The HIV-1 accessory protein Nef is essential for high-titer viral replication and AIDS progression. Nef has no known catalytic activity, and functions by interacting with multiple host cell signaling proteins, including the Src-family kinases Hck, Lyn, and c-Src. During the previous funding period, we established in vitro and cell-based screens to identify small molecules that target the Nef: Hck complex. These screens identified two classes of drug-like compounds with low/sub-micromolar activity against Nef-dependent HIV replication in vitro, providing the first demonstration that small molecules targeting an HIV accessory protein-host cell protein complex have antiretroviral activity. In this renewal application, we will pursue these compounds in terms of their activity against a broader range of HIV strains, including strains resistant to clinical anti- retroviral drugs, in primary human host cells for HIV. We will also explore synergy of these Nef-directed compounds with established anti-HIV therapeutics and investigate their molecular mechanisms of action. The second major goal of this project is to test the hypothesis that the Nef oligomerization interface is a rational target for Nef-directed anti-HIV therapy. Previous studies have established that Nef oligomerization contributes to host cell kinase activation, as well as viral and immune receptor downregulation. Our own recent work shows that Nef mutations that interfere with oligomerization also prevent Nef-mediated enhancement of HIV replication in cell culture. Small molecule inhibitors of Nef oligomerization may therefore block critical Nef functions in HIV- infected cells, thus providing new scaffolds for anti-HIV drug development. A novel fluorescence complementation assay developed during the previous funding period that reports Nef oligomerization in live cells will serve as the basis for development of a high-content, cell-based chemical library screen to identify such compounds.
These studies are focused on discovery and characterization of small molecules that target a unique HIV-1 protein (Nef) that is essential for AIDS progression. Successful completion of these studies will provide new tools for understanding Nef interactions with host cell proteins and identify lead compounds for the development of a new class of anti-HIV therapeutics.
|Shu, Sherry T; Emert-Sedlak, Lori A; Smithgall, Thomas E (2017) Cell-based Fluorescence Complementation Reveals a Role for HIV-1 Nef Protein Dimerization in AP-2 Adaptor Recruitment and CD4 Co-receptor Down-regulation. J Biol Chem 292:2670-2678|
|Moroco, Jamie A; Alvarado, John Jeff; Staudt, Ryan P et al. (2017) Remodeling of HIV-1 Nef Structure by Src-Family Kinase Binding. J Mol Biol :|
|Wales, Thomas E; Poe, Jerrod A; Emert-Sedlak, Lori et al. (2016) Hydrogen Exchange Mass Spectrometry of Related Proteins with Divergent Sequences: A Comparative Study of HIV-1 Nef Allelic Variants. J Am Soc Mass Spectrom 27:1048-61|
|Emert-Sedlak, Lori A; Loughran, H Marie; Shi, Haibin et al. (2016) Synthesis and evaluation of orally active small molecule HIV-1 Nef antagonists. Bioorg Med Chem Lett 26:1480-1484|
|Wales, Thomas E; Hochrein, James M; Morgan, Christopher R et al. (2015) Subtle Dynamic Changes Accompany Hck Activation by HIV-1 Nef and are Reversed by an Antiretroviral Kinase Inhibitor. Biochemistry 54:6382-91|
|Bayer, Avraham; Delorme-Axford, Elizabeth; Sleigher, Christie et al. (2015) Human trophoblasts confer resistance to viruses implicated in perinatal infection. Am J Obstet Gynecol 212:71.e1-71.e8|
|Iyer, Prema C; Zhao, Jielu; Emert-Sedlak, Lori A et al. (2014) Synthesis and structure-activity analysis of diphenylpyrazolodiazene inhibitors of the HIV-1 Nef virulence factor. Bioorg Med Chem Lett 24:1702-6|
|Alvarado, John Jeff; Tarafdar, Sreya; Yeh, Joanne I et al. (2014) Interaction with the Src homology (SH3-SH2) region of the Src-family kinase Hck structures the HIV-1 Nef dimer for kinase activation and effector recruitment. J Biol Chem 289:28539-53|
|Tarafdar, Sreya; Poe, Jerrod A; Smithgall, Thomas E (2014) The accessory factor Nef links HIV-1 to Tec/Btk kinases in an Src homology 3 domain-dependent manner. J Biol Chem 289:15718-28|
|Moroco, Jamie A; Craigo, Jodi K; Iacob, Roxana E et al. (2014) Differential sensitivity of Src-family kinases to activation by SH3 domain displacement. PLoS One 9:e105629|
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