Kinetic Analyses on the Binding of the RhoGEF P‐Rex1 by Gβγ and Potential Small Molecule Inhibitors
Authors: Shost Michael , Cash Jennifer , Tesmer John 
Published: November 2023

Phosphatidylinositol‐3,4,5‐triphosphate (PIP3)‐dependent Rac exchanger (P‐Rex1) is a Rho guanine nucleotide exchange factor (GEF) for Rac GTP‐binding proteins integral to cytoskeletal rearrangement and cell migration. In cases of some metastatic tumors, elevated expression of P‐Rex1 accompanies cancer cell migration. Cell‐membrane associated PIP3 acts to regulate P‐Rex1 by binding a cleft located in the pleckstrin homology (PH) domain. Additional activation and membrane recruitment is provided by the βγ subunits of the heterotrimeric G protein. The position of this activity is still unclear; however, and locating the residues of interest will identify a means by which to modify P‐Rex1 signaling. The second part of our investigation in P‐Rex1 activity is to develop and test a small molecule inhibitor for the established PIP3 activation site on the PH domain. By examining these synergistic paths of P‐Rex1 activation, we hope to arrest cancer metastasis in a clinical setting through regulation of a key cell motility protein. We quantified the interaction between Gβγ and P‐Rex1 using bio‐layer interferometry (BLI). We produced truncated P‐Rex1 constructs for the selective deletion of domains to isolate the regions necessary for Gβγ binding and activation. Results from the truncation of P‐Rex1 after the N‐terminal domains demonstrated a weaker affinity for Gβγ compared to the full‐length protein. This result supports the formation of a tertiary structure involving N‐terminally located domains and their C‐terminally located counterparts. In this conformation, multiple domains of P‐Rex1 contribute to the promotion of enzyme activation by Gβγ. In addition to identifying the site of Gβγ binding, targeting the PIP3 binding site through small molecule inhibition will provide alternate means of P‐Rex1 regulation. High‐throughput screening by differential scanning fluorimetry (DSF) produced candidates to test for P‐Rex1 inhibition. The small molecules that demonstrated thermostabilization of the P‐Rex1 PH domain in DSF were further tested using BLI to determine binding kinetics. Preliminary binding data for these compounds provides affinities in agreement with DSF results. Further analysis counter‐screening with the PH domain of Akt, which also binds PIP3, will allow us to confirm binding to the PIP3 site while developing inhibitors to interact with the P‐Rex1 activation site discriminately. This result supports the affinity of our potential inhibitors for PIP3 binding sites, but suggests that an alteration to increase P‐Rex1 specificity may be necessary. The synergistic activation of P‐Rex1 via Gβγ and PIP3 provides two alternate pathways to the regulation of this cell motility protein. Locating the site for Gβγ binding will provide us a target for future modulation of P‐Rex1 signaling. By developing a small molecule inhibitor for the well‐characterized activation site of PIP3, we hope to halt excessive enzyme activation. Investigating these processes will not only extend our understanding of P‐Rex regulation, but may provide a means by which to manage cancer metastasis.

Support or Funding Information

This work was supported by an American Cancer Society – Michigan Cancer Research Fund Postdoctoral Fellowship (PF‐14‐224‐01‐DMC) to J.C.