Chemical probes of protein tyrosine phosphatase activity

UnrestrictedChemical probes of PTP activity are needed to gain a more complete understanding of their involvement in many physiological pathways and disease states. Two examples of inhibitory chemical probes for PTPs are Pmp, phosphonomethylphenylalanine, and F2Pmp, phosphono(difluoromethyl)phenylalanine. These mimics were synthesized by novel routes so that they were appropriately protected for solid phase peptide synthesis. Pmp was incorporated into two peptides for CD45 and HePTP inhibition studies. F2Pmp was incorporated into nine peptides for crystallization experiments. A very useful chemical probe of PTP activity, called pCAP, was synthesized, incorporated into peptides and used as a substrate to determine PTP subsite specificity in multiply phosphorylated peptide sequences. All three of the above mentioned probes are mimics of phosphotyrosine. Nitrosylating reagents are extremely useful inhibitory chemical probes that are not based on phosphotyrosine. Sodium nitroprusside (SNP) and a peptide-based homocysteine-S-nitroso-thiol (14merHCys-NO) were found to inhibit LYP with low micromolar IC50 values. SNP inhibited by nitrosylating multiple cysteine residues while 14merHCys-NO appeared to only nitrosylate the active site cysteine residue. Due to the importance of reactive oxygen species and reactive nitrogen species in many biological processes, three chemical probes were designed and synthesized to detect these species based on a coumarin scaffold. Two probes contained boron based reporters and were effective at detecting hydrogen peroxide as well as nitric oxide, while the third contained a diamine reporter that was not effective at detecting any oxidant. The boron based probes will be useful in imaging cellular oxidants. All of these chemical probes have aided in gaining a better understanding of the regulation and activity of PTPs