title: Interaction Between HIV-1 Nef and Calnexin: From Modeling to Small Molecule Inhibitors Reversing HIV-Induced Lipid Accumulation.
abstract: HIV-infected patients are at an increased risk of developing atherosclerosis, in part because of downmodulation and functional impairment of ATP-binding cassette A1 (ABCA1) cholesterol transporter by the HIV-1 protein Nef. The mechanism of this effect involves Nef interacting with an ER chaperone calnexin and disrupting calnexin binding to ABCA1, leading to ABCA1 retention in ER, its degradation and resulting suppression of cholesterol efflux. However, molecular details of Nef-calnexin interaction remained unknown, limiting the translational impact of this finding.
  
  
  
  Here, we used molecular modeling and mutagenesis to characterize Nef-calnexin interaction and to identify small molecule compounds that could block it. We demonstrated that the interaction between Nef and calnexin is direct and can be reconstituted using recombinant proteins in vitro with a binding affinity of 89.1 nmol/L measured by surface plasmon resonance. The cytoplasmic tail of calnexin is essential and sufficient for interaction with Nef, and binds Nef with an affinity of 9.4 nmol/L. Replacing lysine residues in positions 4 and 7 of Nef with alanines abrogates Nef-calnexin interaction, prevents ABCA1 downregulation by Nef, and preserves cholesterol efflux from HIV-infected cells. Through virtual screening of the National Cancer Institute library of compounds, we identified a compound, 1[(7-oxo-7H-benz[de]anthracene-3-yl)amino]anthraquinone, which blocked Nef-calnexin interaction, partially restored ABCA1 activity in HIV-infected cells, and reduced foam cell formation in a culture of HIV-infected macrophages.
  
  
  
