Biochemical Characterization of CXCR4 Interactions With HIV -1, Natural Ligands and De Novo Designed Inhibitors

180 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2006.Chemokines and their receptors are implicated in a wide range of human diseases including Acquired Immune Deficiency Syndrome (AIDS) and HIV-associated dementia (HAD). The entry of human immunodeficiency virus type 1 (HIV-1) into the cell is initiated by the interaction of its surface envelope protein with two cell surface components of the target cell, CD4 and a chemokine coreceptor, usually CXCR4 or CCR5. The natural ligand of CXCR4 is stromal cell-derived factor (SDF)-1alpha. Whereas the overlap between HIV-1 and SDF-1alpha functional sites on the extracellular domains of CXCR4 has been well documented, it is yet to be determined whether there are sites in CXCR4 important for HIV-1 and/or SDF-1alpha functions and, if such sites do exist, whether they are overlapping or distinctive for the separate functions of CXCR4. In this study, we found that the mutations of many CXCR4 transmembrane (TM) and the second extracellular loop (ECL2) residues could selectively decrease HIV-1 mediated cell fusion without impairing the normal SDF-1alpha function, which may suggest a mechanistic basis for the discovery of new selective anti-HIV agents. In conjunction with the mutational analysis of CXCR4, the structure-function relationship of two important chemokines, SDF-1alpha and viral macrophage inflammatory protein (vMIP)-II, was studied. To enable the applications of chemokines as probes of receptor biology and inhibitors of pathological processes, a major problem with the lack of receptor selectivity of natural chemokines must be overcome. Here, we combined total protein synthesis with modular modifications to generate a new class of unnatural chemokines, termed SMM (S&barbelow;ynthetically and M&barbelow;odularly M&barbelow;odified)-chemokines, that were chemically engineered with significantly enhanced selectivity and potency for CXCR4 or CCR5, and improved pharmacological profiles. In addition to being valuable chemical probes of receptor biology to study ligand binding and signaling mechanisms, these novel molecules were shown to be promising leads for the development of anti-HIV and -HAD therapeutics, as they were more potent in blocking HIV-1 entry and infection and less toxic than natural chemokines. The mutational mapping analysis also revealed that SMM-chemokines, particularly those unnatural D-amino acid-containing analogs, share many CXCR4 binding sites with HIV-1 gp120 and yet differ from SDF-1alpha.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD