Kinase experiment:

The interaction of COTI-2 with 227 kinases is tested using the AMBIT BIOSCIENCES KINOMESCAN assay. In brief, streptavidin-coated magnetic beads are treated with biotinylated small molecule ligands for 30 min at 25℃ to generate affinity resins for kinase assays. The liganded beads are blocked with excess biotin and washed with blocking buffer (1% BSA, 0.05% Tween 20, 1 mM DTT) to remove unbound ligand and to reduce non-specific binding. Binding reactions are assembled by combining phage lysates, liganded affinity beads, and COTI-2 in 1× binding buffer (20% SeaBlock, 0.17× PBS, 0.05% Tween 20, 6 mM DTT). All reactions are carried out in polystyrene 96-well plates that have been pre-treated with blocking buffer in a final volume of 0.1 mL[2].

Cell experiment:

SHP-77 cells are cultured with various concentrations of COTI-2 for 48 h. Cells are then washed twice with 1× cold PBS and stained with Annexin V and 7AAD according to the manufacturer's instructions. Briefly, 5 μL of Annexin V and 7AAD are added to 1×105 cells and incubated for 15 min at room temperature in the dark. Then 400 μL of the 1× binding buffer (100 mM HEPES, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) is added to the cells. Finally, cells are analyzed using a flow cytometer[2].

Animal experiment:

SHP-77 and HT-29 cells are injected in 50% matrigel into flanks of NCr-nu mice (2×106 cells per injection site) (n=5 mice per group). In the case of SHP-77 xenografts, treatment with COTI-2 begins prior to the appearance of palpable tumors. One day after injection of SHP-77 cells, animals receive 3 mg/kg of COTI-2 (once every two days, up to 38 days). Tumor sizes are estimated at 5, 10, 17, 24, and 38 days, by standard caliper measurements. In the case of HT-29 xenografts, the capacity of COTI-2 to suppress growth of established tumors is assessed. HT-29 xenografts are allowed to grow to 200 mm3 before starting IP treatment with COTI-2 (10 mg/kg, 5 days a week for 7 weeks) or saline IP. Tumor growth is measured every 4 days by caliper measurement[2].

COTI-2 activates mutant forms of p53.

p53 has varoius mechanisms of anticancer function and plays a critical role in genomic stability, apoptosis and inhibition of angiogenesis.

In vitro: Previous study tested the efficacy of COTI-2 against a diverse group of human cancer cell lines with different genetic mutation backgrounds. Results showed that COTI-2 efficiently inhibited the proliferation rate of all the tested cell lines following 72 h of treatment. Most cell lines showed nanomolar sensitivity to COTI-2 treatment. In addition, COTI-2 was significantly more effective at inhibiting tumor cell proliferation than either cetuximab or erlotinib in COLO-205, HCT-15, and SW620 cell lines. Notably, all three lines were insensitive to growth inhibition to any degree in response to low concentrations of cetuximab and erlotinib, but highly sensitive to even low doses of COTI-2 [1].

In vivo: The effects of COTI-2 on inhibiting the growth of HT-29 and SHP-77 xenografts in immunocompromised mice was assessed when intraperitoneally administered. It was found that COTI-2 at 10 mg/kg could significantly inhibit tumor growth in the HT-29 human colorectal tumor xenografts. In addition to reducing tumor volumes at specific times post-treatment, COTI-2 could also delay the time required for tumors to reach specified volumes [1].

Clinical trial: A study of COTI-2 for the treatment of advanced or recurrent gynecologic malignancies is currently recruiting patients [2].

References:
[1] Salim, K. Y.,Vareki, S.M.,Danter, W.R., et al. COTI-2, a novel small molecule that is active against multiple human cancer cell lines in vitro and in vivo. Oncotarget 7(27), (2016).
[2] https://clinicaltrials. gov/ct2/show/NCT02433626term=COTI-2&rank=1