Design and synthesis of improved active-site SHP2 inhibitors with anti-breast cancer cell effects.

The Src homology containing phosphotyrosyl phosphatase 2 (SHP2) is a bona fide oncogene particularly in cancers driven by overexpression of receptor tyrosine kinases (RTKs). As such, there is a growing interest to target SHP2 in cancer. Based on these premises, several active site (type I) and allosteric site (type II) inhibitors have been developed, but no SHP2 targeting therapies have reached the clinic yet. In an effort to fill these gaps, we embarked on producing optimized versions of our parent active-site SHP2 inhibitor CNBDA. The objectives were to produce derivatives with increased inhibitory potential and improved selectivity. Accordingly, we designed derivatives around the CNBDA scaffold and predicted their binding property by in silico molecular modeling. Based on comparative differences in free energy of binding to the SHP2 versus the SHP1 active sites, ten were selected, chemically synthesized, and evaluated by NMR and mass spectroscopy for structural integrity. Among the ten derivatives, BPDA2 was found to be the most potent and highly selective compound, inhibiting the SHP2 enzyme activity with an IC of 92 nM when DiFMUP was used as a substrate and with an IC of 47 nM when pNPP was used as a substrate. Furthermore, enzyme kinetic analyses showed that BPDA2 is a competitive SHP2 inhibitor. Selectivity comparisons in a PTPase assay using DiFMUP as a substrate demonstrated that BPDA2 is more selective to SHP2 than to SHP1 and PTP1B by more than 369-fold and 442-fold, respectively. Evaluation with a cellular thermal shift assay (CETSA) confirmed that BPDA2 binds to wild-type SHP2 in a cellular context, and stabilizes it in solution. Treatment of cells with DBDA2 downregulates mitogenic and cell survival signaling and RTK expression in a concentration dependent manner. Furthermore, treatment of cells with BPDA2 suppresses anchorage independent growth and cancer stem cell properties of breast cancer cells. Overall, data described in this report show that BPDA2 is a more potent derivative of CNBDA with a highly improved selectivity for SHP2.