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McCauley M, Huston M, Condren AR, Pereira F, Cline J, Yaple-Maresh M, Painter MM, Zimmerman GE, Robertson AW, Carney N, Goodall C, Terry V, Müller R, Sherman DH, Collins KL. Structure-Activity Relationships of Natural and Semisynthetic Plecomacrolides Suggest Distinct Pathways for HIV-1 Immune Evasion and Vacuolar ATPase-Dependent Lysosomal Acidification. J Med Chem 2024; 67:4483-4495. [PMID: 38452116 PMCID: PMC10978252 DOI: 10.1021/acs.jmedchem.3c01574] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/09/2024]
Abstract
The human immunodeficiency virus (HIV)-encoded accessory protein Nef enhances pathogenicity by reducing major histocompatibility complex I (MHC-I) cell surface expression, protecting HIV-infected cells from immune recognition. Nef-dependent downmodulation of MHC-I can be reversed by subnanomolar concentrations of concanamycin A (1), a well-known inhibitor of vacuolar ATPase, at concentrations below those that interfere with lysosomal acidification or degradation. We conducted a structure-activity relationship study that assessed 76 compounds for Nef inhibition, 24 and 72 h viability, and lysosomal neutralization in Nef-expressing primary T cells. This analysis demonstrated that the most potent compounds were natural concanamycins and their derivatives. Comparison against a set of new, semisynthetic concanamycins revealed that substituents at C-8 and acylation of C-9 significantly affected Nef potency, target cell viability, and lysosomal neutralization. These findings provide important progress toward understanding the mechanism of action of these compounds and the identification of an advanced lead anti-HIV Nef inhibitory compound.
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Affiliation(s)
- Morgan McCauley
- University of Michigan, Life Sciences Institute, Ann Arbor, MI 48109
| | - Matthew Huston
- University of Michigan, Department of Internal Medicine, Ann Arbor, MI 48109
| | - Alanna R. Condren
- University of Michigan, Life Sciences Institute, Ann Arbor, MI 48109
| | - Filipa Pereira
- University of Michigan, Life Sciences Institute, Ann Arbor, MI 48109
| | - Joel Cline
- University of Michigan, Department of Internal Medicine, Ann Arbor, MI 48109
| | | | - Mark M. Painter
- University of Michigan, Graduate Program in Immunology, Ann Arbor, MI 48109
| | | | - Andrew W. Robertson
- University of Michigan, Life Sciences Institute, Ann Arbor, MI 48109
- University of Michigan Natural Products Discovery Core, Life Sciences Institute, Ann Arbor, MI 48109
| | - Nolan Carney
- University of Michigan, Department of Chemistry, Ann Arbor, MI 48109
| | - Christopher Goodall
- University of Michigan, Department of Internal Medicine, Ann Arbor, MI 48109
| | - Valeri Terry
- University of Michigan, Department of Internal Medicine, Ann Arbor, MI 48109
| | - Rolf Müller
- Helmholtz Institute for Pharmaceutical Research Saarland, Saarbrücken, Germany 66123
| | - David H. Sherman
- University of Michigan, Department of Microbiology & Immunology, Ann Arbor, MI 48109
- University of Michigan, Life Sciences Institute, Ann Arbor, MI 48109
- University of Michigan, Department of Medicinal Chemistry, Ann Arbor, MI 48109
- University of Michigan, Department of Chemistry, Ann Arbor, MI 48109
| | - Kathleen L. Collins
- University of Michigan, Graduate Program in Immunology, Ann Arbor, MI 48109
- University of Michigan, Department of Internal Medicine, Ann Arbor, MI 48109
- University of Michigan, Department of Microbiology & Immunology, Ann Arbor, MI 48109
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Nzimande B, Makhwitine JP, Mkhwanazi NP, Ndlovu SI. Developments in Exploring Fungal Secondary Metabolites as Antiviral Compounds and Advances in HIV-1 Inhibitor Screening Assays. Viruses 2023; 15:v15051039. [PMID: 37243125 DOI: 10.3390/v15051039] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2023] [Revised: 04/16/2023] [Accepted: 04/19/2023] [Indexed: 05/28/2023] Open
Abstract
The emergence of drug-resistant Human Immunodeficiency Virus-1 strains against anti-HIV therapies in the clinical pipeline, and the persistence of HIV in cellular reservoirs remains a significant concern. Therefore, there is a continuous need to discover and develop new, safer, and effective drugs targeting novel sites to combat HIV-1. The fungal species are gaining increasing attention as alternative sources of anti-HIV compounds or immunomodulators that can escape the current barriers to cure. Despite the potential of the fungal kingdom as a source for diverse chemistries that can yield novel HIV therapies, there are few comprehensive reports on the progress made thus far in the search for fungal species with the capacity to produce anti-HIV compounds. This review provides insights into the recent research developments on natural products produced by fungal species, particularly fungal endophytes exhibiting immunomodulatory or anti-HIV activities. In this study, we first explore currently existing therapies for various HIV-1 target sites. Then we assess the various activity assays developed for gauging antiviral activity production from microbial sources since they are crucial in the early screening phases for discovering novel anti-HIV compounds. Finally, we explore fungal secondary metabolites compounds that have been characterized at the structural level and demonstrate their potential as inhibitors of various HIV-1 target sites.
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Affiliation(s)
- Bruce Nzimande
- Discipline of Medical Microbiology, School of Laboratory Medicine and Medical Sciences, Medical School, University of KwaZulu-Natal, Durban 4000, South Africa
| | - John P Makhwitine
- Discipline of Medical Microbiology, School of Laboratory Medicine and Medical Sciences, Medical School, University of KwaZulu-Natal, Durban 4000, South Africa
| | - Nompumelelo P Mkhwanazi
- HIV Pathogenesis Programme, Doris Duke Medical Research Institute, School of Laboratory Medicine and Medical Sciences, University of KwaZulu-Natal, Durban 4000, South Africa
| | - Sizwe I Ndlovu
- Department of Biotechnology and Food Technology, Doornfontein Campus, University of Johannesburg, Johannesburg 2028, South Africa
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3
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Heinrich F, Thomas CE, Alvarado JJ, Eells R, Thomas A, Doucet M, Whitlatch KN, Aryal M, Lösche M, Smithgall TE. Neutron Reflectometry and Molecular Simulations Demonstrate HIV-1 Nef Homodimer Formation on Model Lipid Bilayers. J Mol Biol 2023; 435:168009. [PMID: 36773691 PMCID: PMC10079580 DOI: 10.1016/j.jmb.2023.168009] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2022] [Revised: 01/16/2023] [Accepted: 02/03/2023] [Indexed: 02/12/2023]
Abstract
The HIV-1 Nef protein plays a critical role in viral infectivity, high-titer replication in vivo, and immune escape of HIV-infected cells. Nef lacks intrinsic biochemical activity, functioning instead through interactions with diverse host cell signaling proteins and intracellular trafficking pathways. Previous studies have established an essential role for Nef homodimer formation at the plasma membrane for most if not all its functions. Here we combined neutron reflectometry of full-length myristoylated Nef bound to model lipid bilayers with molecular simulations based on previous X-ray crystal structures of Nef homodimers. This integrated approach provides direct evidence that Nef associates with the membrane as a homodimer with its structured core region displaced from the membrane for partner protein engagement. Parallel studies of a dimerization-defective mutant, Nef-L112D, demonstrate that the helical dimerization interface present in previous crystal structures stabilizes the membrane-bound dimer. X-ray crystallography of the Nef-L112D mutant in complex with the SH3 domain of the Nef-associated host cell kinase Hck revealed a monomeric 1:1 complex instead of the 2:2 dimer complex formed with wild-type Nef. Importantly, the crystal structure of the Nef-L112D core and SH3 interface are virtually identical to the wild-type complex, indicating that this mutation does not affect the overall Nef fold. These findings support the intrinsic capacity of Nef to homodimerize at lipid bilayers using structural features present in X-ray crystal structures of dimeric complexes.
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Affiliation(s)
- Frank Heinrich
- Department of Physics, Carnegie Mellon University, Pittsburgh, PA 15213, USA; NIST Center for Neutron Research, Gaithersburg, MD 20899, USA
| | - Catherine E Thomas
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA
| | - John J Alvarado
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA
| | - Rebecca Eells
- Department of Physics, Carnegie Mellon University, Pittsburgh, PA 15213, USA
| | - Alyssa Thomas
- Department of Physics, Carnegie Mellon University, Pittsburgh, PA 15213, USA
| | - Mathieu Doucet
- Neutron Scattering Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA
| | - Kindra N Whitlatch
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA
| | - Manish Aryal
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA
| | - Mathias Lösche
- Department of Physics, Carnegie Mellon University, Pittsburgh, PA 15213, USA; NIST Center for Neutron Research, Gaithersburg, MD 20899, USA
| | - Thomas E Smithgall
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA.
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4
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Majumder S, Deganutti G, Pipitò L, Chaudhuri D, Datta J, Giri K. Computer-aided de novo design and optimization of novel potential inhibitors of HIV-1 Nef protein. Comput Biol Chem 2023; 104:107871. [PMID: 37084691 DOI: 10.1016/j.compbiolchem.2023.107871] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2022] [Revised: 04/07/2023] [Accepted: 04/14/2023] [Indexed: 04/23/2023]
Abstract
Nef is a small accessory protein pivotal in the HIV-1 viral replication cycle. It is a multifunctional protein and its interactions with kinases in host cells have been well characterized through many in vitro and structural studies. Nef forms a homodimer to activate the kinases and subsequently the phosphorylation pathways. The disruption of its homodimerization represents a valuable approach in the search for novel classes of antiretroviral. However, this research avenue is still underdeveloped as just a few Nef inhibitors have been reported so far, with very limited structural information about their mechanism of action. To address this issue, we have employed an in silico structure-based drug design strategy that combines de novo ligand design with molecular docking and extensive molecular dynamics simulations. Since the Nef pocket involved in homodimerization has high lipophilicity, the initial de novo-designed structures displayed poor drug-likeness and solubility. Taking information from the hydration sites within the homodimerization pocket, structural modifications in the initial lead compound have been introduced to improve the solubility and drug-likeness, without affecting the binding profile. We propose lead compounds that can be the starting point for further optimizations to deliver long-awaited, rationally designed Nef inhibitors.
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Affiliation(s)
| | - Giuseppe Deganutti
- Centre for Sport, Exercise, and Life Sciences, Coventry University, Coventry CV1 5FB, UK
| | - Ludovico Pipitò
- Centre for Sport, Exercise, and Life Sciences, Coventry University, Coventry CV1 5FB, UK
| | | | - Joyeeta Datta
- Department of Life Sciences, Presidency University, Kolkata, India
| | - Kalyan Giri
- Department of Life Sciences, Presidency University, Kolkata, India.
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5
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Aryal M, Lin D, Regan K, Du S, Shi H, Alvarado JJ, Ilina TV, Andreotti AH, Smithgall TE. The HIV-1 protein Nef activates the Tec family kinase Btk by stabilizing an intermolecular SH3-SH2 domain interaction. Sci Signal 2022; 15:eabn8359. [PMID: 36126115 PMCID: PMC9830684 DOI: 10.1126/scisignal.abn8359] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
Abstract
The Nef protein produced by the viruses HIV-1 and SIV drives efficient viral replication partially by inducing constitutive activation of host cell tyrosine kinases, including members of the Src and Tec families. Here, we uncovered the mechanism by which both HIV-1 and SIV Nef enhanced the activity of the Tec family kinase Btk in vitro and in cells. A Nef mutant that could not bind to the SH3 domain of Src family kinases activated Btk to the same extent as did wild-type Nef, demonstrating that Nef activated Src and Tec family kinases by distinct mechanisms. The Btk SH3-SH2 region formed a homodimer requiring the CD loop in the SH2 domain, which was stabilized by the binding of Nef homodimers. Alanine substitution of Pro327 in the CD loop of the Btk SH2 domain destabilized SH3-SH2 dimers, abolished the interaction with Nef, and prevented activation by Nef in vitro. In cells, Nef stabilized and activated wild-type but not P327A Btk homodimers at the plasma membrane. These data reveal that the interaction with Nef stabilizes Btk dimers through the SH3-SH2 interface to promote kinase activity and show that the HIV-1 Nef protein evolved distinct mechanisms to activate Src and Tec family tyrosine kinases to enhance viral replication.
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Affiliation(s)
- Manish Aryal
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh PA, 15219 USA
| | - David Lin
- Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA, 50011 USA
| | - Kiera Regan
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh PA, 15219 USA
| | - Shoucheng Du
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh PA, 15219 USA
| | - Haibin Shi
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh PA, 15219 USA
| | - John J. Alvarado
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh PA, 15219 USA
| | - Tatiana V. Ilina
- Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh PA, 15260 USA
| | - Amy H. Andreotti
- Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA, 50011 USA
| | - Thomas E. Smithgall
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh PA, 15219 USA
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6
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Emert-Sedlak LA, Shi H, Tice CM, Chen L, Alvarado JJ, Shu ST, Du S, Thomas CE, Wrobel JE, Reitz AB, Smithgall TE. Antiretroviral Drug Discovery Targeting the HIV-1 Nef Virulence Factor. Viruses 2022; 14:v14092025. [PMID: 36146831 PMCID: PMC9503669 DOI: 10.3390/v14092025] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2022] [Revised: 09/06/2022] [Accepted: 09/08/2022] [Indexed: 11/16/2022] Open
Abstract
While antiretroviral drugs have transformed the lives of HIV-infected individuals, chronic treatment is required to prevent rebound from viral reservoir cells. People living with HIV also are at higher risk for cardiovascular and neurocognitive complications, as well as cancer. Finding a cure for HIV-1 infection is therefore an essential goal of current AIDS research. This review is focused on the discovery of pharmacological inhibitors of the HIV-1 Nef accessory protein. Nef is well known to enhance HIV-1 infectivity and replication, and to promote immune escape of HIV-infected cells by preventing cell surface MHC-I display of HIV-1 antigens. Recent progress shows that Nef inhibitors not only suppress HIV-1 replication, but also restore sufficient MHC-I to the surface of infected cells to trigger a cytotoxic T lymphocyte response. Combining Nef inhibitors with latency reversal agents and therapeutic vaccines may provide a path to clearance of viral reservoirs.
