Structure-activity relationship studies of allosteric inhibitors of EYA2 tyrosine phosphatase.

Human eyes absent (EYA) proteins possess Tyr phosphatase activity, which is critical for numerous cancer and metastasis promoting activities, making it an attractive target for cancer therapy. In this work, we demonstrate that the inhibitor-bound form of EYA2 does not favour binding to Mg2+ , which is indispensable for the Tyr phosphatase activity. We further describe characterization and optimization of this class of allosteric inhibitors. A series of analogues were synthesized to improve potency of the inhibitors and to elucidate structure-activity relationships. Two co-crystal structures confirm the binding modes of this class of inhibitors. Our medicinal chemical, structural, biochemical, and biophysical studies provide insight into the molecular interactions of EYA2 with these allosteric inhibitors. The compounds derived from this study are useful for exploring the function of the Tyr phosphatase activity of EYA2 in normal and cancerous cells and serve as reference compounds for screening or developing allosteric phosphatase inhibitors. Finally, the co-crystal structures reported in this study will aid in structure-based drug discovery against EYA2.

Structural and Functional Analyses of an Allosteric EYA2 Phosphatase Inhibitor That Has On-Target Effects in Human Lung Cancer Cells.

EYA proteins (EYA1-4) are critical developmental transcriptional cofactors that contain an EYA domain (ED) harboring Tyr phosphatase activity. EYA proteins are largely downregulated after embryogenesis but are reexpressed in cancers, and their Tyr phosphatase activity plays an important role in the DNA damage response and tumor progression. We previously identified a class of small-molecule allosteric inhibitors that specifically inhibit the Tyr phosphatase activity of EYA2. Herein, we determined the crystal structure of the EYA2 ED in complex with NCGC00249987 (a representative compound in this class), revealing that it binds to an induced pocket distant from the active site. NCGC00249987 binding leads to a conformational change of the active site that is unfavorable for Mg2+ binding, thereby inhibiting EYA2's Tyr phosphatase activity. We demonstrate, using genetic mutations, that migration, invadopodia formation, and invasion of lung adenocarcinoma cells are dependent on EYA2 Tyr phosphatase activity, whereas growth and survival are not. Further, we demonstrate that NCGC00249987 specifically targets migration, invadopodia formation, and invasion of lung cancer cells, but that it does not inhibit cell growth or survival. The compound has no effect on lung cancer cells carrying an EYA2 F290Y mutant that abolishes compound binding, indicating that NCGC00249987 is on target in lung cancer cells. These data suggest that the NCGC00249987 allosteric inhibitor can be used as a chemical probe to study the function of the EYA2 Tyr phosphatase activity in cells and may have the potential to be developed into an antimetastatic agent for cancers reliant on EYA2's Tyr phosphatase activity.

Butelase 1: A Versatile Ligase for Peptide and Protein Macrocyclization.

Macrocyclization is a valuable tool for drug design and protein engineering. Although various methods have been developed to prepare macrocycles, a general and efficient strategy is needed. Here we report a highly efficient method using butelase 1 to macrocyclize peptides and proteins ranging in sizes from 26 to >200 residues. We achieved cyclizations that are 20,000 times faster than sortase A, the most widely used ligase for protein cyclization. The reactions completed within minutes with up to 95% yields.

Assessing the Efficacy of Mdm2/Mdm4-Inhibiting Stapled Peptides Using Cellular Thermal Shift Assays.

Previous publications on stapled peptide inhibitors against Mdm2/Mdm4-p53 interactions have established that this new class of drugs have the potential to be easily optimised to attain high binding affinity and specificity, but the mechanisms controlling their cellular uptake and target engagement remain elusive and controversial. To aid in understanding the rules of peptide and staple design, and to enable rapid optimisation, we employed the newly-developed cellular thermal shift assay (CETSA). CETSA was able to validate stapled peptide binding to Mdm2 and Mdm4, and the method was also used to determine the extent of cellular uptake, cellular availability, and intracellular binding of the endogenous target proteins in its native environment. Our data suggest that while the stapled peptides engage their targets intracellularly, more work is needed to improve their cellular entry and target engagement efficiency in vivo. CETSA now provides a valuable tool to optimize such in vivo properties of stapled peptides.