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. 2021 Apr;9(2):e00737.
doi: 10.1002/prp2.737.

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

Rashmi  1 , Shyam K More  1 , Qinggang Wang  1 , Emilie E Vomhof-DeKrey  1   2 , James E Porter  2 , Marc D Basson  1   2   3
Affiliations

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

Rashmi et al. Pharmacol Res Perspect. 2021 Apr.

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 (Vmax ) of FAK. The apparent Km 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 Vmax 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.

Keywords: focal adhesion kinase; migration; mucosal repair; nonsteroidal anti-inflammatory drugs.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIGURE 1
FIGURE 1
Studies of potential interactions between Zn27 and the tyrosine phosphatases that inactivate FAK. (A) Zn27 (10 nM) stimulated FAK‐Tyr‐397 phosphorylation as measured by Western blot in Caco‐2 cells without or with PTP LYP inhibitor (50 μM) (n = 25, *p < .05). (B) Densitometric analysis of the blots described in (A) Because PTP LYP inhibition dramatically accentuated FAK phosphorylation, a Y‐axis break has been placed to be able to visually represent the effects of Zn27 on FAK phosphorylation in both the absence and presence of PTP LYP inhibitor on the same graph. (C) Lack of effect of 10 nM Zn27 on FAK‐PTP1B association in suspended Caco‐2 cells. Cell lysates were immunoprecipitated with anti‐FAK and then blotted for either co‐precipitating PTP1B (top panel) or FAK itself (bottom panel). Input is shown in the first two lanes and the immunoprecipitant in the third and fourth lanes. (D) Densitometric analysis of eight similar studies to the one shown in C demonstrates that treating with Zn27 (10 nM) does not affect the amount of PTP1B co‐precipitating with FAK (n = 8) (E) Lack of effect of Zn27 (100 nM) on SHP2 activity (n = 2). All data are represented as mean ± SE, *p < .05
FIGURE 2
FIGURE 2
Zn27 directly stimulates the activity of FAK. The chemical structures (A) ZINC40099021 (Zn21) and (B) ZINC40099027 (Zn27) drawn using PubChem Sketcher (https://pubchem.ncbi.nlm.nih.gov//edit3/index.html). 84 (C) A schematic diagram represents the different domains of FAK. (D) Zn27 (10 nM) directly stimulates the Tyr‐397 autophosphorylation of full length (125 kDa) (Figure represents pooled data from 9 experiments with 4 replicates in each experiment, n = 9, *p < .05) and 35 kDa FAK (Figure represents pooled data from 8 experiments with 4 replicates in each experiment, n = 8, *p < .05). (E) Zn27 stimulates the conversion of ATP to ADP by highly purified full‐length 125 kDa human FAK in an in vitro kinase assay (Figure represents pooled data from 3 experiments with 5 replicates in each experiment, n = 3, *p < .05) (F) Zn27 binding stimulates the conversion of ATP to ADP by the 35 kDa FAK kinase domain in an in vitro kinase assay compared to negative control Zn21 (Figure represents pooled data from 2 experiments with 4 replicates in each experiment, n = 2, *p < .05). (G) Dose–response curve of Zn27 as an agonist of the 35 kDa kinase domain of FAK. (Figure represents pooled data from 9 experiments with 3 replicates in each experiment, n = 9, *p < .05)
FIGURE 3
FIGURE 3
Competition displacement study (A) The effect of PF573228 (10 μM) on Zn27 (10 nM) induced phosphorylation of FAK‐Tyr‐397 in suspended Caco‐2 cells (p < .05, n = 8, * Zn27 significant vs. DMSO p < .05; * Zn27+PF573228 (10, 100, 1000 nM) significant vs. DMSO, p < .05; # Zn27+PF573228 significant vs. Zn27 p < .05). (B) Dose‐dependent inhibition of Zn27 (10 nM) activation of 35 kDa FAK kinase domain by PF573228 (IC50 8.4 ± 0.04 nM). (Figure represents pooled data from 9 experiments with 4 replicates in each experiment, n = 9, *p < .05). (C) Concentration‐dependent activation of 35 kDa kinase domain of FAK by Zn27 in the absence or presence of PF573228 (10 nM). *’s represents comparison of luminescence at varying concentrations of Zn27 in the absence or presence of 10 nM PF573228. Increasing concentration of Zn27 causes a rightward shift of the concentration–response curve. Figure represents pooled data from 21 experiments with 3 replicates in each experiment, n = 21, *p < .05. (D) EC50 for Zn27 in absence and presence of 10 nM PF573228 (n = 21, *p < .05)
FIGURE 4
FIGURE 4
Saturation‐binding assay for ATP in the presence or absence of FAK for Zn27 (10 nM). The saturation curve representing the total binding, specific binding, and nonspecific‐binding activity of FAK with increasing concentrations (0–300 μM) of ATP (A) DMSO (B) Zn27 (10 nM). (Figure represents pooled data from 6 experiments with 3 replicates in each experiment, n = 6, *p < .05) (C) Specific binding for ATP was determined in the presence or absence of Zn27 (10 nM). Specific binding = Total‐(Nonspecific) (Figure represents pooled data from 6 experiments with 3 replicates in each experiment, n = 6, *p < .05) (D and E) Effect of Zn27 (10 nM) on V max (D) and K m (E). The figure represents pooled data from six experiments with three replicates in each experiment, n = 6, *p < .05
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