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Affiliation(s)
- Lori A. Emert-Sedlak
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA
| | - Haibin Shi
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA
| | - Colin M. Tice
- Fox Chase Chemical Diversity Center, Inc., Pennsylvania Biotechnology Center, Doylestown, PA 18902, USA
| | - Li Chen
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA
| | - John J. Alvarado
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA
| | - Sherry T. Shu
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA
| | - Shoucheng Du
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA
| | - Catherine E. Thomas
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA
| | - Jay E. Wrobel
- Fox Chase Chemical Diversity Center, Inc., Pennsylvania Biotechnology Center, Doylestown, PA 18902, USA
| | - Allen B. Reitz
- Fox Chase Chemical Diversity Center, Inc., Pennsylvania Biotechnology Center, Doylestown, PA 18902, USA
| | - Thomas E. Smithgall
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA
- Correspondence:
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Biradar S, Agarwal Y, Lotze MT, Bility MT, Mailliard RB. The BLT Humanized Mouse Model as a Tool for Studying Human Gamma Delta T Cell-HIV Interactions In Vivo. Front Immunol 2022; 13:881607. [PMID: 35669780 PMCID: PMC9164110 DOI: 10.3389/fimmu.2022.881607] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2022] [Accepted: 04/15/2022] [Indexed: 11/22/2022] Open
Abstract
Gamma-delta (γδ) T cells recognize antigens in a major histocompatibility complex (MHC) independent and have cytotoxic capability. Human immunodeficiency virus (HIV) infection reduces the proportion of the Vδ2 cell subset compared to the Vδ1 cell subset of γδ T cells in the blood in most infected individuals, except for elite controllers. The capacity of Vδ2 T cells to kill HIV-infected targets has been demonstrated in vitro, albeit in vivo confirmatory studies are lacking. Here, we provide the first characterization of γδ T cell-HIV interactions in bone marrow-liver-thymus (BLT) humanized mice and examined the immunotherapeutic potential of Vδ2 T cells in controlling HIV replication in vivo. We demonstrate a reduced proportion of Vδ2 T cells and an increased proportion of Vδ1 T cells in HIV-infected BLT humanized mice, like in HIV-positive individuals. HIV infection in BLT humanized mice also impaired the ex vivo expansion of Vδ2 T cells, like in HIV-positive individuals. Adoptive transfer of activated Vδ2 T cells did not control HIV replication during cell-associated HIV transmission in BLT humanized mice but instead exacerbated viremia, suggesting that Vδ2 T cells may serve as early targets for HIV replication. Our findings demonstrate that BLT humanized mice can model γδ T cell-HIV interactions in vivo.
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Affiliation(s)
- Shivkumar Biradar
- Department of Infectious Diseases and Microbiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA, United States
| | - Yash Agarwal
- Department of Infectious Diseases and Microbiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA, United States
| | - Michael T. Lotze
- Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States
- Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States
- Department of Bioengineering, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States
| | - Moses T. Bility
- Department of Infectious Diseases and Microbiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA, United States
| | - Robbie B. Mailliard
- Department of Infectious Diseases and Microbiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA, United States
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8
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Emert-Sedlak LA, Moukha-Chafiq O, Shi H, Du S, Alvarado JJ, Pathak V, Tanner SG, Hunter RN, Nebane M, Chen L, Ilina TV, Ishima R, Zhang S, Kuzmichev YV, Wonderlich ER, Schader SM, Augelli-Szafran CE, Ptak RG, Smithgall TE. Inhibitors of HIV-1 Nef-Mediated Activation of the Myeloid Src-Family Kinase Hck Block HIV-1 Replication in Macrophages and Disrupt MHC-I Downregulation. ACS Infect Dis 2022; 8:91-105. [PMID: 34985256 PMCID: PMC9274903 DOI: 10.1021/acsinfecdis.1c00288] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023]
Abstract
HIV-1 Nef is an attractive target for antiretroviral drug discovery because of its role in promoting HIV-1 infectivity, replication, and host immune system avoidance. Here, we applied a screening strategy in which recombinant HIV-1 Nef protein was coupled to activation of the Src-family tyrosine kinase Hck, which enhances the HIV-1 life cycle in macrophages. Nef stimulates recombinant Hck activity in vitro, providing a robust assay for chemical library screening. High-throughput screening of more than 730 000 compounds using the Nef·Hck assay identified six unique hit compounds that bound directly to recombinant Nef by surface plasmon resonance (SPR) in vitro and inhibited HIV-1 replication in primary macrophages in the 0.04 to 5 μM range without cytotoxicity. Eighty-four analogs were synthesized around an isothiazolone scaffold from this series, many of which bound to recombinant Nef and inhibited HIV-1 infectivity in the low to submicromolar range. Compounds in this series restored MHC-I to the surface of HIV-infected primary cells and disrupted a recombinant protein complex of Nef with the C-terminal tail of MHC-I and the μ1 subunit of the AP-1 endocytic trafficking protein. Nef inhibitors in this class have the potential to block HIV-1 replication in myeloid cells and trigger recognition of HIV-infected cells by the adaptive immune system in vivo.
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Affiliation(s)
- Lori A. Emert-Sedlak
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, 450 Technology Drive, Pittsburgh, PA 15219
| | - Omar Moukha-Chafiq
- Department of Chemistry, Southern Research Institute, 2000 Ninth Avenue South, Birmingham, AL 35205
| | - Haibin Shi
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, 450 Technology Drive, Pittsburgh, PA 15219
| | - Shoucheng Du
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, 450 Technology Drive, Pittsburgh, PA 15219
| | - John J. Alvarado
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, 450 Technology Drive, Pittsburgh, PA 15219
| | - Vibha Pathak
- Department of Chemistry, Southern Research Institute, 2000 Ninth Avenue South, Birmingham, AL 35205
| | - Samuel G. Tanner
- Department of Chemistry, Southern Research Institute, 2000 Ninth Avenue South, Birmingham, AL 35205
| | - Robert N. Hunter
- Department of Chemistry, Southern Research Institute, 2000 Ninth Avenue South, Birmingham, AL 35205
| | - Miranda Nebane
- Department of High-throughput Screening, Drug Discovery Division, Southern Research Institute, 2000 Ninth Avenue South, Birmingham, AL 35205
| | - Li Chen
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, 450 Technology Drive, Pittsburgh, PA 15219
| | - Tatiana V. Ilina
- Department of Structural Biology, University of Pittsburgh School of Medicine, 3501 Fifth Avenue, Pittsburgh, PA 15260
| | - Rieko Ishima
- Department of Structural Biology, University of Pittsburgh School of Medicine, 3501 Fifth Avenue, Pittsburgh, PA 15260
| | - Sixue Zhang
- Department of High-throughput Screening, Drug Discovery Division, Southern Research Institute, 2000 Ninth Avenue South, Birmingham, AL 35205
| | - Yury V. Kuzmichev
- Department of Infectious Disease Research, Drug Development Division, Southern Research Institute, 431 Aviation Way, Frederick, MD 21701
| | - Elizabeth R. Wonderlich
- Department of Infectious Disease Research, Drug Development Division, Southern Research Institute, 431 Aviation Way, Frederick, MD 21701
| | - Susan M. Schader
- Department of Infectious Disease Research, Drug Development Division, Southern Research Institute, 431 Aviation Way, Frederick, MD 21701
| | | | - Roger G. Ptak
- Department of Infectious Disease Research, Drug Development Division, Southern Research Institute, 431 Aviation Way, Frederick, MD 21701
| | - Thomas E. Smithgall
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, 450 Technology Drive, Pittsburgh, PA 15219
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Shu ST, Li WF, Smithgall TE. Visualization of Host Cell Kinase Activation by Viral Proteins Using GFP Fluorescence Complementation and Immunofluorescence Microscopy. Bio Protoc 2021; 11:e4068. [PMID: 34327265 DOI: 10.21769/bioprotoc.4068] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2020] [Revised: 03/29/2021] [Accepted: 04/01/2021] [Indexed: 11/02/2022] Open
Abstract
Non-receptor protein-tyrosine kinases regulate cellular responses to many external signals and are important drug discovery targets for cancer and infectious diseases. While many assays exist for the assessment of kinase activity in vitro, methods that report changes in tyrosine kinase activity in single cells have the potential to provide information about kinase responses at the cell population level. In this protocol, we combined bimolecular fluorescence complementation (BiFC), an established method for the assessment of protein-protein interactions, and immunofluorescence staining with phosphospecific antibodies to characterize changes in host cell tyrosine kinase activity in the presence of an HIV-1 virulence factor, Nef. Specifically, two Tec family kinases (Itk and Btk) as well as Nef were fused to complementary, non-fluorescent fragments of the Venus variant of YFP. Each kinase was expressed in 293T cells in the presence or absence of Nef and immunostained for protein expression and activity with anti-phosphotyrosine (pTyr) antibodies. Multi-color confocal microscopy revealed the interaction of Nef with each kinase (BiFC), kinase activity, and kinase protein expression. Strong BiFC signals were observed when Nef was co-expressed with both Itk and Btk, indicative of interaction, and a strong anti-pTyr immunoreactivity was also seen. The BiFC, pTyr, and kinase expression signals co-localized to the plasma membrane, consistent with Nef-mediated kinase activation in this subcellular compartment. Image analysis allowed calculation of pTyr-to-kinase protein ratios, which showed a range of responses in individual cells across the population that shifted upward in the presence of Nef and back down in the presence of a kinase inhibitor. This method has the potential to reveal changes in steady-state non-receptor tyrosine kinase activity and subcellular localization in a cell population in response to other protein-kinase interactions, information that is not attainable from immunoblotting or other in vitro methods.
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Affiliation(s)
- Sherry T Shu
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA
| | - Wing Fai Li
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA
| | - Thomas E Smithgall
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA
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10
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Adzhubei AA, Kulkarni A, Tolstova AP, Anashkina AA, Sviridov D, Makarov AA, Bukrinsky MI. Direct interaction between ABCA1 and HIV-1 Nef: Molecular modeling and virtual screening for inhibitors. Comput Struct Biotechnol J 2021; 19:3876-3884. [PMID: 34584633 PMCID: PMC8440812 DOI: 10.1016/j.csbj.2021.06.050] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2021] [Revised: 06/23/2021] [Accepted: 06/30/2021] [Indexed: 12/18/2022] Open
Abstract
HIV-1 infection impairs cellular cholesterol efflux by downmodulating the cholesterol transporter ABCA1, leading to metabolic co-morbidities like cardio-vascular disease. The main mechanism of this effect is impairment by the HIV-1 protein Nef of the ABCA1 interaction with the endoplasmic reticulum chaperone calnexin, which leads to a block in ABCA1 maturation followed by its degradation. However, ABCA1 is also downmodulated by Nef delivered with the extracellular vesicles, suggesting involvement of a direct Nef:ABCA1 interaction at the plasma membrane. Here, we present an optimized model of the Nef:ABCA1 interaction, which identifies interaction sites and provides an opportunity to perform a virtual screening for potential inhibitors. Interestingly, the predicted sites on Nef involved in the ABCA1 interaction overlap with those involved in the interaction with calnexin. The compounds previously shown to block Nef:calnexin interaction were among the top ranking ligands in docking simulations with ABCA1-interacting sites on Nef, suggesting the possibility that both interactions can be inhibited by the same chemical compounds. This study identifies a series of compounds for potential development as inhibitors of Nef-mediated co-morbidities of HIV infection.
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Affiliation(s)
- Alexei A. Adzhubei
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia
| | - Amol Kulkarni
- Howard University College of Pharmacy, Washington, District of Columbia, USA
| | - Anna P. Tolstova
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia
| | | | - Dmitri Sviridov
- Baker Heart and Diabetes Institute, Melbourne, Victoria, Australia
- Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia
| | - Alexander A. Makarov
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia
| | - Michael I. Bukrinsky
- The George Washington University School of Medicine and Health Sciences, Washington, District of Columbia, USA
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11
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Raghuvanshi R, Bharate SB. Recent Developments in the Use of Kinase Inhibitors for Management of Viral Infections. J Med Chem 2021; 65:893-921. [PMID: 33539089 DOI: 10.1021/acs.jmedchem.0c01467] [Citation(s) in RCA: 29] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Kinases are a group of therapeutic targets involved in the progression of numerous diseases, including cancer, rheumatoid arthritis, Alzheimer's disease, and viral infections. The majority of approved antiviral agents are inhibitors of virus-specific targets that are encoded by individual viruses. These inhibitors are narrow-spectrum agents that can cause resistance development. Viruses are dependent on host cellular proteins, including kinases, for progression of their life-cycle. Thus, targeting kinases is an important therapeutic approach to discovering broad-spectrum antiviral agents. As there are a large number of FDA approved kinase inhibitors for various indications, their repurposing for viral infections is an attractive and time-sparing strategy. Many kinase inhibitors, including baricitinib, ruxolitinib, imatinib, tofacitinib, pacritinib, zanubrutinib, and ibrutinib, are under clinical investigation for COVID-19. Herein, we discuss FDA approved kinase inhibitors, along with a repertoire of clinical/preclinical stage kinase inhibitors that possess antiviral activity or are useful in the management of viral infections.
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Affiliation(s)
- Rinky Raghuvanshi
- Medicinal Chemistry Division,CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu 180001, India.,Academy of Scientific & Innovative Research, Ghaziabad 201002, India
| | - Sandip B Bharate
- Medicinal Chemistry Division,CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu 180001, India.,Academy of Scientific & Innovative Research, Ghaziabad 201002, India
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12
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Abstract
Viruses commonly antagonize the antiviral type I interferon response by targeting signal transducer and activator of transcription 1 (STAT1) and STAT2, key mediators of interferon signaling. Other STAT family members mediate signaling by diverse cytokines important to infection, but their relationship with viruses is more complex. Importantly, virus-STAT interaction can be antagonistic or stimulatory depending on diverse viral and cellular factors. While STAT antagonism can suppress immune pathways, many viruses promote activation of specific STATs to support viral gene expression and/or produce cellular conditions conducive to infection. It is also becoming increasingly clear that viruses can hijack noncanonical STAT functions to benefit infection. For a number of viruses, STAT function is dynamically modulated through infection as requirements for replication change. Given the critical role of STATs in infection by diverse viruses, the virus-STAT interface is an attractive target for the development of antivirals and live-attenuated viral vaccines. Here, we review current understanding of the complex and dynamic virus-STAT interface and discuss how this relationship might be harnessed for medical applications.
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13
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Staudt RP, Smithgall TE. Nef homodimers down-regulate SERINC5 by AP-2-mediated endocytosis to promote HIV-1 infectivity. J Biol Chem 2020; 295:15540-15552. [PMID: 32873704 DOI: 10.1074/jbc.ra120.014668] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2020] [Revised: 08/24/2020] [Indexed: 12/13/2022] Open
Abstract
SERINC5 is a multipass intrinsic membrane protein that suppresses HIV-1 infectivity when incorporated into budding virions. The HIV-1 Nef virulence factor prevents viral incorporation of SERINC5 by triggering its down-regulation from the producer cell membrane through an AP-2-dependent endolysosomal pathway. However, the mechanistic basis for SERINC5 down-regulation by Nef remains elusive. Here we demonstrate that Nef homodimers are important for SERINC5 down-regulation, trafficking to late endosomes, and exclusion from newly synthesized viral particles. Based on previous X-ray crystal structures, we mutated three conserved residues in the Nef dimer interface (Leu112, Tyr115, and Phe121) and demonstrated attenuated homodimer formation in a cell-based fluorescence complementation assay. Point mutations at each position reduced the infectivity of HIV-1 produced from transfected 293T cells, the Jurkat TAg T-cell line, and donor mononuclear cells in a SERINC5-dependent manner. In SERINC5-transfected 293T cells, virion incorporation of SERINC5 was increased by dimerization-defective Nef mutants, whereas down-regulation of SERINC5 from the membrane of transfected Jurkat cells by these mutants was significantly reduced. Nef dimer interface mutants also failed to trigger internalization of SERINC5 and localization to Rab7+ late endosomes in T cells. Importantly, fluorescence complementation assays demonstrated that dimerization-defective Nef mutants retained interaction with both SERINC5 and AP-2. These results show that down-regulation of SERINC5 and subsequent enhancement of viral infectivity require Nef homodimers and support a mechanism by which the Nef dimer bridges SERINC5 to AP-2 for endocytosis. Pharmacological disruption of Nef homodimers may control HIV-1 infectivity and viral spread by enhancing virion incorporation of SERINC5.
