Discovery of Small Molecules That Target the Phosphatidylinositol (3,4,5) Trisphosphate (PIP₃)-Dependent Rac Exchanger 1 (P-Rex1) PIP₃-Binding Site and Inhibit P-Rex1–Dependent Functions in Neutrophils

Abstract

Phosphatidylinositol (3,4,5) trisphosphate (PIP 3)-dependent Rac exchanger 1 (P-Rex1) is a Rho guanine-nucleotide exchange factor that was originally discovered in neutrophils and is regulated by G protein bg subunits and the lipid PIP 3 in response to chemoattractants. P-Rex1 has also become increasingly recognized for its role in promoting metastasis of breast cancer, prostate cancer, and melanoma. Recent structural, biochemical, and biologic work has shown that binding of PIP 3 to the pleckstrin homology (PH) domain of P-Rex1 is required for its activation in cells. Here, differential scanning fluorimetry was used in a medium-throughput screen to identify six small molecules that interact with the P-Rex1 PH domain and block binding of and activation by PIP 3. Three of these compounds inhibit N-formylmethionylleucyl-phenylalanine induced spreading of human neutrophils as well as activation of the GTPase Rac2, both of which are downstream effects of P-Rex1 activity. Furthermore, one of these compounds reduces neutrophil velocity and inhibits neutrophil recruitment in response to inflammation in a zebrafish model. These results suggest that the PH domain of P-Rex1 is a tractable drug target and that these compounds might be useful for inhibiting P-Rex1 in other experimental contexts. SIGNIFICANCE STATEMENT A set of small molecules identified in a thermal shift screen directed against the phosphatidylinositol (3,4,5) trisphosphate-dependent Rac exchanger 1 (P-Rex1) pleckstrin homology domain has effects consistent with P-Rex1 inhibition in neutrophils.

<p>Identification of P-Rex1 in the Regulation of Liver Cancer Cell Proliferation and Migration via HGF/c-Met/Akt Pathway</p>

“…39,40 More recent studies have identified a set of small molecules against P-Rex1 by differential scanning fluorimetry, but inhibition of P-Rex1-related cell function needs to be further confirmed. 41 Liver cancer is an extraordinarily heterogeneous malignancy with a poor prognosis and a lack of curative treatments that requires continued research efforts. This study demonstrated that P-Rex1 is involved in liver cancer cell proliferation, migration, and tumor growth by modulating Akt and Erk1/2 phosphorylation via c-Met targeting.…”

P-Rex1 Cooperates With TGFβR2 to Drive Lung Fibroblast Migration in Pulmonary Fibrosis

“…P-Rex1 deficiency attenuated bleomycin-induced lung inflammatory response. A combination of TGF-β1 signal pathway inhibitors with P-Rex1 inhibitor would shed light on therapeutic approaches for better pulmonary fibrosis treatment ( Lucato et al, 2015 ; Cash et al, 2020 ).…”

Identification of G Protein αi Signaling Partners by Proximity Labeling Reveals a Network of Interactions that Includes PDZ-RhoGEF

“…Neutrophils were isolated from human peripheral blood as described previously [56]. Freshly isolated blood was carefully layered on top of 1-step polymorphs (AN221725, Accurate chemicals and scientific corporation) (1:1 Blood and Polymorphs) and centrifuged at 1000× g for 45 min and buffy coat was transferred to fresh tubes.…”