title: Insights into the mechanism of inhibition of CXCR4: identification of Piperidinylethanamine analogs as anti-HIV-1 inhibitors.
abstract: The cellular entry of HIV-1 into CD4(+) T cells requires ordered interactions of HIV-1 envelope glycoprotein with C-X-C chemokine receptor type 4 (CXCR4) receptors. However, such interactions, which should be critical for rational structure-based discovery of new CXCR4 inhibitors, remain poorly understood. Here we first determined the effects of amino acid substitutions in CXCR4 on HIV-1NL 4 - 3 glycoprotein-elicited fusion events using site-directed mutagenesis-based fusion assays and identified 11 potentially key amino acid substitutions, including D97A and E288A, which caused >30% reductions in fusion. We subsequently carried out a computational search of a screening library containing ∼604,000 compounds, in order to identify potential CXCR4 inhibitors. The computational search used the shape of IT1t, a known CXCR4 inhibitor, as a reference and employed various algorithms, including shape similarity, isomer generation, and docking against a CXCR4 crystal structure. Sixteen small molecules were identified for biological assays based on their high shape similarity to IT1t, and their putative binding modes formed hydrogen bond interactions with the amino acids identified above. Three compounds with piperidinylethanamine cores showed activity and were resynthesized. One molecule, designated CX6, was shown to significantly inhibit fusion elicited by X4 HIV-1NL 4 - 3 glycoprotein (50% inhibitory concentration [IC50], 1.9 μM), to inhibit Ca(2+) flux elicited by stromal cell-derived factor 1α (SDF-1α) (IC50, 92 nM), and to exert anti-HIV-1 activity (IC50, 1.5 μM). Structural modeling demonstrated that CX6 bound to CXCR4 through hydrogen bond interactions with Asp97 and Glu288. Our study suggests that targeting CXCR4 residues important for fusion elicited by HIV-1 envelope glycoprotein should be a useful and feasible approach to identifying novel CXCR4 inhibitors, and it provides important insights into the mechanism by which small-molecule CXCR4 inhibitors exert their anti-HIV-1 activities.
authors.affiliation: Experimental Retrovirology Section, HIV and AIDS Malignancy Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA dasd@mail.nih.gov.
authors.forename: Debananda
authors.initials: D
authors.lastname: Das
authors.affiliation: Experimental Retrovirology Section, HIV and AIDS Malignancy Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Kenji
authors.initials: K
authors.lastname: Maeda
authors.affiliation: Experimental Retrovirology Section, HIV and AIDS Malignancy Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Hayashi
authors.affiliation: Departments of Chemistry and Medicinal Chemistry, Purdue University, West Lafayette, Indiana, USA.
authors.forename: Navnath
authors.initials: N
authors.lastname: Gavande
authors.affiliation: Experimental Retrovirology Section, HIV and AIDS Malignancy Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Darshan V
authors.initials: DV
authors.lastname: Desai
authors.affiliation: Experimental Retrovirology Section, HIV and AIDS Malignancy Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Simon B
authors.initials: SB
authors.lastname: Chang
authors.affiliation: Departments of Chemistry and Medicinal Chemistry, Purdue University, West Lafayette, Indiana, USA.
authors.forename: Arun K
authors.initials: AK
authors.lastname: Ghosh
authors.affiliation: Experimental Retrovirology Section, HIV and AIDS Malignancy Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA Departments of Hematology and Infectious Diseases, Kumamoto University Graduate School of Medical and Pharmaceutical Sciences, Kumamoto, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Mitsuya
mesh_terms: D000465:Algorithms; D019943:Amino Acid Substitution; D019380:Anti-HIV Agents / Q000138:chemical synthesis* / Q000494:pharmacology*; D002470:Cell Survival / Q000187:drug effects; D023581:HIV Fusion Inhibitors / Q000138:chemical synthesis* / Q000494:pharmacology*; D015497:HIV-1 / Q000187:drug effects*; D057166:High-Throughput Screening Assays; D006801:Humans; D006860:Hydrogen Bonding; D016297:Mutagenesis, Site-Directed; D010880:Piperidines / Q000138:chemical synthesis* / Q000494:pharmacology*; D019718:Receptors, CXCR4 / Q000037:antagonists & inhibitors* / Q000737:chemistry; D054852:Small Molecule Libraries; D014759:Viral Envelope Proteins / Q000378:metabolism
chemical_list: D019380:Anti-HIV Agents; C000603508:CX6 compound; D023581:HIV Fusion Inhibitors; D010880:Piperidines; D019718:Receptors, CXCR4; D054852:Small Molecule Libraries; D014759:Viral Envelope Proteins