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Affiliation(s)
- Ryan P Staudt
- Department of Microbiology and Molecular Genetics, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania, USA
| | - Thomas E Smithgall
- Department of Microbiology and Molecular Genetics, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania, USA.
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14
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Staudt RP, Alvarado JJ, Emert-Sedlak LA, Shi H, Shu ST, Wales TE, Engen JR, Smithgall TE. Structure, function, and inhibitor targeting of HIV-1 Nef-effector kinase complexes. J Biol Chem 2020; 295:15158-15171. [PMID: 32862141 DOI: 10.1074/jbc.rev120.012317] [Citation(s) in RCA: 37] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2020] [Revised: 08/28/2020] [Indexed: 11/06/2022] Open
Abstract
Antiretroviral therapy has revolutionized the treatment of AIDS, turning a deadly disease into a manageable chronic condition. Life-long treatment is required because existing drugs do not eradicate HIV-infected cells. The emergence of drug-resistant viral strains and uncertain vaccine prospects highlight the pressing need for new therapeutic approaches with the potential to clear the virus. The HIV-1 accessory protein Nef is essential for viral pathogenesis, making it a promising target for antiretroviral drug discovery. Nef enhances viral replication and promotes immune escape of HIV-infected cells but lacks intrinsic enzymatic activity. Instead, Nef works through diverse interactions with host cell proteins primarily related to kinase signaling pathways and endosomal trafficking. This review emphasizes the structure, function, and biological relevance of Nef interactions with host cell protein-tyrosine kinases in the broader context of Nef functions related to enhancement of the viral life cycle and immune escape. Drug discovery targeting Nef-mediated kinase activation has allowed identification of promising inhibitors of multiple Nef functions. Pharmacological inhibitors of Nef-induced MHC-I down-regulation restore the adaptive immune response to HIV-infected cells in vitro and have the potential to enhance immune recognition of latent viral reservoirs as part of a strategy for HIV clearance.
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Affiliation(s)
- Ryan P Staudt
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
| | - John J Alvarado
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
| | - Lori A Emert-Sedlak
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
| | - Haibin Shi
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
| | - Sherry T Shu
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
| | - Thomas E Wales
- Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts, USA
| | - John R Engen
- Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts, USA
| | - Thomas E Smithgall
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
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15
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Li WF, Aryal M, Shu ST, Smithgall TE. HIV-1 Nef dimers short-circuit immune receptor signaling by activating Tec-family kinases at the host cell membrane. J Biol Chem 2020; 295:5163-5174. [PMID: 32144207 DOI: 10.1074/jbc.ra120.012536] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/03/2020] [Revised: 03/04/2020] [Indexed: 01/08/2023] Open
Abstract
The HIV-1 virulence factor Nef promotes high-titer viral replication, immune escape, and pathogenicity. Nef interacts with interleukin-2-inducible T-cell kinase (Itk) and Bruton's tyrosine kinase (Btk), two Tec-family kinases expressed in HIV-1 target cells (CD4 T cells and macrophages, respectively). Using a cell-based bimolecular fluorescence complementation assay, here we demonstrate that Nef recruits both Itk and Btk to the cell membrane and induces constitutive kinase activation in transfected 293T cells. Nef homodimerization-defective mutants retained their interaction with both kinases but failed to induce activation, supporting a role for Nef homodimer formation in the activation mechanism. HIV-1 infection up-regulates endogenous Itk activity in SupT1 T cells and donor-derived peripheral blood mononuclear cells. However, HIV-1 strains expressing Nef variants with mutations in the dimerization interface replicated poorly and were significantly attenuated in Itk activation. We conclude that direct activation of Itk and Btk by Nef at the membrane in HIV-infected cells may override normal immune receptor control of Tec-family kinase activity to enhance the viral life cycle.
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Affiliation(s)
- Wing Fai Li
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, 450 Technology Drive, Pittsburgh, Pennsylvania 15219
| | - Manish Aryal
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, 450 Technology Drive, Pittsburgh, Pennsylvania 15219
| | - Sherry T Shu
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, 450 Technology Drive, Pittsburgh, Pennsylvania 15219
| | - Thomas E Smithgall
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, 450 Technology Drive, Pittsburgh, Pennsylvania 15219
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16
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Shi H, Tice CM, Emert-Sedlak L, Chen L, Li WF, Carlsen M, Wrobel JE, Reitz AB, Smithgall TE. Tight-Binding Hydroxypyrazole HIV-1 Nef Inhibitors Suppress Viral Replication in Donor Mononuclear Cells and Reverse Nef-Mediated MHC-I Downregulation. ACS Infect Dis 2020; 6:302-312. [PMID: 31775511 DOI: 10.1021/acsinfecdis.9b00382] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
Abstract
The HIV-1 Nef accessory factor is critical to the viral life cycle in vivo and promotes immune escape of infected cells via downregulation of cell-surface MHC-I. Previously, we discovered small molecules that bind directly to Nef and block many of its functions, including enhancement of viral infectivity and replication in T cell lines. These compounds also restore cell-surface MHC-I expression in HIV-infected CD4 T cells from AIDS patients, enabling recognition and killing by autologous cytotoxic T lymphocytes (CTLs). In this study, we describe the synthesis and evaluation of a diverse set of analogs based on the original hydroxypyrazole Nef inhibitor core. All analogs were screened for the interaction with recombinant HIV-1 Nef by surface plasmon resonance (SPR) and for antiretroviral activity in TZM-bl reporter cells infected with HIV-1. Active analogs were ranked on the basis of an activity score that integrates three aspects of the SPR data (affinity, residence time, and extent of binding) with antiretroviral activity. The top scoring compounds bound tightly to Nef by SPR, with KD values in the low nM to pM range, and displayed very slow dissociation from their Nef target. These analogs also suppressed HIV-1 replication in donor peripheral blood mononuclear cells (PBMCs) with IC50 values in the 1-10 nM range without cytotoxicity, inhibited Nef-mediated IL-2-inducible tyrosine kinase (Itk) and hematopoietic cell kinase (Hck) activation, and rescued MHC-I downregulation in a Nef-transfected T cell line. The development of Nef inhibitors based on the structure-activity relationships defined here has promise as a new approach to antiretroviral therapy that includes a path to eradication of HIV-infected cells via the adaptive immune response.
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Affiliation(s)
- Haibin Shi
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Suite 523 Bridgeside Point II, 450 Technology Drive, Pittsburgh, Pennsylvania 15219, United States
| | - Colin M. Tice
- Fox Chase Chemical Diversity Center, Inc., Pennsylvania Biotechnology Center, 3805 Old Easton Road, Doylestown, Pennsylvania 18902, United States
| | - Lori Emert-Sedlak
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Suite 523 Bridgeside Point II, 450 Technology Drive, Pittsburgh, Pennsylvania 15219, United States
| | - Li Chen
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Suite 523 Bridgeside Point II, 450 Technology Drive, Pittsburgh, Pennsylvania 15219, United States
| | - Wing Fai Li
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Suite 523 Bridgeside Point II, 450 Technology Drive, Pittsburgh, Pennsylvania 15219, United States
| | - Marianne Carlsen
- Fox Chase Chemical Diversity Center, Inc., Pennsylvania Biotechnology Center, 3805 Old Easton Road, Doylestown, Pennsylvania 18902, United States
| | - Jay E. Wrobel
- Fox Chase Chemical Diversity Center, Inc., Pennsylvania Biotechnology Center, 3805 Old Easton Road, Doylestown, Pennsylvania 18902, United States
| | - Allen B. Reitz
- Fox Chase Chemical Diversity Center, Inc., Pennsylvania Biotechnology Center, 3805 Old Easton Road, Doylestown, Pennsylvania 18902, United States
| | - Thomas E. Smithgall
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Suite 523 Bridgeside Point II, 450 Technology Drive, Pittsburgh, Pennsylvania 15219, United States
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17
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Dorman HR, Close D, Wingert BM, Camacho CJ, Johnston PA, Smithgall TE. Discovery of Non-peptide Small Molecule Allosteric Modulators of the Src-family Kinase, Hck. Front Chem 2019; 7:822. [PMID: 31850311 PMCID: PMC6893557 DOI: 10.3389/fchem.2019.00822] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2019] [Accepted: 11/12/2019] [Indexed: 01/18/2023] Open
Abstract
The eight mammalian Src-family tyrosine kinases are dynamic, multi-domain structures, which adopt distinct “open” and “closed” conformations. In the closed conformation, the regulatory SH3 and SH2 domains pack against the back of the kinase domain, providing allosteric control of kinase activity. Small molecule ligands that engage the regulatory SH3-SH2 region have the potential to modulate Src-family kinase activity for therapeutic advantage. Here we describe an HTS-compatible fluorescence polarization assay to identify small molecules that interact with the unique-SH3-SH2-linker (U32L) region of Hck, a Src-family member expressed exclusively in cells of myeloid lineage. Hck has significant potential as a drug target in acute myeloid leukemia, an aggressive form of cancer with substantial unmet clinical need. The assay combines recombinant Hck U32L protein with a fluorescent probe peptide that binds to the SH3 domain in U32L, resulting in an increased FP signal. Library compounds that interact with the U32L protein and interfere with probe binding reduce the FP signal, scoring as hits. Automated 384-well high-throughput screening of 60,000 compounds yielded Z'-factor coefficients > 0.7 across nearly 200 assay plates, and identified a series of hit compounds with a shared pyrimidine diamine substructure. Surface plasmon resonance assays confirmed direct binding of hit compounds to the Hck U32L target protein as well as near-full-length Hck. Binding was not observed with the individual SH3 and SH2 domains, demonstrating that these compounds recognize a specific three-dimensional conformation of the regulatory regions. This conclusion is supported by computational docking studies, which predict ligand contacts with a pocket formed by the juxtaposition of the SH3 domain, the SH3-SH2 domain connector, and the SH2-kinase linker. Each of the four validated hits stimulated recombinant, near-full-length Hck activity in vitro, providing evidence for allosteric effects on the kinase domain. These results provide a path to discovery and development of chemical scaffolds to target the regulatory regions of Hck and other Src family kinases as a new approach to pharmacological kinase control.
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Affiliation(s)
- Heather R Dorman
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States
| | - David Close
- Department of Pharmaceutical Sciences, University of Pittsburgh School of Pharmacy, Pittsburgh, PA, United States
| | - Bentley M Wingert
- Department of Computational and Systems Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States
| | - Carlos J Camacho
- Department of Computational and Systems Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States
| | - Paul A Johnston
- Department of Pharmaceutical Sciences, University of Pittsburgh School of Pharmacy, Pittsburgh, PA, United States
| | - Thomas E Smithgall
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States
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18
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Liu B, Zhang X, Zhang W, Wu L, Jing S, Liu W, Xia B, Zou F, Lu L, Ma X, He D, Hu Q, Zhang Y, Deng K, Cai W, Tang X, Peng T, Zhang H, Li L. Lovastatin Inhibits HIV-1-Induced MHC-I Downregulation by Targeting Nef-AP-1 Complex Formation: A New Strategy to Boost Immune Eradication of HIV-1 Infected Cells. Front Immunol 2019; 10:2151. [PMID: 31572371 PMCID: PMC6749138 DOI: 10.3389/fimmu.2019.02151] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2019] [Accepted: 08/27/2019] [Indexed: 01/05/2023] Open
Abstract
Current combined antiretroviral therapy (cART) mainly targets 3 of the 15 HIV proteins leaving many potential viral vulnerabilities unexploited. To purge the HIV-1 latent reservoir, various strategies including “shock and kill” have been developed. A key question is how to restore impaired immune surveillance. HIV-1 protein Nef has long been known to mediate the downregulation of cell-surface MHC-I and assist HIV-1 to evade the immune system. Through high throughput screening of Food and Drug Administration (FDA) approved drugs, we identified lovastatin, a statin drug, to significantly antagonize Nef to downregulate MHC-I, CD4, and SERINC5, and inhibit the intrinsic infectivity of virions. In addition, lovastatin boosted autologous CTLs to eradicate the infected cells and effectively inhibit the subsequent viral rebound in CD4+ T-lymphocytes isolated from HIV-1-infected individuals receiving suppressive cART. Furthermore, we found that lovastatin inhibits Nef-induced MHC-I downregulation by directly binding with Nef and disrupting the Nef–AP-1 complex. These results demonstrate that lovastatin is a promising agent for counteracting Nef-mediated downregulation of MHC-I, CD4, and SERINC5. Lovastatin could potentially be used in the clinic to enhance anti-HIV-1 immune surveillance.
