gp120-elicited cell-cell fusion assays with the wild-type CXCR4. Both CX6 and CX11 blocked the fusion with IC50 values of 1.9 and 7.9 microM. Moreover, none of the compounds including CX6 and CX11 inhibited the fusion event as examined with the HIV-1-gp120-elicited cell-cell fusion assays using the cells expressing the wild-type CCR5-derived from HIV-1BaL (R5-HIV-1), suggesting that CX6 and CX11 inhibit the fusion event associated with CXCR4 but not with CCR5. When we examined the three dimensional shape overlay of CX6 with IT1t, as determined by ROCS (version 3.0.0, OpenEye Scientific Software, the molecules had an excellent shape Tanimoto overlap coefficient of 0.73. The imidazothiazole ring of IT1t overlays with the cyclopentylpiperidinyl group of CX6. The interactions of the identified hit compound (CX6) with CXCR4 were deduced by molecular docking. The following amino acid residues of CXCR4 were seen to form the active site for the binding of CX6. Thus, the molecule CX6 bound in the active site predominantly formed by residues from transmembranes 1, 2, 3, 7, and ECL2. As expected, no amino acid residues of TM4, TM5, and TM6 had interactions with CX6. The nitrogens of both piperidine groups were determined to be protonated. For CX6, the protonated nitrogen of cyclopentylpiperidinyl formed hydrogen bond interactions with Glu-288 and the protonated nitrogen of piperidinylethanamine formed hydrogen bond interactions with Asp97. The phenol group of CX6 interacted with Glu-32 located in the N-terminus of CXCR4. Asp97 has been shown to be important for the binding of AMD070 as well as for CXCR4-gp120-elicited fusion. Glu288 is important for the fusion event as it is shown that substitution of Glu288 with alanine results in loss of the CXCR4-gp120-elicited fusion. In sequence alignment, the residue corresponding to Glu288 of CXCR4 is Glu283 of CCR5. Therefore, E283 for CCR5 should be important for the binding of CCR5 antagonist aplaviroc and its analogs, in line with previously published results. Indeed, the substitution of E283 of CCR5 resulted in loss of the CCR5-gp120-elicited fusion event, as previously described.

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