Structural and Functional Studies of the Metastatic Factors P‐Rex1 and P‐Rex2: Toward Small Molecule Inhibitor Development

• Published in: The FASEB journal Vol. 31; no. S1
• Main Authors: Sharma, Prateek, Davis, Ellen M., Cash, Jennifer, Tesmer, John J. G.
• Format: Journal Article
• Language: English
• Published: 01-04-2017
• ISSN: 0892-6638; 1530-6860
• DOI: 10.1096/fasebj.31.1_supplement.619.6

Abstract only
Phosphatidylinositol 3,4,5‐trisphosphate (PIP
3
)‐dependent Rac exchanger 1 (P‐Rex1) is a Rho guanine‐nucleotide exchange factor (RhoGEF) that regulates cytoskeletal rearrangement and cell motility and is linked to tumor metastasis. The P‐Rex family includes P‐Rex1 and P‐Rex2, which are activated by both the lipid PIP
3
, generated by activated receptor tyrosine kinases, and free Gβγ subunits, generated by activated GPCRs. However, the structural basis behind functional regulation with P‐Rex is poorly understood. My objective is to determine the structure of P‐Rex2 PH and characterize the interaction of small molecules with different PH domains through qualitative analysis using differential scanning fluorimetry (DSF) and detailed assessment through X‐ray crystallography. Rosetta (DE3) pLysS E. coli (Novagen) cells were used to overexpress P‐Rex2 PH constructs and recombinant protein was purified using Ni‐NTA resin and size exclusion chromatography. A DSF assay was used to analyze the binding interaction between compounds and PH domains. Crystallographic studies led to a high‐resolution structure (1.90 Å) of the independent P‐Rex2 PH domain. A comparison between the P‐Rex1 PH and P‐Rex2 PH structures shows divergence in the β3/β4 loop, shown by our lab to play a role in non‐specific membrane localization, and β5/β6 loop, a likely protein‐protein interaction site. Using DSF, we identified six small molecules from a medium‐throughput screen that bind the P‐Rex1 PH domain. P‐Rex2 and Akt PH (a kinase PH domain which has been successfully targeted with small molecule inhibitors) were used to further assess compound binding to a variety of PH domains. IP
4
, a soluble analog of PIP
3
, resulted in a 12.3 °C shift in T
m
for P‐Rex1 PH and a 15.4 °C shift for P‐Rex2. Compound #13 (20 μM) was most notable in causing a 15.5 °C shift for P‐Rex1, 22.5 °C for P‐Rex2, and 13.5 °C for Akt PH. We are currently co‐crystallizing P‐Rex1 PH with these small molecules to verify that they target the PIP
3
‐binding site. As PIP
3
binding is necessary for P‐Rex activation, small molecules can be developed to perturb this interaction, resulting in therapeutics with anti‐metastatic properties.
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. and an American Heart Association Undergraduate Student Research Program Award (14UFEL20510027) to E.D.