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Affiliation(s)
- Bingfeng Liu
- Key Laboratory of Tropical Disease Control of Ministry of Education, Guangdong Engineering Research Center for Antimicrobial Agent and Immunotechnology, Zhongshan School of Medicine, Institute of Human Virology, Sun Yat-sen University, Guangzhou, China.,Department of Infectious Diseases, Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou, China
| | - Xu Zhang
- Sino-French Hoffmann Institute, Guangzhou Medical University, Guangzhou, China
| | - Wanying Zhang
- Key Laboratory of Tropical Disease Control of Ministry of Education, Guangdong Engineering Research Center for Antimicrobial Agent and Immunotechnology, Zhongshan School of Medicine, Institute of Human Virology, Sun Yat-sen University, Guangzhou, China
| | - Liyang Wu
- Key Laboratory of Tropical Disease Control of Ministry of Education, Guangdong Engineering Research Center for Antimicrobial Agent and Immunotechnology, Zhongshan School of Medicine, Institute of Human Virology, Sun Yat-sen University, Guangzhou, China
| | - Shuliang Jing
- Key Laboratory of Tropical Disease Control of Ministry of Education, Guangdong Engineering Research Center for Antimicrobial Agent and Immunotechnology, Zhongshan School of Medicine, Institute of Human Virology, Sun Yat-sen University, Guangzhou, China
| | - Weiwei Liu
- Key Laboratory of Tropical Disease Control of Ministry of Education, Guangdong Engineering Research Center for Antimicrobial Agent and Immunotechnology, Zhongshan School of Medicine, Institute of Human Virology, Sun Yat-sen University, Guangzhou, China
| | - Baijin Xia
- Key Laboratory of Tropical Disease Control of Ministry of Education, Guangdong Engineering Research Center for Antimicrobial Agent and Immunotechnology, Zhongshan School of Medicine, Institute of Human Virology, Sun Yat-sen University, Guangzhou, China
| | - Fan Zou
- Key Laboratory of Tropical Disease Control of Ministry of Education, Guangdong Engineering Research Center for Antimicrobial Agent and Immunotechnology, Zhongshan School of Medicine, Institute of Human Virology, Sun Yat-sen University, Guangzhou, China.,Department of Molecular Therapy, Qianyang Biomedical Research Institute, Guangzhou, China.,Guangzhou Women and Children Hospital, Institute of Pediatrics, Guangzhou Medical University, Guangzhou, China
| | - Lijuan Lu
- Key Laboratory of Tropical Disease Control of Ministry of Education, Guangdong Engineering Research Center for Antimicrobial Agent and Immunotechnology, Zhongshan School of Medicine, Institute of Human Virology, Sun Yat-sen University, Guangzhou, China
| | - Xiancai Ma
- Key Laboratory of Tropical Disease Control of Ministry of Education, Guangdong Engineering Research Center for Antimicrobial Agent and Immunotechnology, Zhongshan School of Medicine, Institute of Human Virology, Sun Yat-sen University, Guangzhou, China
| | - Dalian He
- Key Laboratory of Tropical Disease Control of Ministry of Education, Guangdong Engineering Research Center for Antimicrobial Agent and Immunotechnology, Zhongshan School of Medicine, Institute of Human Virology, Sun Yat-sen University, Guangzhou, China
| | - Qifei Hu
- Key Laboratory of Tropical Disease Control of Ministry of Education, Guangdong Engineering Research Center for Antimicrobial Agent and Immunotechnology, Zhongshan School of Medicine, Institute of Human Virology, Sun Yat-sen University, Guangzhou, China.,Department of Molecular Therapy, Qianyang Biomedical Research Institute, Guangzhou, China
| | - Yiwen Zhang
- Key Laboratory of Tropical Disease Control of Ministry of Education, Guangdong Engineering Research Center for Antimicrobial Agent and Immunotechnology, Zhongshan School of Medicine, Institute of Human Virology, Sun Yat-sen University, Guangzhou, China
| | - Kai Deng
- Key Laboratory of Tropical Disease Control of Ministry of Education, Guangdong Engineering Research Center for Antimicrobial Agent and Immunotechnology, Zhongshan School of Medicine, Institute of Human Virology, Sun Yat-sen University, Guangzhou, China
| | - Weiping Cai
- Department of Infectious Diseases, Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou, China
| | - Xiaoping Tang
- Department of Infectious Diseases, Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou, China
| | - Tao Peng
- Sino-French Hoffmann Institute, Guangzhou Medical University, Guangzhou, China
| | - Hui Zhang
- Key Laboratory of Tropical Disease Control of Ministry of Education, Guangdong Engineering Research Center for Antimicrobial Agent and Immunotechnology, Zhongshan School of Medicine, Institute of Human Virology, Sun Yat-sen University, Guangzhou, China.,Department of Molecular Therapy, Qianyang Biomedical Research Institute, Guangzhou, China
| | - Linghua Li
- Department of Infectious Diseases, Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou, China
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19
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Wu M, Alvarado JJ, Augelli-Szafran CE, Ptak RG, Smithgall TE. A single β-octyl glucoside molecule induces HIV-1 Nef dimer formation in the absence of partner protein binding. PLoS One 2018; 13:e0192512. [PMID: 29415006 PMCID: PMC5802939 DOI: 10.1371/journal.pone.0192512] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2017] [Accepted: 01/24/2018] [Indexed: 01/05/2023] Open
Abstract
The HIV-1 Nef accessory protein is essential for viral pathogenicity and AIDS progression. Nef forms complexes with multiple host cell factors to facilitate viral replication and promote immune escape of HIV-infected cells. Previous X-ray crystal structures demonstrate that Nef forms homodimers, the orientation of which are influenced by host cell binding partners. In cell-based fluorescence complementation assays, Nef forms homodimers at the plasma membrane. However, recombinant Nef proteins often exist as monomers in solution, suggesting that membrane interaction may also trigger monomer to dimer transitions. In this study, we show that monomeric Nef core proteins can be induced to form dimers in the presence of low concentrations of the non-ionic surfactant, β-octyl glucoside (βOG). X-ray crystallography revealed that a single βOG molecule is present in the Nef dimer, with the 8-carbon acyl chain of the ligand binding to a hydrophobic pocket formed by the dimer interface. This Nef-βOG dimer interface involves helix αB, as observed in previous dimer structures, as well as a helix formed by N-terminal residues 54-66. Nef dimer formation is stabilized in solution by the addition of βOG, providing biochemical validation for the crystal structure. These observations together suggest that the interaction with host cell lipid mediators or other hydrophobic ligands may play a role in Nef dimerization, which has been previously linked to multiple Nef functions including host cell protein kinase activation, CD4 downregulation, and enhancement of HIV-1 replication.
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Affiliation(s)
- Mousheng Wu
- Department of Chemistry, Drug Discovery Division, Southern Research Institute, Birmingham, AL, United States of America
| | - John J. Alvarado
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States of America
| | - Corinne E. Augelli-Szafran
- Department of Chemistry, Drug Discovery Division, Southern Research Institute, Birmingham, AL, United States of America
| | - Roger G. Ptak
- Department of Infectious Disease Research, Southern Research Institute, Frederick, MD, United States of America
| | - Thomas E. Smithgall
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States of America
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20
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Moroco JA, Alvarado JJ, Staudt RP, Shi H, Wales TE, Smithgall TE, Engen JR. Remodeling of HIV-1 Nef Structure by Src-Family Kinase Binding. J Mol Biol 2018; 430:310-321. [PMID: 29258818 PMCID: PMC5801098 DOI: 10.1016/j.jmb.2017.12.008] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/14/2017] [Revised: 12/07/2017] [Accepted: 12/10/2017] [Indexed: 11/25/2022]
Abstract
The HIV-1 accessory protein Nef controls multiple aspects of the viral life cycle and host immune response, making it an attractive therapeutic target. Previous X-ray crystal structures of Nef in complex with key host cell binding partners have shed light on protein-protein interactions critical to Nef function. Crystal structures of Nef in complex with either the SH3 or tandem SH3-SH2 domains of Src-family kinases reveal distinct dimer conformations of Nef. However, the existence of these Nef dimer complexes in solution has not been established. Here we used hydrogen exchange mass spectrometry (HX MS) to compare the solution conformation of Nef alone and in complexes with the SH3 or the SH3-SH2 domains of the Src-family kinase Hck. HX MS revealed that interaction with the Hck SH3 or tandem SH3-SH2 domains induces protection of the Nef αB-helix from deuterium uptake, consistent with a role for αB in dimer formation. HX MS analysis of a Nef mutant (position Asp123, a site buried in the Nef:SH3 dimer but surface exposed in the Nef:SH3-SH2 complex), showed a Hck-induced conformational change in Nef relative to wild-type Nef. These results support a model in which Src-family kinase binding induces conformational changes in Nef to expose residues critical for interaction with the μ1 subunit of adaptor protein 1 and the major histocompatibility complex-1 tail, and subsequent major histocompatibility complex-1 downregulation and immune escape of HIV-infected cells required for functional interactions with downstream binding partners.
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Affiliation(s)
- Jamie A Moroco
- Department of Chemistry and Chemical Biology, Maildrop 412TF, Northeastern University, 360 Huntington Avenue, Boston, MA 02115, USA.
| | - John Jeff Alvarado
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Bridgeside Point II, Suite 523, 450 Technology Drive, Pittsburgh, PA 15219, USA.
| | - Ryan P Staudt
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Bridgeside Point II, Suite 523, 450 Technology Drive, Pittsburgh, PA 15219, USA.
| | - Haibin Shi
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Bridgeside Point II, Suite 523, 450 Technology Drive, Pittsburgh, PA 15219, USA.
| | - Thomas E Wales
- Department of Chemistry and Chemical Biology, Maildrop 412TF, Northeastern University, 360 Huntington Avenue, Boston, MA 02115, USA.
| | - Thomas E Smithgall
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Bridgeside Point II, Suite 523, 450 Technology Drive, Pittsburgh, PA 15219, USA.
| | - John R Engen
- Department of Chemistry and Chemical Biology, Maildrop 412TF, Northeastern University, 360 Huntington Avenue, Boston, MA 02115, USA.
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21
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HIV-I Nef inhibitors: a novel class of HIV-specific immune adjuvants in support of a cure. AIDS Res Ther 2017; 14:53. [PMID: 28893294 PMCID: PMC5594582 DOI: 10.1186/s12981-017-0175-6] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/21/2017] [Accepted: 08/11/2017] [Indexed: 11/22/2022] Open
Abstract
The success of many current vaccines relies on a formulation that incorporates an immune activating adjuvant. This will hold true for the design of a successful therapeutic HIV vaccine targeted at controlling reactivated virus following cessation of combined antiretroviral therapy (cART). The HIV accessory protein Nef functions by interfering with HIV antigen presentation through the major histocompatibility complex I (MHC-I) pathway thereby suppressing CD8+ cytotoxic T cell (CTL)-mediated killing of HIV infected cells. Thus, this important impediment to HIV vaccine success must be circumvented. This review covers our current knowledge of Nef inhibitors that may serve as immune adjuvants that will specifically restore and enhance CTL-mediated killing of reactivated HIV infected cells as part of an overall vaccine strategy to affect a cure for HIV infection.
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Milhas S, Raux B, Betzi S, Derviaux C, Roche P, Restouin A, Basse MJ, Rebuffet E, Lugari A, Badol M, Kashyap R, Lissitzky JC, Eydoux C, Hamon V, Gourdel ME, Combes S, Zimmermann P, Aurrand-Lions M, Roux T, Rogers C, Müller S, Knapp S, Trinquet E, Collette Y, Guillemot JC, Morelli X. Protein-Protein Interaction Inhibition (2P2I)-Oriented Chemical Library Accelerates Hit Discovery. ACS Chem Biol 2016; 11:2140-8. [PMID: 27219844 DOI: 10.1021/acschembio.6b00286] [Citation(s) in RCA: 28] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
Protein-protein interactions (PPIs) represent an enormous source of opportunity for therapeutic intervention. We and others have recently pinpointed key rules that will help in identifying the next generation of innovative drugs to tackle this challenging class of targets within the next decade. We used these rules to design an oriented chemical library corresponding to a set of diverse "PPI-like" modulators with cores identified as privileged structures in therapeutics. In this work, we purchased the resulting 1664 structurally diverse compounds and evaluated them on a series of representative protein-protein interfaces with distinct "druggability" potential using homogeneous time-resolved fluorescence (HTRF) technology. For certain PPI classes, analysis of the hit rates revealed up to 100 enrichment factors compared with nonoriented chemical libraries. This observation correlates with the predicted "druggability" of the targets. A specific focus on selectivity profiles, the three-dimensional (3D) molecular modes of action resolved by X-ray crystallography, and the biological activities of identified hits targeting the well-defined "druggable" bromodomains of the bromo and extraterminal (BET) family are presented as a proof-of-concept. Overall, our present study illustrates the potency of machine learning-based oriented chemical libraries to accelerate the identification of hits targeting PPIs. A generalization of this method to a larger set of compounds will accelerate the discovery of original and potent probes for this challenging class of targets.
