Effector Kinase Coupling Enables High-Throughput Screens for Direct HIV-1 Nef Antagonists with Antiretroviral Activity

SummaryHIV-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.

Graphical AbstractFigure optionsDownload full-size imageDownload high-quality image (367 K)Download as PowerPoint slideHighlights► Nef is a critical HIV virulence factor lacking biochemical activity amenable to HTS ► HTS for inhibitors of Nef-mediated Hck kinase activation identified Nef antagonists ► Hit compound B9 blocks Nef-dependent HIV replication with submicromolar potency ► B9 binds directly to purified HIV Nef in vitro and blocks Nef dimerization in cells