COTI-2, an orally available thiosemicarbazone, is an activator of mutant forms of the p53 protein with potential antineoplastic activity.

COTI-2 (72 h) potently inhibits the proliferation rate of all the tested cell lines. In COLO-205, HCT-15, and SW620 cell lines, COTI-2 markedly inhibits tumor cell proliferation. Relatively low concentrations of COTI-2 are active against all human glioblastoma cell lines tested (U87-MG, SNB-19, SF-268, and SF-295). In SHP-77 cells, COTI-2 treatment with IC50 concentrations causes the induction of early apoptosis among 40 to 47% of total cells.

In the HT-29 human colorectal tumor xenografts, COTI-2 (10 mg/kg) markedly inhibits tumor growth. Except for reducing tumor volumes at specific times post-treatment, COTI-2 also increases the time required for tumors to reach specified volumes. In the SHP-77 SCLC xenograft model, COTI-2 (3 mg/kg) also markedly inhibits tumor growth. COTI-2 can reduce U87-MG tumor volumes at specific times post-treatment and lengthen the time required for U87-MG xenografts to grow in nude mice. Control tumors in mice treated with vehicle alone take only 5 days to reach an average volume of 828 mm3 while tumors in animals treated with COTI-2 take double that time (10 days) to reach a similar mean volume (857 mm3). Regardless of the route of administration, COTI-2 potently inhibits OVCAR-3 xenograft growth.

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°C 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.

COTI-2 is dissolved in 100% dimethyl sulfoxide stock solution and diluted in medium plus FBS such that final DMSO concentrations are 0.5 to 1.0% depending on the 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.

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 the 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.