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Affiliation(s)
- Sabine Milhas
- CNRS, INSERM, Aix-Marseille Université, Institut Paoli-Calmettes, Centre de Recherche en Cancérologie de Marseille, CS30059, 13273 Marseille Cedex 9, France
- CNRS, Aix-Marseille
Université, Screening Platform AD2P, AFMB UMR7257, 13288, Marseille, France
| | - Brigitt Raux
- CNRS, INSERM, Aix-Marseille Université, Institut Paoli-Calmettes, Centre de Recherche en Cancérologie de Marseille, CS30059, 13273 Marseille Cedex 9, France
| | - Stéphane Betzi
- CNRS, INSERM, Aix-Marseille Université, Institut Paoli-Calmettes, Centre de Recherche en Cancérologie de Marseille, CS30059, 13273 Marseille Cedex 9, France
| | - Carine Derviaux
- CNRS, INSERM, Aix-Marseille Université, Institut Paoli-Calmettes, Centre de Recherche en Cancérologie de Marseille, CS30059, 13273 Marseille Cedex 9, France
| | - Philippe Roche
- CNRS, INSERM, Aix-Marseille Université, Institut Paoli-Calmettes, Centre de Recherche en Cancérologie de Marseille, CS30059, 13273 Marseille Cedex 9, France
| | - Audrey Restouin
- CNRS, INSERM, Aix-Marseille Université, Institut Paoli-Calmettes, Centre de Recherche en Cancérologie de Marseille, CS30059, 13273 Marseille Cedex 9, France
| | - Marie-Jeanne Basse
- CNRS, INSERM, Aix-Marseille Université, Institut Paoli-Calmettes, Centre de Recherche en Cancérologie de Marseille, CS30059, 13273 Marseille Cedex 9, France
| | - Etienne Rebuffet
- CNRS, INSERM, Aix-Marseille Université, Institut Paoli-Calmettes, Centre de Recherche en Cancérologie de Marseille, CS30059, 13273 Marseille Cedex 9, France
| | - Adrien Lugari
- CNRS, INSERM, Aix-Marseille Université, Institut Paoli-Calmettes, Centre de Recherche en Cancérologie de Marseille, CS30059, 13273 Marseille Cedex 9, France
| | - Marion Badol
- Cisbio Bioassays, R&D, Parc Marcel Boiteux, BP 84175, 30200 Codolet, France
| | - Rudra Kashyap
- CNRS, INSERM, Aix-Marseille Université, Institut Paoli-Calmettes, Centre de Recherche en Cancérologie de Marseille, CS30059, 13273 Marseille Cedex 9, France
- Department of Human Genetics, KU Leuven, B-3000 Leuven, Belgium
| | - Jean-Claude Lissitzky
- CNRS, INSERM, Aix-Marseille Université, Institut Paoli-Calmettes, Centre de Recherche en Cancérologie de Marseille, CS30059, 13273 Marseille Cedex 9, France
| | - Cécilia Eydoux
- CNRS, Aix-Marseille
Université, Screening Platform AD2P, AFMB UMR7257, 13288, Marseille, France
| | - Véronique Hamon
- CNRS, INSERM, Aix-Marseille Université, Institut Paoli-Calmettes, Centre de Recherche en Cancérologie de Marseille, CS30059, 13273 Marseille Cedex 9, France
| | | | - Sébastien Combes
- CNRS, INSERM, Aix-Marseille Université, Institut Paoli-Calmettes, Centre de Recherche en Cancérologie de Marseille, CS30059, 13273 Marseille Cedex 9, France
| | - Pascale Zimmermann
- CNRS, INSERM, Aix-Marseille Université, Institut Paoli-Calmettes, Centre de Recherche en Cancérologie de Marseille, CS30059, 13273 Marseille Cedex 9, France
- Department of Human Genetics, KU Leuven, B-3000 Leuven, Belgium
| | - Michel Aurrand-Lions
- CNRS, INSERM, Aix-Marseille Université, Institut Paoli-Calmettes, Centre de Recherche en Cancérologie de Marseille, CS30059, 13273 Marseille Cedex 9, France
| | - Thomas Roux
- Cisbio Bioassays, R&D, Parc Marcel Boiteux, BP 84175, 30200 Codolet, France
| | - Catherine Rogers
- Target
Discovery Institute, University of Oxford, NDM Research Building, Roosevelt
Drive, Oxford OX3 7FZ, U.K
- Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ, U.K
| | - Susanne Müller
- Target
Discovery Institute, University of Oxford, NDM Research Building, Roosevelt
Drive, Oxford OX3 7FZ, U.K
- Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ, U.K
| | - Stefan Knapp
- Target
Discovery Institute, University of Oxford, NDM Research Building, Roosevelt
Drive, Oxford OX3 7FZ, U.K
- Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ, U.K
- Goethe-University, Institute
for Pharmaceutical Chemistry and Buchmann
Institute for Life Science, Campus Riedberg, Max-von Laue Str. 9, 60438 Frankfurt am Main, Germany
| | - Eric Trinquet
- Cisbio Bioassays, R&D, Parc Marcel Boiteux, BP 84175, 30200 Codolet, France
| | - Yves Collette
- CNRS, INSERM, Aix-Marseille Université, Institut Paoli-Calmettes, Centre de Recherche en Cancérologie de Marseille, CS30059, 13273 Marseille Cedex 9, France
| | - Jean-Claude Guillemot
- CNRS, Aix-Marseille
Université, Screening Platform AD2P, AFMB UMR7257, 13288, Marseille, France
| | - Xavier Morelli
- CNRS, INSERM, Aix-Marseille Université, Institut Paoli-Calmettes, Centre de Recherche en Cancérologie de Marseille, CS30059, 13273 Marseille Cedex 9, France
- CNRS, Aix-Marseille
Université, Screening Platform AD2P, AFMB UMR7257, 13288, Marseille, France
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Bavi R, Kumar R, Rampogu S, Kim Y, Kwon YJ, Park SJ, Lee KW. Novel virtual lead identification in the discovery of hematopoietic cell kinase (HCK) inhibitors: application of 3D QSAR and molecular dynamics simulation. J Recept Signal Transduct Res 2016; 37:224-238. [DOI: 10.1080/10799893.2016.1212376] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/21/2022]
Affiliation(s)
- Rohit Bavi
- Division of Applied Life Science (BK21 Plus Program), Systems and Synthetic Agrobiotech Center (SSAC), Plant Molecular Biology and Biotechnology Research Center (PMBBRC), Research Institute of Natural Science (RINS), Gyeongsang National University (GNU), Jinju, Republic of Korea
| | - Raj Kumar
- Division of Applied Life Science (BK21 Plus Program), Systems and Synthetic Agrobiotech Center (SSAC), Plant Molecular Biology and Biotechnology Research Center (PMBBRC), Research Institute of Natural Science (RINS), Gyeongsang National University (GNU), Jinju, Republic of Korea
| | - Shailima Rampogu
- Division of Applied Life Science (BK21 Plus Program), Systems and Synthetic Agrobiotech Center (SSAC), Plant Molecular Biology and Biotechnology Research Center (PMBBRC), Research Institute of Natural Science (RINS), Gyeongsang National University (GNU), Jinju, Republic of Korea
| | - Yongseong Kim
- Department of Science Education, Kyungnam University, Masan, Republic of Korea
| | - Yong Jung Kwon
- Department of Chemical Engineering, Kangwon National University, Chunchon, Republic of Korea
| | - Seok Ju Park
- Department of Internal Medicine, College of Medicine, Busan Paik Hospital, Inje University, Republic of Korea
| | - Keun Woo Lee
- Division of Applied Life Science (BK21 Plus Program), Systems and Synthetic Agrobiotech Center (SSAC), Plant Molecular Biology and Biotechnology Research Center (PMBBRC), Research Institute of Natural Science (RINS), Gyeongsang National University (GNU), Jinju, Republic of Korea
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24
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Roberts JM, Tarafdar S, Joseph RE, Andreotti AH, Smithgall TE, Engen JR, Wales TE. Dynamics of the Tec-family tyrosine kinase SH3 domains. Protein Sci 2016; 25:852-64. [PMID: 26808198 DOI: 10.1002/pro.2887] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2015] [Revised: 01/13/2016] [Accepted: 01/19/2016] [Indexed: 11/10/2022]
Abstract
The Src Homology 3 (SH3) domain is an important regulatory domain found in many signaling proteins. X-ray crystallography and NMR structures of SH3 domains are generally conserved but other studies indicate that protein flexibility and dynamics are not. We previously reported that based on hydrogen exchange mass spectrometry (HX MS) studies, there is variable flexibility and dynamics among the SH3 domains of the Src-family tyrosine kinases and related proteins. Here we have extended our studies to the SH3 domains of the Tec family tyrosine kinases (Itk, Btk, Tec, Txk, Bmx). The SH3 domains of members of this family augment the variety in dynamics observed in previous SH3 domains. Txk and Bmx SH3 were found to be highly dynamic in solution by HX MS and Bmx was unstructured by NMR. Itk and Btk SH3 underwent a clear EX1 cooperative unfolding event, which was localized using pepsin digestion and mass spectrometry after hydrogen exchange labeling. The unfolding was localized to peptide regions that had been previously identified in the Src-family and related protein SH3 domains, yet the kinetics of unfolding were not. Sequence alignment does not provide an easy explanation for the observed dynamics behavior, yet the similarity of location of EX1 unfolding suggests that higher-order structural properties may play a role. While the exact reason for such dynamics is not clear, such motions can be exploited in intra- and intermolecular binding assays of proteins containing the domains.
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Affiliation(s)
- Justin M Roberts
- Department of Chemistry & Chemical Biology, Northeastern University, Boston, Massachusetts, 02115
| | - Sreya Tarafdar
- Department of Microbiology & Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, 15219
| | - Raji E Joseph
- Roy J. Carver Department of Biochemistry, Biophysics & Molecular Biology, Iowa State University, Ames, Iowa, 50011
| | - Amy H Andreotti
- Roy J. Carver Department of Biochemistry, Biophysics & Molecular Biology, Iowa State University, Ames, Iowa, 50011
| | - Thomas E Smithgall
- Department of Microbiology & Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, 15219
| | - John R Engen
- Department of Chemistry & Chemical Biology, Northeastern University, Boston, Massachusetts, 02115
| | - Thomas E Wales
- Department of Chemistry & Chemical Biology, Northeastern University, Boston, Massachusetts, 02115
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25
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Synthesis and evaluation of orally active small molecule HIV-1 Nef antagonists. Bioorg Med Chem Lett 2016; 26:1480-1484. [PMID: 26852364 DOI: 10.1016/j.bmcl.2016.01.043] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2015] [Revised: 01/13/2016] [Accepted: 01/15/2016] [Indexed: 01/31/2023]
Abstract
The HIV-1 Nef accessory factor enhances viral replication and promotes immune system evasion of HIV-infected cells, making it an attractive target for drug discovery. Recently we described a novel class of diphenylpyrazolodiazene compounds that bind directly to Nef in vitro and inhibit Nef-dependent HIV-1 infectivity and replication in cell culture. However, these first-generation Nef antagonists have several structural liabilities, including an azo linkage that led to poor oral bioavailability. The azo group was therefore replaced with either a one- or two-carbon linker. The resulting set of non-azo analogs retained nanomolar binding affinity for Nef by surface plasmon resonance, while inhibiting HIV-1 replication with micromolar potency in cell-based assays without cytotoxicity. Computational docking studies show that these non-azo analogs occupy the same predicted binding site within the HIV-1 Nef dimer interface as the original azo compound. Computational methods also identified a hot spot for inhibitor binding within this site that is defined by conserved HIV-1 Nef residues Asp108, Leu112, and Pro122. Pharmacokinetic evaluation of the non-azo B9 analogs in mice showed that replacement of the azo linkage dramatically enhanced oral bioavailability without substantially affecting plasma half-life or clearance. The improved oral bioavailability of non-azo diphenylpyrazolo Nef antagonists provides a starting point for further drug lead optimization in support of future efficacy testing in animal models of HIV/AIDS.
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26
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Wales TE, Hochrein JM, Morgan CR, Emert-Sedlak LA, Smithgall TE, Engen JR. Subtle Dynamic Changes Accompany Hck Activation by HIV-1 Nef and are Reversed by an Antiretroviral Kinase Inhibitor. Biochemistry 2015; 54:6382-91. [PMID: 26440750 PMCID: PMC4615603 DOI: 10.1021/acs.biochem.5b00875] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
Abstract
The HIV-1 virulence factor Nef interacts with the macrophage Src-family kinase Hck, resulting in constitutive kinase activation that contributes to viral replication and immune escape. Previous chemical library screens identified the diphenylfuranopyrimdine kinase inhibitor DFP-4AB, which selectively inhibits Nef-dependent Hck activity in biochemical assays and potently blocks HIV replication in vitro. In the present study, hydrogen exchange mass spectrometry (HX MS) was used to study conformational changes in downregulated Hck that result from Nef binding, as well as the impact of DFP-4AB on these changes. Remarkably, interaction with Nef induced only subtle changes in deuterium uptake by Hck, with the most significant changes in the N-lobe of the kinase domain adjacent to the docking site for Nef on the SH3 domain. No changes in hydrogen exchange were observed in the Hck SH2 domain or C-terminal tail, indicating that this regulatory interaction is unaffected by Nef binding. When HX MS was performed in the presence of DFP-4AB, the effect of Nef on Hck N-lobe dynamics was completely reversed. These results show that constitutive activation of Hck by HIV-1 Nef requires only modest changes to the conformational dynamics of the overall kinase structure. DFP-4AB reverses these effects, consistent with its activity against this Nef-induced signaling event in HIV-infected cells.
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Affiliation(s)
- Thomas E. Wales
- Department of Chemistry & Chemical Biology, Northeastern University, Boston, MA 02115
| | - James M. Hochrein
- Department of Chemistry & Chemical Biology, Northeastern University, Boston, MA 02115
| | - Christopher R. Morgan
- Department of Chemistry & Chemical Biology, Northeastern University, Boston, MA 02115
| | - Lori A. Emert-Sedlak
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219
| | - Thomas E. Smithgall
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219
| | - John R. Engen
- Department of Chemistry & Chemical Biology, Northeastern University, Boston, MA 02115
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27
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Pirrone GF, Emert-Sedlak LA, Wales TE, Smithgall TE, Kent MS, Engen JR. Membrane-Associated Conformation of HIV-1 Nef Investigated with Hydrogen Exchange Mass Spectrometry at a Langmuir Monolayer. Anal Chem 2015; 87:7030-5. [PMID: 26133569 PMCID: PMC4509969 DOI: 10.1021/acs.analchem.5b01725] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
In the companion paper to this work, we described development of a new type of hydrogen exchange (HX) mass spectrometry (MS) measurement that integrates Langmuir monolayers. With Langmuir monolayers, the lipid packing density can be reproducibly controlled and changed as desired. Analysis of HX in proteins that may undergo conformational changes as a function of lipid packing (for example, conformational rearrangements after insertion into a lipid layer) are then possible. We previously used neutron reflection to characterize just such a conformational change in the myristoylated HIV-1 Nef protein (myrNef): at high lipid packing density, myrNef could not insert into the lipids and maintained a compact conformation adjacent to the monolayer, whereas at lower lipid packing density, myrNef was able to insert N-terminal arm residues, causing displacement of the core domain away from the monolayer. In order to locate where conformation may have been altered by lipid association, we applied the HX MS Langmuir monolayer method to myrNef associated with monolayers of packing densities identical to those used for the prior neutron reflection measurements. The results show that the N-terminal region and the C-terminal unstructured loop undergo conformational changes when associated with a low density lipid monolayer. The results are not consistent with the hypothesis of myrNef dimerization upon membrane association in the absence of other myrNef binding partners. The HX MS Langmuir monolayer method provides new and meaningful information for myrNef that helps explain necessary conformational changes required for function at the membrane.
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Affiliation(s)
- Gregory F. Pirrone
- Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115
| | - Lori A. Emert-Sedlak
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219
| | - Thomas E. Wales
- Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115
| | - Thomas E. Smithgall
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219
| | - Michael S. Kent
- Bioenergy and Defense Technologies, Sandia National Laboratories, Albuquerque, NM 87185
| | - John R. Engen
- Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115
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28
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Viral bimolecular fluorescence complementation: a novel tool to study intracellular vesicular trafficking pathways. PLoS One 2015; 10:e0125619. [PMID: 25915798 PMCID: PMC4411132 DOI: 10.1371/journal.pone.0125619] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2015] [Accepted: 03/24/2015] [Indexed: 01/23/2023] Open
Abstract
The Human Immunodeficiency Virus type 1 (HIV-1) accessory protein Nef interacts with a multitude of cellular proteins, manipulating the host membrane trafficking machinery to evade immune surveillance. Nef interactions have been analyzed using various in vitro assays, co-immunoprecipitation studies, and more recently mass spectrometry. However, these methods do not evaluate Nef interactions in the context of viral infection nor do they define the sub-cellular location of these interactions. In this report, we describe a novel bimolecular fluorescence complementation (BiFC) lentiviral expression tool, termed viral BiFC, to study Nef interactions with host cellular proteins in the context of viral infection. Using the F2A cleavage site from the foot and mouth disease virus we generated a viral BiFC expression vector capable of concurrent expression of Nef and host cellular proteins; PACS-1, MHC-I and SNX18. Our studies confirmed the interaction between Nef and PACS-1, a host membrane trafficking protein involved in Nef-mediated immune evasion, and demonstrated co-localization of this complex with LAMP-1 positive endolysosomal vesicles. Furthermore, we utilized viral BiFC to localize the Nef/MHC-I interaction to an AP-1 positive endosomal compartment. Finally, viral BiFC was observed between Nef and the membrane trafficking regulator SNX18. This novel demonstration of an association between Nef and SNX18 was localized to AP-1 positive vesicles. In summary, viral BiFC is a unique tool designed to analyze the interaction between Nef and host cellular proteins by mapping the sub-cellular locations of their interactions during viral infection.
