Cdc14 phosphatases use an intramolecular pseudosubstrate motif to stimulate and regulate catalysis.

Cdc14 phosphatases are related structurally and mechanistically to protein tyrosine phosphatases (PTP) but evolved a unique specificity for phosphoSer-Pro-X-Lys/Arg sites primarily deposited by cyclin-dependent kinases. This specialization is widely conserved in eukaryotes. The evolutionary reconfiguration of the Cdc14 active site to selectively accommodate phosphoSer-Pro likely required modification to the canonical PTP catalytic cycle. While studying Saccharomyces cerevisiae Cdc14 we discovered a short sequence in the disordered C-terminus, distal to the catalytic domain, that mimics an optimal substrate. Kinetic analyses demonstrated this pseudosubstrate binds the active site and strongly stimulates rate-limiting phosphoenzyme hydrolysis, and we named it "substrate-like catalytic enhancer" (SLiCE). The SLiCE motif is found in all Dikarya fungal Cdc14 orthologs and contains an invariant glutamine, which we propose is positioned via substrate-like contacts to assist orientation of the hydrolytic water, similar to a conserved active site glutamine in other PTPs that Cdc14 lacks. AlphaFold2 predictions revealed vertebrate Cdc14 orthologs contain a conserved C-terminal alpha helix bound to the active site. Although apparently unrelated to the fungal sequence, this motif also makes substrate-like contacts and has an invariant glutamine in the catalytic pocket. Altering these residues in human Cdc14A and Cdc14B demonstrated that it functions by the same mechanism as the fungal motif. However, the fungal and vertebrate SLiCE motifs were not functionally interchangeable, illuminating potential active site differences during catalysis. Finally, we show that the fungal SLiCE motif is a target for phosphoregulation of Cdc14 activity. Our study uncovered evolution of an unusual stimulatory pseudosubstrate motif in Cdc14 phosphatases.

Development of Novel Phosphonodifluoromethyl-Containing Phosphotyrosine Mimetics and a First-In-Class, Potent, Selective, and Bioavailable Inhibitor of Human CDC14 Phosphatases.

Together with protein tyrosine kinases, protein tyrosine phosphatases (PTPs) control protein tyrosine phosphorylation and regulate numerous cellular functions. Dysregulated PTP activity is associated with the onset of multiple human diseases. Nevertheless, understanding of the physiological function and disease biology of most PTPs remains limited, largely due to the lack of PTP-specific chemical probes. In this study, starting from a well-known nonhydrolyzable phosphotyrosine (pTyr) mimetic, phosphonodifluoromethyl phenylalanine (F2Pmp), we synthesized 7 novel phosphonodifluoromethyl-containing bicyclic/tricyclic aryl derivatives with improved cell permeability and potency toward various PTPs. Furthermore, with fragment- and structure-based design strategies, we advanced compound 9 to compound 15, a first-in-class, potent, selective, and bioavailable inhibitor of human CDC14A and B phosphatases. This study demonstrates the applicability of the fragment-based design strategy in creating potent, selective, and bioavailable PTP inhibitors and provides a valuable probe for interrogating the biological roles of hCDC14 phosphatases and assessing their potential for therapeutic interventions.