The AlphaLISA assay is used to screen the ChemDiv library.

<p>A, The assay identifies SMIs that inhibit the TG2-FN42 interaction by >50% at 10 μM. B, Dose dependent inhibition of TG2-FN42 interaction by selected compounds. Bars represent means +/− SD of triplicate measurements. Asterisks denote <i>p <</i>0.05. C, Selected compounds are grouped in two categories based on chemical structure: diamino-pyrimidines and pyrolydinyl-pyrimidines.</p

Characteristics of the AlphaLISA^TM assay developed to measure the TG2-FN42 interaction.

<p>A, Design of the AlphaLISA assay that measures the TG2-FN interaction. Donor beads coated with streptavidin and acceptor nickel chelate beads were used to capture biotinylated FN42 and His tagged TG2 protein, respectively. After excitation at 680 nm singlet oxygen is transferred from donor to acceptor beads coming within a distance of 200 nm, resulting in a chemiluminescence signal. B, Cross-titration was performed to optimize detection of the TG2-FN interaction by the assay. Saturation isotherms of FN42 binding to TG2 were generated. The K_d was 2.43 nM. C, Titration curves represented with GraphPad Prism demonstrate reaching the hook point at 3 nM biotinylated FN42 and 10 nM TG2-His.</p

Small Molecule Inhibitors Target the Tissue Transglutaminase and Fibronectin Interaction

<div><p>Tissue transglutaminase (TG2) mediates protein crosslinking through generation of ε−(γ-glutamyl) lysine isopeptide bonds and promotes cell adhesion through interaction with fibronectin (FN) and integrins. Cell adhesion to the peritoneal matrix regulated by TG2 facilitates ovarian cancer dissemination. Therefore, disruption of the TG2-FN complex by small molecules may inhibit cell adhesion and metastasis. A novel high throughput screening (HTS) assay based on AlphaLISA™ technology was developed to measure the formation of a complex between His-TG2 and the biotinylated FN fragment that binds TG2 and to discover small molecules that inhibit this protein-protein interaction. Several hits were identified from 10,000 compounds screened. The top candidates selected based on >70% inhibition of the TG2/FN complex formation were confirmed by using ELISA and bioassays measuring cell adhesion, migration, invasion, and proliferation. In conclusion, the AlphaLISA bead format assay measuring the TG2-FN interaction is robust and suitable for HTS of small molecules. One compound identified from the screen (TG53) potently inhibited ovarian cancer cell adhesion to FN, cell migration, and invasion and could be further developed as a potential inhibitor for ovarian cancer dissemination.</p></div

ELISA-based approach measures the TG2-FN interaction.

<p><b>A</b>, Design of the ELISA measuring the TG2-FN interaction. His tagged TG2 is captured by the anti-His antibody coating the wells. Biotinylated FN42 interacting with TG2 is recognized by streptavidin-HRP, which reacts with a TMB substrate. The signal is abrogated if the TG2-FN interaction is disrupted. <b>B</b>, Specificity of the assay is demonstrated by incubating His-tagged TG2 with increasing concentrations of biotinylated FN42 (from 0 nM to 16 nM) in the presence of unlabeled FN42 (from 0 nM to 16 nM). <b>C</b>, ELISA in the presence of the competitive inhibitor 4G3, an anti-TG2 antibody against the FN-binding domain of TG2 (5 μg/ml), and in the presence of unlabeled FN42 (3 nM). <b>D</b>, ELISA measures inhibition of the TG2-FN42 interaction by SMIs selected from the AlphaLISA HTS. Bars represent means +/− SD of triplicate measurements. Asterisks denote <i>p <</i>0.05. <b>E</b>, ELISA measures dose dependent inhibition of TG2-FN42 interaction by TG53. <b>F</b>, Saturation curves of FN42 in the presence of increasing concentrations of TG53 were used to calculate the Ki (4.15 μM) of TG53 for TG2. Inset corresponds to representative Lineweaver-Burk plots showing that TG53 competes for the same binding site in TG2 as FN42.</p

Effect of TG53 on SKOV3 cell migration, invasion, and proliferation.

<p><b>A</b>, Representative phase-contrast microscopy images of cells migrating into the wounded area in an <i>in vitro</i> scratch wound healing assay. SKOV3 cells incubated in serum-free media supplemented with 1% DMSO or TG53 diluted to 10 μM in DMSO at time 0 (0.5 h) and 24 hours after wounding. <b>B</b>, Wound closure was assessed as the distance between the wound edges, calculated and quantified using Image Pro-Express software 4.01. The percentage of wound closure was evaluated using the formula (wound size at 24 hrs/ initial wound size) × 100. Data are shown as means ± SD of triplicate experiments. <b>C</b>, Cell migration rate (μm/h) was quantified as the difference between wound edges at 0 and 24 h, divided by 24. Results are means ± SD of three independent experiments. <b>D</b>, Numbers of SKOV3 cells invading through matrigel coated transwells were counted in 5 random fields. Results are means ± SD of duplicate experiments. <b>E</b>, Effects of TG53 (1–30 μM) on TGase activity, measured as described and compared to control (DMSO). <b>F</b>, Effects of top inhibitors selected from the AlphaLISA on SKOV3 cell proliferation measured by CCK-8 assay. Dose-response curves representing percentage of surviving cells were plotted using GraphPad Prism against the logarithmic concentrations of drugs used during 48 h treatment. Four SMIs were tested: TG37, TG53, TG63 and TG64.</p

List of top hits from the HTS using AlphaLISA and their predicted physico-chemical characteristics.

<p>List of top hits from the HTS using AlphaLISA and their predicted physico-chemical characteristics.</p

Top SMIs selected from the AlphaLISA based HTS inhibit cell adhesion to FN.

<p><b>A</b>, Effects of top compounds (25 μM) on SKOV3 cells adhesion to wells coated by 5 μg/mL of FN. <b>B</b>, Effects of top compounds (25 μM) on IGROV1 cells adhesion to FN. <b>C</b>, Dose-dependent effects of compound TG53 (1–25 μM) on SKOV3 cells adhesion to FN. <b>D</b>, Comparison between TG53 (25 μM) and a structurally similar, but inactive compound (TG288, 25 μM), on SKOV3 cells adhesion. <b>E</b>, Effects of TG53 (25 μM) on SKOV3 cells adhesion to wells coated with collagen type I (20 µg/ml). Bars represent means +/− s.e.m. of quadruplicate measurements. Asterisks denote <i>p <</i>0.05.</p