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29
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Pawlak EN, Dikeakos JD. HIV-1 Nef: a master manipulator of the membrane trafficking machinery mediating immune evasion. Biochim Biophys Acta Gen Subj 2015; 1850:733-41. [PMID: 25585010 DOI: 10.1016/j.bbagen.2015.01.003] [Citation(s) in RCA: 57] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2014] [Revised: 12/09/2014] [Accepted: 01/06/2015] [Indexed: 11/25/2022]
Abstract
BACKGROUND Many viral genomes encode a limited number of proteins, illustrating their innate efficiency in bypassing host immune surveillance. This concept of genomic efficiency is exemplified by the 9 kb RNA genome of human immunodeficiency virus 1 (HIV-1), encoding 15 proteins sub-divided according to function. The enzymatic group includes proteins such as the drug targets reverse transcriptase and protease. In contrast, the accessory proteins lack any known enzymatic or structural function, yet are essential for viral fitness and HIV-1 pathogenesis. Of these, the HIV-1 accessory protein Nef is a master manipulator of host cellular processes, ensuring efficient counterattack against the host immune response, as well as long-term evasion of immune surveillance. In particular, the ability of Nef to downmodulate major histocompatibility complex class I (MHC-I) is a key cellular event that enables HIV-1 to bypass the host's defenses by evading the adaptive immune response. SCOPE OF REVIEW In this article, we briefly review how various pathogenic viruses control cell-surface MHC-I, and then focus on the mechanisms and implications of HIV-1 Nef-mediated MHC-I downregulation via modulation of the host membrane trafficking machinery. CONCLUSION The extensive interaction network formed between Nef and numerous membrane trafficking regulators suggests that Nef's role in evading the immune surveillance system intersects multiple host membrane trafficking pathways. SIGNIFICANCE Nef's ability to evade the immune surveillance system is linked to AIDS pathogenesis. Thus, a complete understanding of the molecular pathways that are subverted by Nef in order to downregulate MHC-I will enhance our understanding of HIV-1's progression to AIDS.
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Affiliation(s)
- Emily N Pawlak
- Department of Microbiology and Immunology, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, Ontario, Canada, N6A 5C1
| | - Jimmy D Dikeakos
- Department of Microbiology and Immunology, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, Ontario, Canada, N6A 5C1.
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30
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Moroco JA, Baumgartner MP, Rust HL, Choi HG, Hur W, Gray NS, Camacho CJ, Smithgall TE. A Discovery Strategy for Selective Inhibitors of c-Src in Complex with the Focal Adhesion Kinase SH3/SH2-binding Region. Chem Biol Drug Des 2014; 86:144-55. [PMID: 25376742 DOI: 10.1111/cbdd.12473] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2014] [Revised: 09/29/2014] [Accepted: 10/29/2014] [Indexed: 11/30/2022]
Abstract
The c-Src tyrosine kinase co-operates with the focal adhesion kinase to regulate cell adhesion and motility. Focal adhesion kinase engages the regulatory SH3 and SH2 domains of c-Src, resulting in localized kinase activation that contributes to tumor cell metastasis. Using assay conditions where c-Src kinase activity required binding to a tyrosine phosphopeptide based on the focal adhesion kinase SH3-SH2 docking sequence, we screened a kinase-biased library for selective inhibitors of the Src/focal adhesion kinase peptide complex versus c-Src alone. This approach identified an aminopyrimidinyl carbamate compound, WH-4-124-2, with nanomolar inhibitory potency and fivefold selectivity for c-Src when bound to the phospho-focal adhesion kinase peptide. Molecular docking studies indicate that WH-4-124-2 may preferentially inhibit the 'DFG-out' conformation of the kinase active site. These findings suggest that interaction of c-Src with focal adhesion kinase induces a unique kinase domain conformation amenable to selective inhibition.
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Affiliation(s)
- Jamie A Moroco
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, 450 Technology Drive, Pittsburgh, PA, 15219, USA
| | - Matthew P Baumgartner
- Department of Computational and Systems Biology, University of Pittsburgh School of Medicine, 3501 Fifth Avenue, Pittsburgh, PA, 15260, USA
| | - Heather L Rust
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, 450 Technology Drive, Pittsburgh, PA, 15219, USA
| | - Hwan Geun Choi
- Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School , 250 Longwood Avenue, Boston, MA, 02115, USA.,Department of Cancer Biology, Dana-Farber Cancer Institute, 250 Longwood Avenue, Boston, MA, 02115, USA
| | - Wooyoung Hur
- Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School , 250 Longwood Avenue, Boston, MA, 02115, USA.,Department of Cancer Biology, Dana-Farber Cancer Institute, 250 Longwood Avenue, Boston, MA, 02115, USA
| | - Nathanael S Gray
- Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School , 250 Longwood Avenue, Boston, MA, 02115, USA.,Department of Cancer Biology, Dana-Farber Cancer Institute, 250 Longwood Avenue, Boston, MA, 02115, USA
| | - Carlos J Camacho
- Department of Computational and Systems Biology, University of Pittsburgh School of Medicine, 3501 Fifth Avenue, Pittsburgh, PA, 15260, USA
| | - Thomas E Smithgall
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, 450 Technology Drive, Pittsburgh, PA, 15219, USA
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31
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Smithgall TE, Thomas G. Small molecule inhibitors of the HIV-1 virulence factor, Nef. DRUG DISCOVERY TODAY. TECHNOLOGIES 2014; 10:e523-9. [PMID: 24451644 DOI: 10.1016/j.ddtec.2013.07.002] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
Although antiretroviral therapy has revolutionized the clinical management of AIDS, life-long treatment is required because these drugs do not eradicate HIV- infected cells. Chronic antiretroviral therapy may not protect AIDS patients from cognitive impairment, raising important quality of life issues. Because of the rise of HIV strains resistant to current drugs and uncertain vaccine prospects, an urgent need exists for the discovery and development of new therapeutic approaches. This review is focused on one such approach, which involves targeting HIV-1 Nef, a viral accessory protein essential for AIDS pathogenesis.
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Alvarado JJ, Tarafdar S, Yeh JI, Smithgall TE. 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 2014; 289:28539-53. [PMID: 25122770 DOI: 10.1074/jbc.m114.600031] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022] Open
Abstract
HIV-1 Nef supports high titer viral replication in vivo and is essential for AIDS progression. Nef function depends on interactions with multiple host cell effectors, including Hck and other Src-family kinases. Here we describe the x-ray crystal structure of Nef in complex with the Hck SH3-SH2 regulatory region to a resolution of 1.86 Å. The complex crystallized as a dimer of complexes, with the conserved Nef PXXPXR motif engaging the Hck SH3 domain. A new intercomplex contact was found between SH3 Glu-93, and Nef Arg-105. Mutagenesis of Hck SH3 Glu-93 interfered with Nef·Hck complex formation and kinase activation in cells. The Hck SH2 domains impinge on the N-terminal region of Nef to stabilize a dimer conformation that exposes Asp-123, a residue critical for Nef function. Our results suggest that in addition to serving as a kinase effector for Nef, Hck binding may reorganize the Nef dimer for functional interaction with other signaling partners.
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Affiliation(s)
- John Jeff Alvarado
- From the Departments of Microbiology and Molecular Genetics and Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15219 and
| | - Sreya Tarafdar
- From the Departments of Microbiology and Molecular Genetics and Department of Infectious Diseases and Microbiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
| | - Joanne I Yeh
- Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15219 and
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Protein-protein interactions and human cellular cofactors as new targets for HIV therapy. Curr Opin Pharmacol 2014; 18:1-8. [PMID: 24993074 DOI: 10.1016/j.coph.2014.06.005] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2014] [Revised: 06/04/2014] [Accepted: 06/05/2014] [Indexed: 01/24/2023]
Abstract
Two novel approaches for the development of new drugs against AIDS are summarized each leading to the achievement of important discoveries in anti-HIV therapy. Despite the success of HAART in reducing mortality, resistant strains continue to emerge in the clinic, underscoring the importance of developing next-generation drugs. Protein-protein interactions and human cellular cofactors represent the new targets of tomorrow in HIV research. The most relevant results obtained in the last few years by the two new strategies are described herein.
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Tarafdar S, Poe JA, Smithgall TE. The accessory factor Nef links HIV-1 to Tec/Btk kinases in an Src homology 3 domain-dependent manner. J Biol Chem 2014; 289:15718-28. [PMID: 24722985 DOI: 10.1074/jbc.m114.572099] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
The HIV-1 Nef virulence factor interacts with multiple host cell-signaling proteins. Nef binds to the Src homology 3 domains of Src family kinases, resulting in kinase activation important for viral infectivity, replication, and MHC-I down-regulation. Itk and other Tec family kinases are also present in HIV target cells, and Itk has been linked to HIV-1 infectivity and replication. However, the molecular mechanism linking Itk to HIV-1 is unknown. In this study, we explored the interaction of Nef with Tec family kinases using a cell-based bimolecular fluorescence complementation assay. In this approach, interaction of Nef with a partner kinase juxtaposes nonfluorescent YFP fragments fused to the C terminus of each protein, resulting in YFP complementation and a bright fluorescent signal. Using bimolecular fluorescence complementation, we observed that Nef interacts with the Tec family members Bmx, Btk, and Itk but not Tec or Txk. Interaction with Nef occurs through the kinase Src homology 3 domains and localizes to the plasma membrane. Allelic variants of Nef from all major HIV-1 subtypes interacted strongly with Itk in this assay, demonstrating the highly conserved nature of this interaction. A selective small molecule inhibitor of Itk kinase activity (BMS-509744) potently blocked wild-type HIV-1 infectivity and replication, but not that of a Nef-defective mutant. Nef induced constitutive Itk activation in transfected cells that was sensitive to inhibitor treatment. Taken together, these results provide the first evidence that Nef interacts with cytoplasmic tyrosine kinases of the Tec family and suggest that Nef provides a mechanistic link between HIV-1 and Itk signaling in the viral life cycle.
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Affiliation(s)
- Sreya Tarafdar
- From the Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15219 and the Department of Infectious Diseases and Microbiology, University of Pittsburgh Graduate School of Public Health, Pittsburgh, Pennsylvania 15261
| | - Jerrod A Poe
- From the Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15219 and
| | - Thomas E Smithgall
- From the Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15219 and
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Iyer PC, Zhao J, Emert-Sedlak LA, Moore KK, Smithgall TE, Day BW. Synthesis and structure-activity analysis of diphenylpyrazolodiazene inhibitors of the HIV-1 Nef virulence factor. Bioorg Med Chem Lett 2014; 24:1702-6. [PMID: 24650642 DOI: 10.1016/j.bmcl.2014.02.045] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/02/2014] [Revised: 02/12/2014] [Accepted: 02/18/2014] [Indexed: 10/25/2022]
Abstract
HIV-1 Nef is a critical AIDS progression factor yet underexplored target for antiretroviral drug discovery. A recent high-throughput screen for pharmacological inhibitors of Nef-dependent Src-family kinase activation identified a diphenylpyrazolodiazene hit compound with submicromolar potency in HIV-1 replication assays against a broad range of primary Nef variants. This compound, known as 'B9', binds directly to Nef and inhibits its dimerization in cells as a possible mechanism of action. Here were synthesized a diverse set of B9 analogs and identified structural features essential to antiretroviral activity. Chemical modifications to each of the three rings present in the parent compound were identified that did not compromise antiviral action. These analogs will guide the development of next-generation compounds with appropriate pharmacological profiles for assessment of antiretroviral activity in vivo.
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Affiliation(s)
- Prema C Iyer
- Department of Pharmaceutical Sciences, University of Pittsburgh School of Pharmacy, Pittsburgh, PA 15261, USA; University of Pittsburgh Drug Discovery Institute, Pittsburgh, PA 15261, USA
| | - Jielu Zhao
- Department of Pharmaceutical Sciences, University of Pittsburgh School of Pharmacy, Pittsburgh, PA 15261, USA; University of Pittsburgh Drug Discovery Institute, Pittsburgh, PA 15261, USA
| | - Lori A Emert-Sedlak
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Bridgeside Point II, Suite 523, 450 Technology Drive, Pittsburgh, PA 15219, USA
| | - Kerry K Moore
- Department of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260, USA
| | - Thomas E Smithgall
- University of Pittsburgh Drug Discovery Institute, Pittsburgh, PA 15261, USA; Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Bridgeside Point II, Suite 523, 450 Technology Drive, Pittsburgh, PA 15219, USA.
| | - Billy W Day
- Department of Pharmaceutical Sciences, University of Pittsburgh School of Pharmacy, Pittsburgh, PA 15261, USA; University of Pittsburgh Drug Discovery Institute, Pittsburgh, PA 15261, USA; Department of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260, USA
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Dürr R, Keppler O, Christ F, Crespan E, Garbelli A, Maga G, Dietrich U. Targeting Cellular Cofactors in HIV Therapy. TOPICS IN MEDICINAL CHEMISTRY 2014. [DOI: 10.1007/7355_2014_45] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
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Trible RP, Narute P, Emert-Sedlak LA, Alvarado JJ, Atkins K, Thomas L, Kodama T, Yanamala N, Korotchenko V, Day BW, Thomas G, Smithgall TE. Discovery of a diaminoquinoxaline benzenesulfonamide antagonist of HIV-1 Nef function using a yeast-based phenotypic screen. Retrovirology 2013; 10:135. [PMID: 24229420 PMCID: PMC3874621 DOI: 10.1186/1742-4690-10-135] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2013] [Accepted: 10/31/2013] [Indexed: 11/10/2022] Open
Abstract
BACKGROUND HIV-1 Nef is a viral accessory protein critical for AIDS progression. Nef lacks intrinsic catalytic activity and binds multiple host cell signaling proteins, including Hck and other Src-family tyrosine kinases. Nef binding induces constitutive Hck activation that may contribute to HIV pathogenesis by promoting viral infectivity, replication and downregulation of cell-surface MHC-I molecules. In this study, we developed a yeast-based phenotypic screen to identify small molecules that inhibit the Nef-Hck complex. RESULTS Nef-Hck interaction was faithfully reconstituted in yeast cells, resulting in kinase activation and growth arrest. Yeast cells expressing the Nef-Hck complex were used to screen a library of small heterocyclic compounds for their ability to rescue growth inhibition. The screen identified a dihydrobenzo-1,4-dioxin-substituted analog of 2-quinoxalinyl-3-aminobenzene-sulfonamide (DQBS) as a potent inhibitor of Nef-dependent HIV-1 replication and MHC-I downregulation in T-cells. Docking studies predicted direct binding of DQBS to Nef which was confirmed in differential scanning fluorimetry assays with recombinant purified Nef protein. DQBS also potently inhibited the replication of HIV-1 NL4-3 chimeras expressing Nef alleles representative of all M-group HIV-1 clades. CONCLUSIONS Our findings demonstrate the utility of a yeast-based growth reversion assay for the identification of small molecule Nef antagonists. Inhibitors of Nef function discovered with this assay, such as DQBS, may complement the activity of current antiretroviral therapies by enabling immune recognition of HIV-infected cells through the rescue of cell surface MHC-I.
