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).