1. Determination of binding mode of newly developed CCR5 inhibitors with CCR5 and elucidation of structure-activity relationships. We previously examined the interactions between CCR5 and CCR5 inhibitors containing the SDP scaffold, AK530 and AK317, both of which were docked into the hydrophobic cavity located between the upper transmembrane domain and ECL2 of CCR5. The data showed the lipophilic potential mapped on the binding cavity of CCR5 and relative binding modes of AK530 and AK317. Molecular dynamics simulations for inhibitor-unbound CCR5 showed hydrogen bond interactions among transmembrane residues Y108, E283, and Y251, which were crucial for HIV-1-gp120/sCD4 complex binding and HIV-1 fusion. The data should not only help delineate the dynamics of CCR5 following inhibitor binding but also aid in designing CCR5 inhibitors that are more potent against HIV-1 and prevent or delay the emergence of resistant HIV-1 variants. We continued the design, synthesis, and evaluation of different CCR5 inhibitors in collaboration with Professor Ghosh of Purdue University. We initially started from a published CCR5 antagonist from the literature, and built structural models of its interaction with CCR5. Over the last five years, the design, synthesis and biological evaluation of more than 100 inhibitors has been carried out and several novel and potent inhibitors of CCR5 have been discovered in our study. Identified CCR5 antagonists include GRL-117, GRL-10007, and GRL-10018, which proved to be active against R5-HIV-1 with antiretroviral IC50 values of 0.6 nM, 1.4 nM and 2.9 nM, respectively. We built structural models of such newly identified CCR5 inhibitors to gain insight to the mechanism of potency of such inhibitors. In a model of the interaction of GRL117, GRL117 binds in a cavity formed within the transmembrane helices and ECL2. The models suggest that GRL117 has polar interactions with Y37, C178, K191, and T195. Y37 is located in transmembrane-1 (TM-1), K191 and T195 are located in TM-5. C178 is located in ECL2 and is highly conserved amongst class-A GPCRs. These residues are also important for gp120 fusion, and for the binding of APL and other CCR5 inhibitors. We postulated that interactions of inhibitors with CCR5 residues that are important for gp120 fusion may cause conformation change in CCR5, and may represent a salient molecular mechanism enabling allosteric inhibition. The interactions of GRL117 with such residues might be responsible for its potent antiretroviral IC50 of 0.6 nM. 2. Study of impacts of amino acid substitutions on CD4/gp120-CXCR4-induced fusion and identification of lead CXCR4 inhibitors. We attempted to examine the impacts of amino acid substitution on CD4/gp120-CXCR4-induced fusion by introducing amino acid substitutions into human wild-type CXCR4 (CXCR4WT) and determined the changes in fusion levels, compared to the fusion activity of CXCR4WT (referenced as 100%). We found that amino acid substitutions D97A, D262A, and E288A located in different transmembrane domains resulted in substantial loss of fusion activity. These residues have also been shown to be important for the binding of different CXCR4 inhibitors. As expected, various amino acid substitutions in ECL2, such as A175F, D182A, D187A, R188A and Y190A also resulted in compromised fusion. The availability of the crystal structure of CXCR4 with a small molecule inhibitor greatly aided our efforts to identify lead CXCR4 inhibitors. Out of an initial selection of 16 molecules with virtual screening of 622,000 different molecules, we identified cyclopentane-piperidine analogues as having anti-HIV activity (Das, Maeda, Hayashi, and Mitsuya: manuscript in preparation). 3. Optimization and development of potent CXCR4 inhibitors specific to CXCR4. CX-6 and other derivatives that inhibited HIV replication in MTT assay also blocked the biding of SDF-1alpha to CXCR4, suggesting that the derivatives inhibit HIV replication through CXCR4. Hence, we started collaboration with Professor Ghosh in optimizing the CX-6 scaffold. As of now, the group of Dr. Ghosh has synthesized more than 100 derivatives as candidates of CXCR4 inhibitors and all molecules were examined for their anti-HIV activity using MTT assay with X4-HIV (HIV-1NL4-3). Among them, 60 molecules that have cyclopentane-piperidine scaffold were tested and several compounds had decent anti-HIV activity with IC50 values of less than 1 micro M. We also started to synthesize molecules that have some other scaffolds as core structures. After optimization, we have identified several potent CXCR4 inhibitors that have tetrahydroquinoline scaffolds as a core structure. Among them, GRL-13004CX was most potent with IC50 value of 0.2 nM in MTT assay, followed by GRL-12066CX (IC50: 3 nM) and GRL-13006 (IC50: 3 nM). We also identified several compounds that have IC50 values less than 100 nM. GRL-13004CX and other derivatives were examined for other anti-HIV assays. They showed activity in MAGI assay and cell-cell fusion assay using X4-HIV-1 or an X4-HIV envelope (gp120), but they were not active when R5-HIV was used for MAGI assay, suggesting that these compounds are active only for X4-HIV-1. The ability of chemokine binding inhibition was also tested and it was found that all tested compounds inhibited the SDF-1alpha binding to CXCR4. From these findings, we concluded that the GRL-13004CX and other derivatives are CXCR4-spesific anti-HIV inhibitors. Moreover, it is considered that the potency of GRL-13004CX is the strongest among all CXCR4 inhibitors currently reported.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Investigator-Initiated Intramural Research Projects (ZIA)
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National Cancer Institute Division of Basic Sciences
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