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PhoRC—a New Approach Inducing Dephosphorylation of Target Proteins


Post-translational modifications (PTM), including ubiquitination, phosphorylation, methylation, lipidation and glycosylation, are important processes in protein maturity, which can influence the localization, activity, and stability of their conjugated proteins. In the past decade, proteolysis targeting chimera (PROTAC), a heterobifunctional small molecule consisting of a targeted protein binder, an E3 ligase binder and a linker, has been proved as a powerful chemical tool to degrade specific proteins. However, except ubiquitination, methods to manipulate other PTMs of proteins remain elusive. A recent paper in J. Med. Chem described a pioneering work of using a novel heterobifunctional molecule to induce dephosphorylation of a targeted kinase.

Phosphorylation is the prerequisite for the activities of kinases and their downstream signaling pathways. In contrast, dephosphorylation of kinases is an effective approach to inhibit kinase activities. In this paper, the researchers conjugated a reported PP1-activating synthetic peptide (PDP1), which binds a ubiquitously expressed phosphatase PP1, with a Protein kinase B (AKT) inhibitor via a PEG linker. This heterobifunctional molecule 3 greatly decreased the pAKTT308 levels in a recombinant biochemical system in a PPI-dose dependent manner. 

However, this molecule failed to change the cellular pAKTT308 levels due to its poor permeability or potential proteolytic instability. To address this problem, the authors synthesized another heterobifunctional molecule 4a with a shorter and better permeable PPI peptide. As anticipated, 4a greatly decreased the level of pAKTT308 and pAKTS473 in prostate cancer LNCaP cells at 10 µM, whereas the negative control 4b did not. Consistent with the previous biochemical assay, PP1 binder 6 could restore the levels of pAKTT308 and pAKTS473 through disrupting the ternary complex formation.


To test the generality of this targeted dephosphorylation strategy, the authors synthesized a bifunctional molecule 7 with a covalent EGFR inhibitor and a HaloTag reactive chloroalkane. As expected, 7 substantially decreased the pEGFRY1068 level in the HaloTag-PP1-FLAG transfected cells. 

Although the in vitro activities of the compounds developed in this study were not highly potent due to their poor permeabilities, this work expanded the applications of heterobifunctional molecule in manipulating PTMs of proteins and provided a good starting point to develop more potent phosphatase recruiting chimeras (PhoRCs). 
Reference: Yamazoe S, et. al. JHeterobifunctional Molecules Induce Dephosphorylation of Kinases-A Proof of Concept Study. J. Med. Chem. 2020, 63, 2807−2813.

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