| Description |
Protein tyrosine phosphatases (PTPs) play critical roles in cell signaling processes. The aberrant activities of PTPs are highly associated with many diseases such as type-1 diabetes, rheumatoid arthritis, and cancer. Despite their importance, the biological roles and regulation of individual PTPs are poorly understood due to the shortage of sufficient detection methods. To facilitate PTP research, two approaches were used to develop selective, peptide-based PTP chemical probes. The first approach was a rational design method, in which we developed a SHP2- selective fluorescent substrate based on the physiological PTP substrate TIE2 with a high micromolar affinity (KM = 0.7 ± 0.2 mM) towards SHP2. We further optimized the probe based on substrate sequence information available in the literature, which resulted in a 7- fold increase in reactivity. The second approach involved the use of a combinatorial substrate library, in which we designed, built, and screened a diverse library of peptides using an inverse alanine screening approach and obtained substrate positional preference profiles for 11 PTPs at 8 positions surrounding the phosphotyrosine. Based on the preference profile of LMWPTP and PTPμ, we developed two fluorescent probes and achieved up to a 6-fold increase on reactivity compared to a negative control substrate. The preference profile can be used for future probe development and optimization for the 11 PTPs. In addition to fluorescent substrates, we also developed two activity-based probes using a similar approach by incorporating a covalent inhibitor, 4-(azidomethyl) iv phenylvinyl sulfonate, at the phosphotyrosine position of the peptide sequence. Both probes exhibited time- and concentration-dependent inhibitory activity towards PTPN22 and SHP2. The EDNE probe has an IC50 of 130 ± 6 μM towards SHP2 after a one-hour incubation, while the LDLL probe has an IC50 of 80 ± 10 μM under the same condition. The EDNE probe exhibits up to 32-fold selectivity over PTP1B, PTP-PEST, TCPTP, and YopH. Furthermore, the EDNE probe shows selectivity towards SHP2 over other cysteine-based enzymes in a SHP2-expressing lysate. Both the substrates and inhibitors we developed add to the toolkit of chemical probes used to help better understand the regulation and physiological roles of PTPs. |