  This study identifies potential targets that can be exploited to block the pathogenic effect of HIV infection on cholesterol metabolism and prevent atherosclerosis in HIV-infected subjects.
authors.affiliation: From the George Washington University School of Medicine and Health Sciences, Washington, DC (R.H., L.D., T.P., B.B., A.A.A., M.B.); University of Alabama School of Medicine and Dentistry, Birmingham, (M.V., D.V.); Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia (D.S.); Engelhardt Institute of Molecular Biology RAS, Moscow, Russia (A.A. Anashkina, A.A. Adzhubei); and Georgetown University Medical Center, Lombardi Comprehensive Cancer Center, Washington, DC (A.Ü).
authors.forename: Ruth
authors.initials: R
authors.lastname: Hunegnaw
authors.affiliation: From the George Washington University School of Medicine and Health Sciences, Washington, DC (R.H., L.D., T.P., B.B., A.A.A., M.B.); University of Alabama School of Medicine and Dentistry, Birmingham, (M.V., D.V.); Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia (D.S.); Engelhardt Institute of Molecular Biology RAS, Moscow, Russia (A.A. Anashkina, A.A. Adzhubei); and Georgetown University Medical Center, Lombardi Comprehensive Cancer Center, Washington, DC (A.Ü).
authors.forename: Marina
authors.initials: M
authors.lastname: Vassylyeva
authors.affiliation: From the George Washington University School of Medicine and Health Sciences, Washington, DC (R.H., L.D., T.P., B.B., A.A.A., M.B.); University of Alabama School of Medicine and Dentistry, Birmingham, (M.V., D.V.); Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia (D.S.); Engelhardt Institute of Molecular Biology RAS, Moscow, Russia (A.A. Anashkina, A.A. Adzhubei); and Georgetown University Medical Center, Lombardi Comprehensive Cancer Center, Washington, DC (A.Ü).
authors.forename: Larisa
authors.initials: L
authors.lastname: Dubrovsky
authors.affiliation: From the George Washington University School of Medicine and Health Sciences, Washington, DC (R.H., L.D., T.P., B.B., A.A.A., M.B.); University of Alabama School of Medicine and Dentistry, Birmingham, (M.V., D.V.); Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia (D.S.); Engelhardt Institute of Molecular Biology RAS, Moscow, Russia (A.A. Anashkina, A.A. Adzhubei); and Georgetown University Medical Center, Lombardi Comprehensive Cancer Center, Washington, DC (A.Ü).
authors.forename: Tatiana
authors.initials: T
authors.lastname: Pushkarsky
authors.affiliation: From the George Washington University School of Medicine and Health Sciences, Washington, DC (R.H., L.D., T.P., B.B., A.A.A., M.B.); University of Alabama School of Medicine and Dentistry, Birmingham, (M.V., D.V.); Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia (D.S.); Engelhardt Institute of Molecular Biology RAS, Moscow, Russia (A.A. Anashkina, A.A. Adzhubei); and Georgetown University Medical Center, Lombardi Comprehensive Cancer Center, Washington, DC (A.Ü).
authors.forename: Dmitri
authors.initials: D
authors.lastname: Sviridov
authors.affiliation: From the George Washington University School of Medicine and Health Sciences, Washington, DC (R.H., L.D., T.P., B.B., A.A.A., M.B.); University of Alabama School of Medicine and Dentistry, Birmingham, (M.V., D.V.); Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia (D.S.); Engelhardt Institute of Molecular Biology RAS, Moscow, Russia (A.A. Anashkina, A.A. Adzhubei); and Georgetown University Medical Center, Lombardi Comprehensive Cancer Center, Washington, DC (A.Ü).
authors.forename: Anastasia A
authors.initials: AA
authors.lastname: Anashkina
authors.affiliation: From the George Washington University School of Medicine and Health Sciences, Washington, DC (R.H., L.D., T.P., B.B., A.A.A., M.B.); University of Alabama School of Medicine and Dentistry, Birmingham, (M.V., D.V.); Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia (D.S.); Engelhardt Institute of Molecular Biology RAS, Moscow, Russia (A.A. Anashkina, A.A. Adzhubei); and Georgetown University Medical Center, Lombardi Comprehensive Cancer Center, Washington, DC (A.Ü).
authors.forename: Aykut
authors.initials: A
authors.lastname: Üren
authors.affiliation: From the George Washington University School of Medicine and Health Sciences, Washington, DC (R.H., L.D., T.P., B.B., A.A.A., M.B.); University of Alabama School of Medicine and Dentistry, Birmingham, (M.V., D.V.); Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia (D.S.); Engelhardt Institute of Molecular Biology RAS, Moscow, Russia (A.A. Anashkina, A.A. Adzhubei); and Georgetown University Medical Center, Lombardi Comprehensive Cancer Center, Washington, DC (A.Ü).
authors.forename: Beda
authors.initials: B
authors.lastname: Brichacek
authors.affiliation: From the George Washington University School of Medicine and Health Sciences, Washington, DC (R.H., L.D., T.P., B.B., A.A.A., M.B.); University of Alabama School of Medicine and Dentistry, Birmingham, (M.V., D.V.); Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia (D.S.); Engelhardt Institute of Molecular Biology RAS, Moscow, Russia (A.A. Anashkina, A.A. Adzhubei); and Georgetown University Medical Center, Lombardi Comprehensive Cancer Center, Washington, DC (A.Ü).
authors.forename: Dmitry G
authors.initials: DG
authors.lastname: Vassylyev
authors.affiliation: From the George Washington University School of Medicine and Health Sciences, Washington, DC (R.H., L.D., T.P., B.B., A.A.A., M.B.); University of Alabama School of Medicine and Dentistry, Birmingham, (M.V., D.V.); Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia (D.S.); Engelhardt Institute of Molecular Biology RAS, Moscow, Russia (A.A. Anashkina, A.A. Adzhubei); and Georgetown University Medical Center, Lombardi Comprehensive Cancer Center, Washington, DC (A.Ü). mbukrins@gwu.edu alexei.adzhubei@eimb.ru.
authors.forename: Alexei A
authors.initials: AA
authors.lastname: Adzhubei
authors.affiliation: From the George Washington University School of Medicine and Health Sciences, Washington, DC (R.H., L.D., T.P., B.B., A.A.A., M.B.); University of Alabama School of Medicine and Dentistry, Birmingham, (M.V., D.V.); Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia (D.S.); Engelhardt Institute of Molecular Biology RAS, Moscow, Russia (A.A. Anashkina, A.A. Adzhubei); and Georgetown University Medical Center, Lombardi Comprehensive Cancer Center, Washington, DC (A.Ü). mbukrins@gwu.edu alexei.adzhubei@eimb.ru.
authors.forename: Michael
authors.initials: M
authors.lastname: Bukrinsky
mesh_terms: D064286:ATP Binding Cassette Transporter 1 / Q000378:metabolism; D000880:Anthraquinones / Q000737:chemistry / Q000494:pharmacology*; D050197:Atherosclerosis / Q000378:metabolism / Q000517:prevention & control* / Q000821:virology; D001692:Biological Transport; D037281:Calnexin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism*; D017076:Computer-Aided Design; D015195:Drug Design; D005487:Foam Cells / Q000187:drug effects / Q000378:metabolism; D057809:HEK293 Cells; D015658:HIV Infections / Q000188:drug therapy* / Q000378:metabolism / Q000821:virology; D006801:Humans; D000960:Hypolipidemic Agents / Q000737:chemistry / Q000494:pharmacology*; D008239:Lysine; D062105:Molecular Docking Simulation; D009154:Mutation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D013329:Structure-Activity Relationship; D014162:Transfection; D054311:nef Gene Products, Human Immunodeficiency Virus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: C577836:ABCA1 protein, human; D064286:ATP Binding Cassette Transporter 1; D000880:Anthraquinones; D000960:Hypolipidemic Agents; D054311:nef Gene Products, Human Immunodeficiency Virus; C514837:nef protein, Human immunodeficiency virus 1; D037281:Calnexin; D002784:Cholesterol; D008239:Lysine
keywords: HIV-1; Nef; calnexin; cholesterol efflux; foam cell; molecular modeling; virtual screening