Small Molecules Target the Interaction between Tissue Transglutaminase and Fibronectin

Abstract

Tissue transglutaminase (TG2) is a multifunctional protein with enzymatic, GTP-ase, and scaffold properties. TG2 interacts with fibronectin (FN) through its N-terminus domain, stabilizing integrin complexes, which regulate cell adhesion to the matrix. Through this mechanism, TG2 participates in key steps involved in metastasis in ovarian and other cancers. High-throughput screening identified several small molecule inhibitors (SMI) for the TG2/FN complex. Rational medicinal chemistry optimization of the hit compound (TG53) led to second-generation analogues (MT1-6). ELISA demonstrated that these analogues blocked TG2/FN interaction, and bio-layer interferometry (BLI) showed that the SMIs bound to TG2. The compounds also potently inhibited cancer cell adhesion to FN and decreased outside-in signaling mediated through the focal adhesion kinase. Blockade of TG2/FN interaction by the small molecules caused membrane ruffling, delaying the formation of stable focal contacts and mature adhesions points and disrupted organization of the actin cytoskeleton. In an in vivo model measuring intraperitoneal dissemination, MT4 and MT6 inhibited the adhesion of ovarian cancer cells to the peritoneum. Pretreatment with MT4 also sensitized ovarian cancer cells to paclitaxel. The data support continued optimization of the new class of SMIs that block the TG2/FN complex at the interface between cancer cells and the tumor niche.

Figure 1.. Chemical structures

of TG53 (parent compound) and of the 6 derivatives targeting the TG2-FN protein-protein interaction (MT1-MT6).

Figure 2.. TG53 derivatives inhibit TG2-FN interaction.

A, ELISA measured the interaction between His-tagged TG2 and biotinylated FN42 in the presence of vehicle (DMSO, control), TG53 and MT1-MT6 at 10μM and 25μM concentrations. Bars represent means ± SD (n=3). B, Bio-layer Interferometry (BLI) sensograms monitor the real time association and dissociation kinetics of FN45 (captured on streptavidin-coated sensors) and TG2. The TG2 concentrations used at the association step are indicated. The nonlinear regression fits from 1:1 global analysis are shown as thin black lines; k_a = 5320 M⁻¹s⁻¹ (±0.2%), k_d =0.0016 s⁻¹ (±0.4%), yielding K_D = 0.30 nM; goodness of fit: R²= 0.996084. C, Concentration-dependent inhibitory effect of MT4 on the FN45-TG2 interaction. For each MT4 concentration tested, the extent of binding (Rmax) was corrected for nonspecific binding of (TG2+MT4) to bare sensors. D, Bio-layer Interferometry (BLI) sensograms show the real time association and dissociation kinetics of FN45 (20 μg/ml captured on streptavidin-coated sensors) and 1 μM TG2 pre-incubated with MT4 at various concentrations. E, Solid phase assay measured SKOV3 cells’ adhesion to FN (5μg/mL) in the presence of TG53 and MT1–6 (n = 4). F-H, Dose-dependent effect of MT4 (2μM-25μM) on SKOV3 (F), OVCAR433 (G), and OVCAR5 (H) cells adhesion onto FN-coated plates was measured by a solid phase assay (see SM). For all experiments, results represent the mean and SD of at least triplicate samples: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 3.. TG53 inhibits formation of a complex with integrin β1 and activation of FAK and c-Src during SKOV3 cell attachment onto FN.

A–D, Confocal microscopy analysis of TG2 (Cy5, red; ×600) co-localization with integrin β1, p-FAK (Y576/577), c-Src, and p-c-Src (Y416) (Alexa Fluor 488, green) in the presence of either 1μM TG53 (lower panels) or DMSO vehicle (upper panels). Graphs quantifying co-localization of the two proteins are presented. *p<0.05, **p<0.01.

Figure 4.. MT-4 inhibits early OVCAR5 cell attachment onto FN by interfering with the formation of stable focal contacts.

A–C, IF staining of adhesion complex factors in OVCAR5 cells upon cell adhesion to FN. A, Confocal imaging of intracellular localization of vinculin-positive focal adhesion points (white, upper panel), pFAK (white, lower panel), and actin filaments (green, upper and lower panel) at 30 minutes after seeding of OVCAR5 cells onto FN coated chamber slides in the presence or absence of inhibitors (TG53 and MT-4 at 1μM concentration) or vehicle (DMSO). Cells were treated with the SMIs for 72 hours prior to seeding and during the assay. Insets provide detailed view of the vinculin-positive structures. Scale bar=10μm. B, Analysis of the actin cytoskeleton assessed morphological differences during cell attachment. Scale bar=10μm. C, Grayscale images of actin cytoskeleton in TG53- (left) and MT4- treated cells (right). Scale bar=20μm. D, TIRF microscopy analysis of OVCAR5 cells pre-treated with SMIs for 1h to determine the distribution of integrin β1 (red, upper panels) at the interface with FN during cell attachment. Actin cytoskeleton is depicted in green (lower panels). Scale bar=10μm.

Figure 5.. Effects of SMIs targeting the TG2/FN complex on “outside-in” signaling and adhesion to mesothelial surfaces.

A, Western blot analysis of total and phosphorylated FAK and ERK was performed using cell lysates extracted from OVCAR cells plated on FN for 15 to 45 minutes in the presence of TG53 (left) or MT-4 (right) at 5μM concentration, as compared to DMSO. GAPDH was used as a control. OVCAR5 cells had been pretreated with the inhibitors for 72 hours. Densitometry analysis used the Quantity One software and results were normalized to GAPDH loading controls. DMSO 0 min time point was considered as 1 for comparative analysis. Values are shown for each band. B, A modified cell adhesion assay measured the effects of TG53, MT-4, MT-5, and MT-6 (25μM) on SKOV3 cells’ adhesion onto semi-confluent LP9 monolayers. C, Dose-dependent effects of TG53, MT-4, MT-5, and MT-6 on OVCAR433 cells adhesion onto semi-confluent LP9 monolayers. D–E, Wound healing assay measures migration of SKOV3 and OVCAR5 cells in the presence of MT-4, MT-5, MT-6 at a concentration of 8 μM (black bars) or vehicle (open bar). Migration rate was determined over a period of 24 hours by measuring the average distance between the wound borders at the beginning and at the end of the assay interval (n=4). F–G, SKOV3-GFP OC cells were injected ip in NSG mice in the presence of either MT-4 (10μg/kg) (F) or MT-6 (10μg/kg) (G) and allowed to attach to the peritoneal wall. After 2 hours, the cell suspension was recovered from the peritoneal cavity through peritoneal washings and the non-attached OC cells recovered were counted. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 6.. Priming of OVCAR5 cells with MT-4 sensitizes cells to paclitaxel (PTX).

A–B, The CCK8 assay was used to measure cell viability of SKOV3 cells treated with increasing concentrations of TG53 and MT1–6 for 72 hours. C–D, Colony formation from OVCAR-5 cells pre-treated with 1μM MT-4 or 1μM TG53 for 72 hours before seeding in the presence or absence of 5nM PTX (n=6; 10 days incubation to allow colonies formation). Colonies were stained with 0.4% crystal violet and counted using ImageJ. E–F, Sphere formation assay measured proliferation of OVCAR-5 cells pre-treated with 1μM MT-4 or 1μM TG53. Cells were allowed to form spheroids in the presence or absence of 5nM PTX (n = 3). Spheres were counted after ten days. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.