ZINC40099027 activates human focal adhesion kinase by accelerating the enzymatic activity of the FAK kinase domain

Abstract

Focal adhesion kinase (FAK) regulates gastrointestinal epithelial restitution and healing. ZINC40099027 (Zn27) activates cellular FAK and promotes intestinal epithelial wound closure in vitro and in mice. However, whether Zn27 activates FAK directly or indirectly remains unknown. We evaluated Zn27 potential modulation of the key phosphatases, PTP‐PEST, PTP1B, and SHP2, that inactivate FAK, and performed in vitro kinase assays with purified FAK to assess direct Zn27‐FAK interaction. In human Caco‐2 cells, Zn27‐stimulated FAK‐Tyr‐397 phosphorylation despite PTP‐PEST inhibition and did not affect PTP1B‐FAK interaction or SHP2 activity. Conversely, in vitro kinase assays demonstrated that Zn27 directly activates both full‐length 125 kDa FAK and its 35 kDa kinase domain. The ATP‐competitive FAK inhibitor PF573228 reduced basal and ZN27‐stimulated FAK phosphorylation in Caco‐2 cells, but Zn27 increased FAK phosphorylation even in cells treated with PF573228. Increasing PF573228 concentrations completely prevented activation of 35 kDa FAK in vitro by a normally effective Zn27 concentration. Conversely, increasing Zn27 concentrations dose‐dependently activated kinase activity and overcame PF573228 inhibition of FAK, suggesting the direct interactions of Zn27 with FAK may be competitive. Zn27 increased the maximal activity ( V max ) of FAK. The apparent K m of the substrate also increased under laboratory conditions less relevant to intracellular ATP concentrations. These results suggest that Zn27 is highly potent and enhances FAK activity via allosteric interaction with the FAK kinase domain to increase the V max of FAK for ATP. Understanding Zn27 enhancement of FAK activity will be important to redesign and develop a clinical drug that can promote mucosal wound healing.

An αvβ3 integrin checkpoint is critical for efficient TH2 cell cytokine polarization and potentiation of antigen-specific immunity

“…Next, we assessed whether enforced FAK signalling could rescue the cytokine defect in αvβ3-deficient T H 2 cells. Itgav iCD4KO T H 2 cells were cultured in the presence of tamoxifen to induce αvβ3 deletion, and additionally with either DMSO (vehicle) or ZINC40099027 (Zn27), an activator of FAK which is reported to interact with the FAK kinase domain and enhance its enzymatic activity for ATP 25 , 26 . Notably, culture of αvβ3-deficient T H 2 cells with Zn27 resulted in an increase in IL-13 and IL-5 production as compared to vehicle controls ( Fig.…”

The Critical Role of Growth Factors in Gastric Ulcer Healing: The Cellular and Molecular Mechanisms and Potential Clinical Implications

“…It provides a way to stabilize and control the delivery of growth factors necessary for optimal tissue repair and healing. Another promising group of treatments for accelerating the healing of GI ulcers are the recently developed small molecule FAK activators that promote human intestinal epithelial cell migration, monolayer wound closure, and mouse ulcer healing [101][102][103]. Regarding potential clinical translation, several growth factors, e.g., PDGF and recombinant human EGF, are used to treat diabetic foot ulcers by local injection into the wound sites, and several other growth factors are in clinical trials for wound healing [100,[104][105][106][107][108].…”

A novel drug-like water-soluble small molecule Focal Adhesion Kinase (FAK) activator promotes intestinal mucosal healing

“…We previously reported that the FAK activator ZN40099027 enhances FAK activity via interaction with the FAK kinase domain to increase the Vmax of FAK for ATP( RashmiVomhof-DeKrey et al., 2021 ). ZN40099027 at 10 ​nM activates FAK by 14.8% in suspended Caco-2 ​cells ( Wang et al., 2019b ).…”