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Affiliation(s)
- Ronald P Trible
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Bridgeside Point II, Suite 523, 15219, Pittsburgh, PA USA
| | - Purushottam Narute
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Bridgeside Point II, Suite 523, 15219, Pittsburgh, PA USA
| | - Lori A Emert-Sedlak
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Bridgeside Point II, Suite 523, 15219, Pittsburgh, PA USA
| | - John Jeff Alvarado
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Bridgeside Point II, Suite 523, 15219, Pittsburgh, PA USA
| | - Katelyn Atkins
- School of Medicine, Oregon Health and Science University, 97239, Portland, OR, USA
| | - Laurel Thomas
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Bridgeside Point II, Suite 523, 15219, Pittsburgh, PA USA
| | - Toshiaki Kodama
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Bridgeside Point II, Suite 523, 15219, Pittsburgh, PA USA
| | - Naveena Yanamala
- Department of Structural Biology, University of Pittsburgh School of Medicine, 15261, Pittsburgh, PA USA
| | - Vasiliy Korotchenko
- Department of Pharmaceutical Sciences, University of Pittsburgh School of Pharmacy, 15261, Pittsburgh, PA USA
| | - Billy W Day
- Department of Pharmaceutical Sciences, University of Pittsburgh School of Pharmacy, 15261, Pittsburgh, PA USA
| | - Gary Thomas
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Bridgeside Point II, Suite 523, 15219, Pittsburgh, PA USA
| | - Thomas E Smithgall
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Bridgeside Point II, Suite 523, 15219, Pittsburgh, PA USA
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Cornall A, Mak J, Greenway A, Tachedjian G. HIV-1 infection of T cells and macrophages are differentially modulated by virion-associated Hck: a Nef-dependent phenomenon. Viruses 2013; 5:2235-52. [PMID: 24051604 PMCID: PMC3798898 DOI: 10.3390/v5092235] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2013] [Revised: 09/01/2013] [Accepted: 09/12/2013] [Indexed: 12/18/2022] Open
Abstract
The proline repeat motif (PxxP) of Nef is required for interaction with the SH3 domains of macrophage-specific Src kinase Hck. However, the implication of this interaction for viral replication and infectivity in macrophages and T lymphocytes remains unclear. Experiments in HIV-1 infected macrophages confirmed the presence of a Nef:Hck complex which was dependent on the Nef proline repeat motif. The proline repeat motif of Nef also enhanced both HIV-1 infection and replication in macrophages, and was required for incorporation of Hck into viral particles. Unexpectedly, wild-type Hck inhibited infection of macrophages, but Hck was shown to enhance infection of primary T lymphocytes. These results indicate that the interaction between Nef and Hck is important for Nef-dependent modulation of viral infectivity. Hck-dependent enhancement of HIV-1 infection of T cells suggests that Nef-Hck interaction may contribute to the spread of HIV-1 infection from macrophages to T cells by modulating events in the producer cell, virion and target cell.
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Affiliation(s)
- Alyssa Cornall
- Centre for Biomedical Research, Macfarlane Burnet Institute for Medical Research and Public Health, Melbourne 3004, Victoria, Australia; E-Mails: (A.C.); (A.G.)
- Regional HPV Labnet Reference Laboratory, Department of Microbiology and Infectious Diseases, The Royal Women’s Hospital, Parkville 3052, Victoria, Australia
- Murdoch Children’s Research Institute, Parkville 3052, Victoria, Australia
| | - Johnson Mak
- School of Medicine, Faculty of Health, Deakin University, Geelong 3220, Victoria, Australia; E-Mail:
- Commonwealth Scientific and Industrial Research Organisation, Livestock Industries, Australian Animal Health Laboratory, Geelong 3220, Victoria, Australia
| | - Alison Greenway
- Centre for Biomedical Research, Macfarlane Burnet Institute for Medical Research and Public Health, Melbourne 3004, Victoria, Australia; E-Mails: (A.C.); (A.G.)
| | - Gilda Tachedjian
- Centre for Biomedical Research, Macfarlane Burnet Institute for Medical Research and Public Health, Melbourne 3004, Victoria, Australia; E-Mails: (A.C.); (A.G.)
- Department of Microbiology, Monash University, Clayton 3168, Victoria, Australia
- Department of Infectious Diseases, Monash University, Melbourne 3004, Victoria, Australia
- Author to whom correspondence should be addressed; E-Mail: ; Tel.: +61-3-9282-2256; Fax: +61-3-9282-2100
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Perrone R, Nadai M, Poe JA, Frasson I, Palumbo M, Palù G, Smithgall TE, Richter SN. Formation of a unique cluster of G-quadruplex structures in the HIV-1 Nef coding region: implications for antiviral activity. PLoS One 2013; 8:e73121. [PMID: 24015290 PMCID: PMC3754912 DOI: 10.1371/journal.pone.0073121] [Citation(s) in RCA: 90] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2013] [Accepted: 07/24/2013] [Indexed: 01/10/2023] Open
Abstract
G-quadruplexes are tetraplex structures of nucleic acids that can form in G-rich sequences. Their presence and functional role have been established in telomeres, oncogene promoters and coding regions of the human chromosome. In particular, they have been proposed to be directly involved in gene regulation at the level of transcription. Because the HIV-1 Nef protein is a fundamental factor for efficient viral replication, infectivity and pathogenesis in vitro and in vivo, we investigated G-quadruplex formation in the HIV-1 nef gene to assess the potential for viral inhibition through G-quadruplex stabilization. A comprehensive computational analysis of the nef coding region of available strains showed the presence of three conserved sequences that were uniquely clustered. Biophysical testing proved that G-quadruplex conformations were efficiently stabilized or induced by G-quadruplex ligands in all three sequences. Upon incubation with a G-quadruplex ligand, Nef expression was reduced in a reporter gene assay and Nef-dependent enhancement of HIV-1 infectivity was significantly repressed in an antiviral assay. These data constitute the first evidence of the possibility to regulate HIV-1 gene expression and infectivity through G-quadruplex targeting and therefore open a new avenue for viral treatment.
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Affiliation(s)
- Rosalba Perrone
- Department of Molecular Medicine, University of Padua, Padua, Italy
| | - Matteo Nadai
- Department of Molecular Medicine, University of Padua, Padua, Italy
| | - Jerrod A. Poe
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America
| | - Ilaria Frasson
- Department of Molecular Medicine, University of Padua, Padua, Italy
| | - Manlio Palumbo
- Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Padua, Italy
| | - Giorgio Palù
- Department of Molecular Medicine, University of Padua, Padua, Italy
| | - Thomas E. Smithgall
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America
| | - Sara N. Richter
- Department of Molecular Medicine, University of Padua, Padua, Italy
- * E-mail:
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Witkowski W, Verhasselt B. Contributions of HIV-1 Nef to immune dysregulation in HIV-infected patients: a therapeutic target? Expert Opin Ther Targets 2013; 17:1345-56. [PMID: 23967871 DOI: 10.1517/14728222.2013.830712] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
Abstract
INTRODUCTION HIV accessory protein Nef is a factor responsible for many of the viral pathogenic effects. Progression to AIDS is dramatically delayed and in some well-documented cases completely abolished on infection with naturally occurring HIV strains lacking intact nef sequences in their genomes. The topic of this review is the contribution of Nef to the immune pathology as a possible target in HIV-infected patients. AREAS COVERED An overview of known Nef functions accounting for its role in pathogenesis is presented, emphasizing interactions with dendritic cells and macrophages, and Nef-induced exosome secretion, all involved in immune dysregulation during the course of HIV infection. Current approaches to Nef inhibition by different classes of compounds are reviewed. EXPERT OPINION Blocking Nef for therapeutic purposes is a challenging endeavor mainly due to intrinsic properties of this HIV accessory protein. Nef has multiple interfaces to interact with host proteins and lacks a catalytic domain. Potential benefits arising from the development of successful inhibitors could however prove beneficial for reducing gradual deterioration of immune system in chronically infected patients in absence of functional cure.
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Affiliation(s)
- Wojciech Witkowski
- Department of Clinical Chemistry, Microbiology and Immunology of Ghent University , Gent , Belgium +32 93323658 ; +32 93323659 ;
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Perrone R, Nadai M, Frasson I, Poe JA, Butovskaya E, Smithgall TE, Palumbo M, Palù G, Richter SN. A dynamic G-quadruplex region regulates the HIV-1 long terminal repeat promoter. J Med Chem 2013; 56:6521-30. [PMID: 23865750 DOI: 10.1021/jm400914r] [Citation(s) in RCA: 147] [Impact Index Per Article: 12.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
G-Quadruplexes, noncanonical nucleic acid structures, act as silencers in the promoter regions of human genes; putative G-quadruplex forming sequences are also present in promoters of other mammals, yeasts, and prokaryotes. Here we show that also the HIV-1 LTR promoter exploits G-quadruplex-mediated transcriptional regulation with striking similarities to eukaryotic promoters and that treatment with a G-quadruplex ligand inhibits HIV-1 infectivity. Computational analysis on 953 HIV-1 strains substantiated a highly conserved G-rich sequence corresponding to Sp1 and NF-κB binding sites. Biophysical/biochemical analysis proved that two mutually exclusive parallel-like intramolecular G-quadruplexes, stabilized by small molecule ligands, primarily fold in this region. Mutations disrupting G-quadruplex formation enhanced HIV promoter activity in cells, whereas treatment with a G-quadruplex ligand impaired promoter activity and displayed antiviral effects. These findings disclose the possibility of inhibiting the HIV-1 LTR promoter by G-quadruplex-interacting small molecules, providing a new pathway to development of anti-HIV-1 drugs with unprecedented mechanism of action.
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Affiliation(s)
- Rosalba Perrone
- Department of Molecular Medicine, University of Padua, Italy
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Effector kinase coupling enables high-throughput screens for direct HIV-1 Nef antagonists with antiretroviral activity. ACTA ACUST UNITED AC 2013; 20:82-91. [PMID: 23352142 DOI: 10.1016/j.chembiol.2012.11.005] [Citation(s) in RCA: 47] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2012] [Revised: 11/19/2012] [Accepted: 11/21/2012] [Indexed: 12/12/2022]
Abstract
HIV-1 Nef, a critical AIDS progression factor, represents an important target protein for antiretroviral drug discovery. Because Nef lacks intrinsic enzymatic activity, we developed an assay that couples Nef to the activation of Hck, a Src family member and Nef effector protein. Using this assay, we screened a large, diverse chemical library and identified small molecules that block Nef-dependent Hck activity with low micromolar potency. Of these, a diphenylpyrazolo compound demonstrated submicromolar potency in HIV-1 replication assays against a broad range of primary Nef variants. This compound binds directly to Nef via a pocket formed by the Nef dimerization interface and disrupts Nef dimerization in cells. Coupling of nonenzymatic viral accessory factors to host cell effector proteins amenable to high-throughput screening may represent a general strategy for the discovery of new antimicrobial agents.
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Tintori C, Laurenzana I, La Rocca F, Falchi F, Carraro F, Ruiz A, Esté JA, Kissova M, Crespan E, Maga G, Biava M, Brullo C, Schenone S, Botta M. Identification of Hck inhibitors as hits for the development of antileukemia and anti-HIV agents. ChemMedChem 2013; 8:1353-60. [PMID: 23813855 DOI: 10.1002/cmdc.201300204] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2013] [Indexed: 12/16/2022]
Abstract
Hematopoietic cell kinase (Hck) is a member of the Src family of non-receptor protein tyrosine kinases. High levels of Hck are associated with drug resistance in chronic myeloid leukemia. Furthermore, Hck activity has been connected with HIV-1. Herein, structure-based drug design efforts were aimed at identifying novel Hck inhibitors. First, an in-house library of pyrazolo[3,4-d]pyrimidine derivatives, which were previously shown to be dual Abl and c-Src inhibitors, was analyzed by docking studies within the ATP binding site of Hck to select the best candidates to be tested in a cell-free assay. Next, the same computational protocol was applied to screen a database of commercially available compounds. As a result, most of the selected compounds were found active against Hck, with Ki values ranging from 0.14 to 18.4 μM, confirming the suitability of the computational approach adopted. Furthermore, selected compounds showed an interesting antiproliferative activity profile against the human leukemia cell line KU-812, and one compound was found to block HIV-1 replication at sub-toxic concentrations.
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Affiliation(s)
- Cristina Tintori
- Dipartimento Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, Via A. De Gasperi 2, 53100 Siena, Italy
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Pagano MA, Tibaldi E, Palù G, Brunati AM. Viral proteins and Src family kinases: Mechanisms of pathogenicity from a “liaison dangereuse”. World J Virol 2013; 2:71-78. [PMID: 24175231 PMCID: PMC3785045 DOI: 10.5501/wjv.v2.i2.71] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/05/2012] [Revised: 01/07/2013] [Accepted: 01/24/2013] [Indexed: 02/05/2023] Open
Abstract
To complete their life cycle and spread, viruses interfere with and gain control of diverse cellular processes, this most often occurring through interaction between viral proteins (VPs) and resident protein partners. Among the latter, Src family kinases (SFKs), a class of non-receptor tyrosine kinases that contributes to the conversion of extracellular signals into intracellular signaling cascades and is involved in virtually all cellular processes, have recently emerged as critical mediators between the cell’s infrastructure and the viral demands. In this scenario, structural or ex novo synthesized VPs are able to bind to the different domains of these enzymes through specific short linear motifs present along their sequences. Proline-rich motifs displaying the conserved minimal consensus PxxP and recognizing the SFK Src homology (SH)3 domain constitute a cardinal signature for the formation of multiprotein complexes and this interaction may promote phosphorylation of VPs by SFKs, thus creating phosphotyrosine motifs that become a docking site for the SH2 domains of SFKs or other SH2 domain-bearing signaling molecules. Importantly, the formation of these assemblies also results in a change in the activity and/or location of SFKs, and these events are critical in perturbing key signaling pathways so that viruses can utilize the cell’s machinery to their own benefit. In the light of these observations, although VPs as such, especially those with enzyme activity, are still regarded as valuable targets for therapeutic strategies, multiprotein complexes composed of viral and host cell proteins are increasingly becoming objects of investigation with a view to deeply characterize the structural aspects that favor their formation and to develop new compounds able to contrast viral diseases in an alternative manner.
