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

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 sub-micromolar 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 non-enzymatic 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.

Pharmacologic HIV-1 Nef blockade promotes CD8 T cell–mediated elimination of latently HIV-1–infected cells in vitro

“…Previous studies have shown that, unlike WT HIV-1 NL4-3, infectivity and replication of the ΔNef mutant are unaffected by Nef inhibitor treatment (29)(30)(31). Coculture of ex vivo autologous CD8 T cells (n = 6) or HIV-1 peptide-expanded CD8 T cells (n = 10-11) with these NL4-3 ΔNef latently infected CD4 T cells did not result in a significant difference between control and either of the Nef inhibitor-treated samples, as reflected in Figure 3, G and H. Of interest is that baseline levels of IFN-γ in the no inhibitor cocultures with expanded CD8 T cells were elevated if resting CD4 T cells were infected with Nef-deficient virus relative to targets that were infected with Nef-competent virus (median 179.3 pg/ ml vs. 119.1 pg/ml, respectively), supporting the role of Nef in preventing recognition of latently infected cells.…”

“…To test this hypothesis, we employed the use of small molecule inhibitors previously shown to bind HIV-1 Nef directly and block its functions related to HIV-1 infectivity and replication enhancement (29)(30)(31). The structures of the 4 inhibitors used in this study are shown in Supplemental Figure 1 (supplemental material available online with this article; https://doi.org/10.1172/jci.insight.93684DS1).…”

“…Inhibitors DQBS and B9 were added to latently infected cell cultures at 5 μM each, whereas B9 analogs JZ-96-21 and JZ-97-21 were used at a final concentration of 0.5 μM each. The inhibitor concentration employed for compound B9 is based on the IC 50 for inhibition of Nef-mediated Src-family kinase activation and HIV-1 infectivity enhancement in vitro (29), whereas DQBS was tested at 5 μM due to its ability to prevent MHC-I downregulation at this concentration in Nef-transfected cells (30). JZ-96-21 and JZ-97-21 are orally bioavailable, non-azo derivatives of compound B9 with reduced cytotoxicity and are active at the indicated concentrations in HIV-1 replication assays (ref.…”

“…To test this idea, we investigated whether small molecule inhibitors of Nef from 2 distinct chemical classes, previously shown to bind directly to Nef and to block its activity in HIV-1-infected cell lines (29)(30)(31), could abrogate Nef-mediated MHC-I downregulation and allow autologous CD8 T cells to recognize and kill primary CD4 T cells latently infected with HIV-1. In combination with Nef blockade, we also tested whether prior expansion of HIV-specific CD8 T cells, as has been reported (32), could augment elimination of these latently HIV-1-infected cells.…”

Dynamics of the Tec‐family tyrosine kinase SH3 domains

“…Nef-dependent activation of Hck contributes to HIV-1 replication, in that selective inhibitors of this pathway interfere with viral replication in vitro. [42][43][44] More recent studies have suggested a similar role for the Tec-family kinase Itk in HIV-1 pathogenicity as well, although the connection between the virus and Itk were not initially apparent. 45 Subsequent cell-based bimolecular fluorescence complementation (BiFC) assays revealed selective interaction of HIV-1 Nef with the Tecfamily kinases Itk, Btk, and Bmx.…”