Discovery and Assessment of Chemical Probes Targeting Regulatory Surfaces of P‐Rex1, a Metastatic RhoGEF
Authors: Cash Jennifer N , Sharma Prateek , Shost Michael , Davis Ellen , Tesmer John 
Published: November 2023

Phosphatidylinositol 3,4,5‐trisphosphate (PIP3)‐dependent Rac exchanger 1 (P‐Rex1) is a Rho guanine‐nucleotide exchange factor (RhoGEF) that regulates cell motility and is strongly associated with cancer metastasis. P‐Rex1 is synergistically recruited to the cell membrane and activated by PIP3 and heterotrimeric G protein βγ (Gβγ) subunits, positioning the enzyme downstream of multiple classes of cell surface receptors that control processes such as cytoskeleton rearrangement and cell migration. P‐Rex1 has thus become an attractive therapeutic target for the suppression of cancer metastasis. However, development of selective inhibitors against P‐Rex1 is hindered by the fact that its regulatory mechanisms are poorly understood. Our primary goal is to define the molecular basis for regulation of P‐Rex1 by PIP3 and Gβγ using X‐ray crystallography and biochemical and cell‐based assays, with the aim of identifying the surfaces of P‐Rex1 that are important for interaction with these molecules as well as their mechanisms of activation. We have structurally characterized the P‐Rex1 tandem Dbl homology (DH)/pleckstrin homology (PH) domain catalytic core and elucidated how the PH domain interacts with PIP3. Using site‐directed mutagenesis, we have shown that the PH domain is necessary and sufficient for PIP3‐dependent activation, and we believe that PIP3 activates P‐Rex1 through an allosteric mechanism as opposed to simple recruitment to the cell membrane. Our structural analysis led to the identification of a likely protein‐protein interaction site on the PH domain that may be important to regulation of P‐Rex1, either by other domains within the enzyme or by other molecules. In support of this, mutations at this site result in increased P‐Rex1 activity in cells, and, in addition, we show that domains outside the catalytic core are involved in autoinhibition. Furthermore, P‐Rex1 domain truncation along with bio‐layer interferometry experiments using Gβγ have helped to elucidate which domains of P‐Rex1 participate in its regulation by Gβγ. These studies have also opened the door to medium‐throughput screens that led to the identification of several small molecules that bind the PH domain of P‐Rex isoforms. Differential scanning fluorimetry and crystallographic experiments support that these molecules engage residues involved in PIP3 binding, suggesting that they may compete with this physiological activator of P‐Rex1. We are currently testing these small molecules for inhibition of P‐Rex‐dependent cell migration. Ultimately, these compounds may be modified to create high affinity, specific small molecules that could be used therapeutically as P‐Rex inhibitors to combat cancer metastasis.Support or Funding InformationThis work was supported by an American Cancer Society – Michigan Cancer Research Fund Postdoctoral Fellowship (PF‐14‐224‐01‐DMC) to J.C. and an American Heart Association Undergraduate Student Research Program Award (14UFEL20510027) to E.D.