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Jin YJ, Cai CY, Mezei M, Ohlmeyer M, Sanchez R, Burakoff SJ. Identification of a novel binding site between HIV type 1 Nef C-terminal flexible loop and AP2 required for Nef-mediated CD4 downregulation. AIDS Res Hum Retroviruses 2013; 29:725-31. [PMID: 23151229 DOI: 10.1089/aid.2012.0286] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
HIV-1 Nef is an accessory protein necessary for HIV-1 virulence and rapid AIDS development. Nef promotes viral replication and infection by connecting CD4 and several other cell surface receptors to the clathrin adaptor protein AP2, resulting in the internalization and degradation of the receptors interacting with Nef. We investigated how Nef can mediate constitutive receptor endocytosis through the interaction of the dileucine motif in its C-terminal flexible loop (C-loop) with AP2, whereas AP2 binding of the transmembrane receptors usually results in an equilibrated (recycled) endocytosis. Our results indicated that in addition to the dileucine motif, there is a second motif in the Nef C-loop involved in the Nef-AP2 interaction. Nef-mediated CD4 downregulation was impaired when the residue in the hydrophobic region in the Nef C-loop (LL165HPMSLHGM173) was mutated to a basic residue K/R or an acidic residue E/D or to the rigid residue P, or when M168L170, L170H171, or G172M173 was mutated to AA. A pull-down assay indicated that AP2 was not coprecipitated with Nef mutants that did not downregulate CD4. Molecular modeling of the Nef C-terminal flexible loop in complex with AP2 suggests that M168L170 occupies a pocket in the AP2 σ2 subunit. Our data suggest a new model in the Nef-AP2 interaction in which the hydrophobic region in the Nef C-loop with the dileucine (L164L165) motif and M168L170 motif binds to AP2(σ2), while the acidic motif E174 and D175 binds to AP2(α), which explains how Nef through the flexible loop connects CD4 to AP2 for constitutive CD4 downregulation.
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Affiliation(s)
- Yong-Jiu Jin
- Department of Oncological Sciences, Cancer Institute, Mount Sinai School of Medicine, New York, New York
| | - Catherine Yi Cai
- Department of Oncological Sciences, Cancer Institute, Mount Sinai School of Medicine, New York, New York
| | - Mihaly Mezei
- Department of Structural and Chemical Biology, Cancer Institute, Mount Sinai School of Medicine, New York, New York
- Experimental Therapeutics Institute, Cancer Institute, Mount Sinai School of Medicine, New York, New York
| | - Michael Ohlmeyer
- Department of Structural and Chemical Biology, Cancer Institute, Mount Sinai School of Medicine, New York, New York
- Experimental Therapeutics Institute, Cancer Institute, Mount Sinai School of Medicine, New York, New York
| | - Roberto Sanchez
- Department of Structural and Chemical Biology, Cancer Institute, Mount Sinai School of Medicine, New York, New York
- Experimental Therapeutics Institute, Cancer Institute, Mount Sinai School of Medicine, New York, New York
| | - Steven J. Burakoff
- Department of Oncological Sciences, Cancer Institute, Mount Sinai School of Medicine, New York, New York
- Cancer Institute, Mount Sinai School of Medicine, New York, New York
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Hellwig S, Miduturu CV, Kanda S, Zhang J, Filippakopoulos P, Salah E, Deng X, Choi HG, Zhou W, Hur W, Knapp S, Gray NS, Smithgall TE. Small-molecule inhibitors of the c-Fes protein-tyrosine kinase. CHEMISTRY & BIOLOGY 2012; 19:529-40. [PMID: 22520759 PMCID: PMC3334838 DOI: 10.1016/j.chembiol.2012.01.020] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/02/2011] [Revised: 01/12/2012] [Accepted: 01/30/2012] [Indexed: 11/21/2022]
Abstract
The c-Fes protein-tyrosine kinase modulates cellular signaling pathways governing differentiation, the innate immune response, and vasculogenesis. Here, we report the identification of types I and II kinase inhibitors with potent activity against c-Fes both in vitro and in cell-based assays. One of the most potent inhibitors is the previously described anaplastic lymphoma kinase inhibitor TAE684. The crystal structure of TAE684 in complex with the c-Fes SH2-kinase domain showed excellent shape complementarity with the ATP-binding pocket and a key role for the gatekeeper methionine in the inhibitory mechanism. TAE684 and two pyrazolopyrimidines with nanomolar potency against c-Fes in vitro were used to establish a role for this kinase in osteoclastogenesis, illustrating the value of these inhibitors as tool compounds to probe the diverse biological functions associated with this unique kinase.
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Affiliation(s)
- Sabine Hellwig
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA USA
| | - Chandra V. Miduturu
- Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School and Department of Cancer Biology, Dana-Farber Cancer Institute, 250 Longwood Avenue, Boston, MA 02115, USA
| | - Shigeru Kanda
- Department of Molecular Microbiology and Immunology, Nagasaki University Graduate School of Biomedical Sciences, 4-12-1 Sakamoto, Nagasaki 852-8523, and Department of Experimental and Clinical Laboratory Medicine, National Hospital Organization, Nagasaki Hospital, 41-6 Sakuragi-machi, Nagasaki 850-8523, Japan
| | - Jianming Zhang
- Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School and Department of Cancer Biology, Dana-Farber Cancer Institute, 250 Longwood Avenue, Boston, MA 02115, USA
| | | | - Eidarus Salah
- Nuffield Department of Clinical Medicine, SGC, University of Oxford, Oxford, UK
| | - Xianming Deng
- Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School and Department of Cancer Biology, Dana-Farber Cancer Institute, 250 Longwood Avenue, Boston, MA 02115, USA
| | - Hwan Geun Choi
- Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School and Department of Cancer Biology, Dana-Farber Cancer Institute, 250 Longwood Avenue, Boston, MA 02115, USA
| | - Wenjun Zhou
- Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School and Department of Cancer Biology, Dana-Farber Cancer Institute, 250 Longwood Avenue, Boston, MA 02115, USA
| | - Wooyoung Hur
- Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School and Department of Cancer Biology, Dana-Farber Cancer Institute, 250 Longwood Avenue, Boston, MA 02115, USA
| | - Stefan Knapp
- Nuffield Department of Clinical Medicine, SGC, University of Oxford, Oxford, UK
| | - Nathanael S. Gray
- Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School and Department of Cancer Biology, Dana-Farber Cancer Institute, 250 Longwood Avenue, Boston, MA 02115, USA
| | - Thomas E. Smithgall
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA USA
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Pene-Dumitrescu T, Shu ST, Wales TE, Alvarado JJ, Shi H, Narute P, Moroco JA, Yeh JI, Engen JR, Smithgall TE. HIV-1 Nef interaction influences the ATP-binding site of the Src-family kinase, Hck. BMC CHEMICAL BIOLOGY 2012; 12:1. [PMID: 22420777 PMCID: PMC3328272 DOI: 10.1186/1472-6769-12-1] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 09/23/2011] [Accepted: 03/15/2012] [Indexed: 12/13/2022]
Abstract
Background Nef is an HIV-1 accessory protein essential for viral replication and AIDS progression. Nef interacts with a multitude of host cell signaling partners, including members of the Src kinase family. Nef preferentially activates Hck, a Src-family kinase (SFK) strongly expressed in macrophages and other HIV target cells, by binding to its regulatory SH3 domain. Recently, we identified a series of kinase inhibitors that preferentially inhibit Hck in the presence of Nef. These compounds also block Nef-dependent HIV replication, validating the Nef-SFK signaling pathway as an antiretroviral drug target. Our findings also suggested that by binding to the Hck SH3 domain, Nef indirectly affects the conformation of the kinase active site to favor inhibitor association. Results To test this hypothesis, we engineered a "gatekeeper" mutant of Hck with enhanced sensitivity to the pyrazolopyrimidine tyrosine kinase inhibitor, NaPP1. We also modified the RT loop of the Hck SH3 domain to enhance interaction of the kinase with Nef. This modification stabilized Nef:Hck interaction in solution-based kinase assays, as a way to mimic the more stable association that likely occurs at cellular membranes. Introduction of the modified RT loop rendered Hck remarkably more sensitive to activation by Nef, and led to a significant decrease in the Km for ATP as well as enhanced inhibitor potency. Conclusions These observations suggest that stable interaction with Nef may induce Src-family kinase active site conformations amenable to selective inhibitor targeting.
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Affiliation(s)
- Teodora Pene-Dumitrescu
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA.
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Narute PS, Smithgall TE. Nef alleles from all major HIV-1 clades activate Src-family kinases and enhance HIV-1 replication in an inhibitor-sensitive manner. PLoS One 2012; 7:e32561. [PMID: 22393415 PMCID: PMC3290594 DOI: 10.1371/journal.pone.0032561] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2011] [Accepted: 02/01/2012] [Indexed: 01/04/2023] Open
Abstract
The HIV-1 accessory factor Nef is essential for high-titer viral replication and AIDS progression. Nef function requires interaction with many host cell proteins, including specific members of the Src kinase family. Here we explored whether Src-family kinase activation is a conserved property of Nef alleles from a wide range of primary HIV-1 isolates and their sensitivity to selective pharmacological inhibitors. Representative Nef proteins from the major HIV-1 subtypes A1, A2, B, C, F1, F2, G, H, J and K strongly activated Hck and Lyn as well as c-Src to a lesser extent, demonstrating for the first time that Src-family kinase activation is a highly conserved property of primary M-group HIV-1 Nef isolates. Recently, we identified 4-amino substituted diphenylfuropyrimidines (DFPs) that selectively inhibit Nef-dependent activation of Src-family kinases as well as HIV replication. To determine whether DFP compounds exhibit broad-spectrum Nef-dependent antiretroviral activity against HIV-1, we first constructed chimeric forms of the HIV-1 strain NL4-3 expressing each of the primary Nef alleles. The infectivity and replication of these Nef chimeras was indistinguishable from that of wild-type virus in two distinct cell lines (U87MG astroglial cells and CEM-T4 lymphoblasts). Importantly, the 4-aminopropanol and 4-aminobutanol derivatives of DFP potently inhibited the replication of all chimeric forms of HIV-1 in both U87MG and CEM-T4 cells in a Nef-dependent manner. The antiretroviral effects of these compounds correlated with inhibition of Nef-dependent activation of endogenous Src-family kinases in the HIV-infected cells. Our results demonstrate that the activation of Hck, Lyn and c-Src by Nef is highly conserved among all major clades of HIV-1 and that selective targeting of this pathway uniformly inhibits HIV-1 replication.
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Affiliation(s)
- Purushottam S. Narute
- Department of Infectious Disease and Microbiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America
- Department of Microbiology and Molecular Genetics, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America
| | - Thomas E. Smithgall
- Department of Microbiology and Molecular Genetics, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America
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Chutiwitoonchai N, Hiyoshi M, Mwimanzi P, Ueno T, Adachi A, Ode H, Sato H, Fackler OT, Okada S, Suzu S. The identification of a small molecule compound that reduces HIV-1 Nef-mediated viral infectivity enhancement. PLoS One 2011; 6:e27696. [PMID: 22110726 PMCID: PMC3217016 DOI: 10.1371/journal.pone.0027696] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2011] [Accepted: 10/22/2011] [Indexed: 12/14/2022] Open
Abstract
Nef is a multifunctional HIV-1 protein that accelerates progression to AIDS, and enhances the infectivity of progeny viruses through a mechanism that is not yet understood. Here, we show that the small molecule compound 2c reduces Nef-mediated viral infectivity enhancement. When added to viral producer cells, 2c did not affect the efficiency of viral production itself. However, the infectivity of the viruses produced in the presence of 2c was significantly lower than that of control viruses. Importantly, an inhibitory effect was observed with Nef(+) wild-type viruses, but not with viruses produced in the absence of Nef or in the presence of proline-rich PxxP motif-disrupted Nef, both of which displayed significantly reduced intrinsic infectivity. Meanwhile, the overexpression of the SH3 domain of the tyrosine kinase Hck, which binds to a PxxP motif in Nef, also reduced viral infectivity. Importantly, 2c inhibited Hck SH3-Nef binding, which was more marked when Nef was pre-incubated with 2c prior to its incubation with Hck, indicating that both Hck SH3 and 2c directly bind to Nef and that their binding sites overlap. These results imply that both 2c and the Hck SH3 domain inhibit the interaction of Nef with an unidentified host protein and thereby reduce Nef-mediated infectivity enhancement. The first inhibitory compound 2c is therefore a valuable chemical probe for revealing the underlying molecular mechanism by which Nef enhances the infectivity of HIV-1.
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Affiliation(s)
| | | | - Philip Mwimanzi
- Center for AIDS Research, Kumamoto University, Kumamoto, Japan
| | - Takamasa Ueno
- Center for AIDS Research, Kumamoto University, Kumamoto, Japan
| | - Akio Adachi
- Department of Microbiology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan
| | - Hirotaka Ode
- Pathogen Genomics Center, National Institute of Infectious Diseases, Tokyo, Japan
| | - Hironori Sato
- Pathogen Genomics Center, National Institute of Infectious Diseases, Tokyo, Japan
| | - Oliver T. Fackler
- Department of Infectious Diseases, Virology, University of Heidelberg, Heidelberg, Germany
| | - Seiji Okada
- Center for AIDS Research, Kumamoto University, Kumamoto, Japan
| | - Shinya Suzu
- Center for AIDS Research, Kumamoto University, Kumamoto, Japan
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Marcsisin SR, Narute PS, Emert-Sedlak LA, Kloczewiak M, Smithgall TE, Engen JR. On the solution conformation and dynamics of the HIV-1 viral infectivity factor. J Mol Biol 2011; 410:1008-22. [PMID: 21763503 PMCID: PMC3139145 DOI: 10.1016/j.jmb.2011.04.053] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2011] [Revised: 04/20/2011] [Accepted: 04/21/2011] [Indexed: 02/07/2023]
Abstract
Human immunodeficiency virus-1 (HIV-1) has evolved a cunning mechanism to circumvent the antiviral activity of the APOBEC3 family of host cell enzymes. HIV-1 Vif [viral (also called virion) infectivity factor], one of several HIV accessory proteins, targets APOBEC3 proteins for proteasomal degradation and downregulates their expression at the mRNA level. Despite the importance of Vif for HIV-1 infection, there is little conformational data on Vif alone or in complex with other cellular factors due to incompatibilities with many structural techniques and difficulties in producing suitable quantities of the protein for biophysical analysis. As an alternative, we have turned to hydrogen exchange mass spectrometry (HX MS), a conformational analysis method that is well suited for proteins that are difficult to study using X-ray crystallography and/or NMR. HX MS was used to probe the solution conformation of recombinant full-length HIV-1 Vif. Vif specifically interacted with the previously identified binding partner Hck and was able to cause kinase activation, suggesting that the Vif studied by HX MS retained a biochemically competent conformation relevant to Hck interaction. HX MS analysis of Vif alone revealed low deuteration levels in the N-terminal portion, indicating that this region contained structured or otherwise protected elements. In contrast, high deuteration levels in the C-terminal portion of Vif indicated that this region was likely unstructured in the absence of cellular interacting proteins. Several regions within Vif displayed conformational heterogeneity in solution, including the APOBEC3G/F binding site and the HCCH zinc finger. Taken together, these HX MS results provide new insights into the solution conformation of Vif.
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Affiliation(s)
- Sean R. Marcsisin
- Department of Chemistry & Chemical Biology and the Barnett Institute of Chemical & Biological Analysis, Northeastern University, Boston, MA 02115, USA
| | - Purushottam S. Narute
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA
| | - Lori A. Emert-Sedlak
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA
| | | | - Thomas E. Smithgall
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA
| | - John R. Engen
- Department of Chemistry & Chemical Biology and the Barnett Institute of Chemical & Biological Analysis, Northeastern University, Boston, MA 02115, USA
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