EXCEED THE SPACE PROVIDED. The Nef gene of HIV-1 encodes a small dimeric protein essential for AIDS progression. Despite intensive investigation of Nef structure and function, few attempts have been made to target Nef or its downstream signaling partners with small molecule inhibitors. This application proposes novel high-throughput screens (HTS) to identify inhibitors of Nef signaling complexes. One major focus will be the protein-tyrosine I-Ick, a member of the Src kinase family selectively expressed in macrophages. Nef binding induces constitutive Hck kinase activation and contributes to survival signaling in macrophages, an HIV target cell and viral reservoir. In addition, genetic studies support a requirement for Hck in a mouse model of Nef-induced AIDS progression. Discovery of small- molecule inhibitors of Nef signaling will involve complementary in vitro and cell-based approaches:
Aim 1 : Test the hypothesis that the Hck-Nef complex represents a unique target for selective inhibitor discovery.
This Aim proposes a screen for selective inhibitors of the Hck-Nef complex in vitro. Use of the Hck-Nef complex as the source of active kinase in the assay will provide a rich source of molecular targets for inhibitor binding.
Aim 2 : Develop a yeast-based screen for conformationally selective inhibitors of the Hck-Nef complex. This approach will identify compounds that selectively bind to the inactive form of Hck prior to interaction with Nef.
Aim 3 : Develop a cell-based screen to identify inhibitors of Nef survival signaling in an HIV target cell lineage. This system will use an inducible form of Nef to identify selective inhibitors of Nef-dependent proliferation and survival signaling in a macrophage precursor cell line.
Aim 4 : Develop phosphoproteomics approaches to define the tyrosine ldnase signaling networks affected by Nef in myeloid cells and identify mechanisms of inhibitor action in vivo.
This Aim will employ high-resolution 2D gels, anti-phosphotyrosine immunoblotting and mass spectrometry to produce a global picture of Nef-tyrosine kinase signaling in macrophage precursor cells, providing a powerful tool to define the molecular targets for newly discovered small molecule inhibitors. Identification of a selective inhibitor of Hck-Nef signaling will provide an invaluable tool to test the role of this protein complex in AIDS progression as well as an important lead for future drug discovery. PERFORMANCE SITE ========================================Section End===========================================

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
Project #
Application #
Study Section
AIDS and Related Research 8 (AARR)
Program Officer
Miller, Roger H
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Pittsburgh
Schools of Medicine
United States
Zip Code
Moroco, Jamie A; Alvarado, John Jeff; Staudt, Ryan P et al. (2018) Remodeling of HIV-1 Nef Structure by Src-Family Kinase Binding. J Mol Biol 430:310-321
Shi, Jing; Xiong, Ran; Zhou, Tao et al. (2018) HIV-1 Nef Antagonizes SERINC5 Restriction by Downregulation of SERINC5 via the Endosome/Lysosome System. J Virol 92:
Wu, Mousheng; Alvarado, John J; Augelli-Szafran, Corinne E et al. (2018) A single ?-octyl glucoside molecule induces HIV-1 Nef dimer formation in the absence of partner protein binding. PLoS One 13:e0192512
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
Mujib, Shariq; Saiyed, Aamir; Fadel, Saleh et al. (2017) Pharmacologic HIV-1 Nef blockade promotes CD8 T cell-mediated elimination of latently HIV-1-infected cells in vitro. JCI Insight 2:
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; 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
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
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

Showing the most recent 10 out of 34 publications