We searched a general screening library from ChemBridgeTM, which has more than six hundred thousand molecules, to select molecules for assays as potential inhibitors of CXCR4. Previous studies have shown that determination of molecular similarity plays a critical role in analyzing large compound databases in chemical and pharmaceutical research. Therefore, we determined the shape similarity of the molecules, from the ChemBridgeTM library to IT1t, a molecule that binds with high affinity to CXCR4 and demonstrates anti-HIV-1 activity. Seven-hundred-fifty-three (753) unique molecules with one-thousand-five (1,005) configurations/conformations had a shape Tanimoto coefficient of at least 0.70. The binding mode and interactions of these molecules with CXCR4 were determined by molecular docking 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. Based on the hypothesis described above, we selected sixteen compounds (named CX1 to CX16) for biological assays from ChemBridgeTM general screening library. We asked whether the sixteen selected compounds bound to CXCR4 by blocking the intracellular Ca2+ mobilization induced by SDF-1alpha, whose primary receptor is CXCR4. One of the compounds, described as CX6 blocked the SDF-1alpha-induced Ca2+ mobilization in Molt4 cells with an EC50 value of 92 nM. In the same assay, AMD3100 showed inhibition of SDF-1alpha-induced Ca2+ mobilization with an EC50 value of around 10 nM. Two other compounds, CX11 and CX13, both piperidinylethanamine (PEA) derivatives like CX6, blocked SDF-1alpha-induced Ca2+ mobilization in Molt4 cells with EC50 values of 161 and 149 nM, respectively. None of the other compounds bound to CXCR4. Of note, all three PEA derivatives (CX6, CX11 and CX13) failed to block the Ca2+ mobilization induced with RANTES, whose receptor is CCR5, indicating that these compounds did not bind to CCR5. When we asked if these three compounds had agonistic effects to induce Ca2+ mobilization in CXCR4+ cells, none induced Ca2+ mobilization, suggesting that CX6, CX11, and CX13 were antagonists of CXCR4. In summary, the above data strongly suggest that the PEA derivatives bound to CXCR4 with specificity and were antagonists of CXCR4. The fusion event elicited by the interactions of HIV viral envelope glycoprotein with CXCR4 enables the virus to gain entry to cells, eventually leading to viral replication. The sixteen compounds were selected through docking simulations suggesting that they bound to a potential orthosteric binding site of CXCR4. These compounds 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 EC50 values of 1.9 microM 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). This suggested that CX6 and CX11 inhibited the fusion event associated with CXCR4 but not with CCR5. CX6 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 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. Glutamic acid at position 6 in TM7 is a highly conserved amino acid residue in chemokine receptors and has been demonstrated to be important for the binding of non-peptidic ligands to chemokine receptors. Our current study suggests that Glu288 is an important residue to target for rational structure based design and discovery of CXCR4 inhibitors. We determined the anti-HIV-1 activity and cytotoxicity of CX6 with the MTT assay using X4-HIV-1NL4-3 and MT4 as target cells. CX6 exerted substantial activity against HIV-1NL4-3 and HIV-1LAI, both of which are X4-HIV-1 strains, with EC50 values of 1.5 and 3.0 microM, respectively. The cytotoxicity CC50 of CX6 was 58 microM with a therapeutic index of 39. CX6 blocked the fusion event as examined in the fusion inhibition assay using HIV-1NL-4-3-derived envelope protein. In a Ca2+-flux inhibition assay, in which SDF-1alpha-elicited Ca2+-flux levels are determined in the presence or absence of a potential small molecule inhibitor, CX6 was found to block the flux at an EC50 value of 92 nM. However, as expected, CX6 failed to block the infectivity and replication of an R5-HIV-1 (HIV-1BaL)(EC50: 10 microM) as examined in an anti-HIV assay using HIV-1BaL and MAGI cells. CX11 and CX13 similarly exerted activity against X4-HIV-1NL4-3 and X4-HIV-1LAI, but failed to block the infectivity and replication of R5-HIV-1BaL.
