Small Molecules Targeted to a Non-Catalytic “RVxF” Binding Site of Protein Phosphatase-1 Inhibit HIV-1
Figure 5
1H4 compound competes with RVxF motif.
A. Superimposition of pRb-Tat peptide on the complex of PP1 with Spinophilin. PP1 surface is colored after the atom types. The spinophilin peptide is shown as magenta ribbon and the pRb-Tat peptide as orange ribbon. The Val25 and Phe27 residues of pRb-Tat and the Ile449 and Phe 451 residues of Spinophilin are shown as sticks. The 2-(N-morpholino)-ethanesulfonic acid bound in the active site of PP1 is shown in green spheres. The phosphorylated Ser6 residue of pRb-Tat peptide is shown as sticks. B. Phosphorylated Ser 6 residue binds to the active site of PP1. Comparative superimposition of pRb-Tat peptide over the crystal structure of MES bound in the active site of PP1. Catalytic resides are shown as sticks. MES is shown as green sticks, and the phosphorylated Ser6 residue of pRb-Tat peptide is shown as orange sticks. The pRb-Tat peptide is shown as orange ribbon. C. 1H4 inhibits kinetics of pRb-Tat peptide dephosphorylation by PP1α. Recombinant PP1α was assayed with pRb-Tat (WT or QACA mutant, 120 µM) in the absence or presence of 1H4 as indicated. The reactions were stopped at indicated time points and the phosphate release was quantified by malachite green assay. Initial velocity was calculated by linear regression in Prism. D. 1H4 inhibits kinetics of pRb-cdNIPP dephosphorylation by PP1α. Recombinant PP1α was assayed with pRb-cdNIPP1 in the absence or presence of 1H4 as indicated. Mutant pRb cdNIPP1 pA-RATA was used as negative control. The reactions were stopped at indicated time points and the phosphate release was quantified by malachite green assay. Initial velocity was calculated by linear regression in Prism. E and F. 1H4 competitively inhibits pRb-Tat peptide dephosphorylation by PP1α. Initial rates of pRb-Tat peptide dephosphorylation by PP1α were assayed at the indicated concentrations of the substrate in the absence or presence of 300 µM 1H4 or non-HIV-1 inhibitory 1G3. The amount of the released phosphate was quantified with malachite green. The VMAX and Km were calculated by non-linear regression analysis in Prism with the assumption that 25% of the substrate contained the phosphate group. Transformation of the data to Lineweaver-Burk plot (panel E) showed competitive inhibition of pRb-Tat dephosphorylation.