Effector kinase coupling enables high-throughput screens for direct HIV-1 Nef antagonists with antiretroviral activity.
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.
Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA.
Lori A
LA
Emert-Sedlak
Purushottam
P
Narute
Sherry T
ST
Shu
Jerrod A
JA
Poe
Haibin
H
Shi
Naveena
N
Yanamala
John Jeff
JJ
Alvarado
John S
JS
Lazo
Joanne I
JI
Yeh
Paul A
PA
Johnston
Thomas E
TE
Smithgall
D044966:Anti-Retroviral Agents / Q000737:chemistry* / Q000494:pharmacology*; D002460:Cell Line; D015658:HIV Infections / Q000188:drug therapy; D015497:HIV-1 / Q000187:drug effects* / Q000502:physiology; D006801:Humans; D062105:Molecular Docking Simulation; D055503:Protein Multimerization / Q000187:drug effects; D051572:Proto-Oncogene Proteins c-hck / Q000378:metabolism; D011720:Pyrazoles / Q000737:chemistry / Q000494:pharmacology; D054852:Small Molecule Libraries / Q000737:chemistry* / Q000494:pharmacology*; D014779:Virus Replication / Q000187:drug effects; D054311:nef Gene Products, Human Immunodeficiency Virus / Q000037:antagonists & inhibitors* / Q000378:metabolism
D044966:Anti-Retroviral Agents; D011720:Pyrazoles; D054852:Small Molecule Libraries; D054311:nef Gene Products, Human Immunodeficiency Virus; D051572:Proto-Oncogene Proteins c-hck