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. 2009 Nov 20;4(11):939-47.
doi: 10.1021/cb900195c.

Chemical library screens targeting an HIV-1 accessory factor/host cell kinase complex identify novel antiretroviral compounds

Lori Emert-Sedlak  1 , Toshiaki Kodama, Edwina C Lerner, Weixiang Dai, Caleb Foster, Billy W Day, John S Lazo, Thomas E Smithgall
Affiliations

Chemical library screens targeting an HIV-1 accessory factor/host cell kinase complex identify novel antiretroviral compounds

Lori Emert-Sedlak et al. ACS Chem Biol. .

Abstract

Nef is an HIV-1 accessory protein essential for AIDS progression and an attractive target for drug discovery. Lack of a catalytic function makes Nef difficult to assay in chemical library screens. We developed a high-throughput screening assay for inhibitors of Nef function by coupling it to one of its host cell binding partners, the Src-family kinase Hck. Hck activation is dependent upon Nef in this assay, providing a direct readout of Nef activity in vitro. Using this screen, a unique diphenylfuropyrimidine was identified as a strong inhibitor of Nef-dependent Hck activation. This compound also exhibited remarkable antiretroviral effects, blocking Nef-dependent HIV replication in cell culture. Structurally related analogs were synthesized and shown to exhibit similar Nef-dependent antiviral activity, identifying the diphenylfuropyrimidine substructure as a new lead for antiretroviral drug development. This study demonstrates that coupling noncatalytic HIV accessory factors with host cell target proteins addressable by high-throughput assays may afford new avenues for the discovery of anti-HIV agents.

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Figures

Figure 1
Figure 1
Screening for Nef:Hck inhibitors using the Z’-Lyte assay. (a) Nef stimulates Hck protein-tyrosine kinase activity in vitro. Recombinant Hck was purified from Sf9 insect cells in its downregulated form (Hck-YEEI; see text) and assayed with a FRET-peptide substrate and ATP as described under Materials and Methods. Reactions were run in the presence of increasing amounts of Hck-YEEI either alone (open circles) or in the presence of a 10-fold molar excess of purified recombinant HIV-1 Nef (closed circles). Each condition was repeated in quadruplicate, and the extent of phosphorylation is expressed as mean percent of phosphorylation relative to a control phosphopeptide ± S.D. Chemical library screens were performed under conditions where Hck-YEEI activation is dependent upon Nef (arrow). (b) Chemical libraries (10,000 compounds total) were screened for inhibitors of Nef-induced Hck kinase activity. Shown are representative emission ratios for 350 compounds from one plate which includes a hit (DFP-4AP; see text for details). The average emission ratio and 50% inhibition cutoff are indicated by the horizontal lines. Clusters of control points include wells with no ATP (open circles) and DMSO vehicle control (open triangles).
Figure 2
Figure 2
Inhibition of Nef-induced Hck activation and HIV-1 replication by DFP-4-aminopropanol (DFP-4AP). (a) Nef-dependence of HIV-1 replication in U87MG cells. Cells were infected with wild-type HIV strain NL4-3 (WT) or a mutant that fails to express Nef (ΔNef) over the range of viral titers shown. Relative HIV p24 levels were determined by ELISA 5 days later. (b) Inhibition of Nef-induced Hck activation by DFP-4AP. The Nef:Hck complex was assayed in vitro with a peptide substrate in the presence of DFP-4AP over the range of concentrations shown. Each concentration was assayed in triplicate and data are expressed as percent inhibition relative to control reactions run in the presence of solvent only. The data were best-fit by non-linear regression analysis (GraphPad Prism Software), yielding an IC50 value of 4 µM. (c) Inhibition of HIV-1 replication by DFP. U87MG cells were infected with HIV strain NL4-3 in the presence of DFP-4AP over the range of concentrations shown. Release of viral p24 was determined by ELISA 5 days later. The data were best-fit by non-linear regression analysis, yielding an IC50 value of 6 µM.
Figure 3
Figure 3
Structures of DFP-4-aminopropanol and related 4-amino derivatives.
Figure 4
Figure 4
Selective inhibition of Nef-induced Hck activation by diphenylfuropyrimidines. The kinase activities of the Nef:Hck complex and Hck alone were assayed in vitro with a peptide substrate in the presence of DFP-4AP and the three analogs shown in Figure 3 over the range of concentrations shown. Each concentration was assayed in triplicate and data are expressed as percent inhibition relative to control reactions run in the absence of compound. Where possible, non-linear regression analysis was used to estimate IC50 values shown (GraphPad Prism).
Figure 5
Figure 5
Inhibition of HIV-1 replication by diphenylfuropyrimidines requires Nef. U87MG cells were infected in 96-well plates (200 µl final volume) with equal titers (100 pg p24) of wild-type HIV strain NL4-3 (WT) or a mutant that fails to express Nef (ΔNef) in the presence of the DFP analogs (Figure 3) over the range of concentrations shown. HIV p24 levels were determined by ELISA 5 days later. Data are presented as percent of p24 release observed in the absence of compound ± S.D.
Figure 6
Figure 6
DFP analogs fail to inhibit HIV-1 replication in Jurkat cells. (a) HIV-1 replication is Nef-independent in Jurkat T-cells. Cells were infected in 96-well plates (200 µl final volume) with wild-type HIV strain NL4-3 (WT) or a mutant that fails to express Nef (ΔNef) over the range of viral titers shown. Relative HIV p24 levels were determined by ELISA 5 days later. (b) Jurkat cells were infected with equal titers of wild-type HIV (WT) or the Nef-defective mutant (ΔNef) in the presence of 5 µM of the DFP analogs (Figure 3). HIV p24 release was determined by ELISA 5 days later. Data are presented as percent of p24 release observed in the absence of compound ± S.D.
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