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Purity: ≥98%
COTI-2 is an orally available, third-generation small molecule activator of the mutant p53 protein (a tumor suppressor). It has potential anti-cancer activity because it can restore the mutant p53 protein to its wild-type conformation. An additional AKT2 inhibitor is COTI-2. Both in vitro and in vivo testing has shown that COTI-2 is highly effective against numerous cancer cell lines from a variety of human cancers. Additionally, it showed better efficacy when compared to conventional chemotherapeutic and targeted-therapeutic agents and a low toxicity profile in mice. Early research on COTI-2's mechanism of action shows that it is neither a conventional kinase inhibitor nor an inhibitor of the Hsp90 protein, but instead kills cancer cells by inducing apoptosis. Clinical trials are currently being conducted with COTI-2.
| Targets |
p53
Mutant p53; PI3K/AKT/mTOR pathway [1] Mutant p53; PI3K/AKT/mTOR pathway [2] |
|---|---|
| ln Vitro |
The thiosemicarbazone COTI-2 is said to encourage the refolding of mutant p53 and restore the function of wild-type p53. At nanomolar concentrations, it is effective against human tumor cell lines originating from a variety of sources and causes cell death through apoptosis[1]. Both in vitro and in vivo, COTI-2 is incredibly effective against a variety of cancer cell lines from a wide range of human cancers. Over 200 kinases from important cancer-related kinase pathways evaluated in both kinase assays were not significantly inhibited by COTI-2, and COTI-2 did not inhibit Hsp90's ATPase activity, a widely distributed molecular chaperone crucial to cell survival and cell cycle regulation[2].
COTI-2 is a small molecule that targets mutant p53 and negatively modulates the PI3K/AKT/mTOR pathway, showing potential as an anticancer agent [1] COTI-2 exhibited potent anti-proliferative activity against a diverse panel of human cancer cell lines (covering colorectal, lung, glioblastoma, ovarian, breast, leukemia, endometrial, and pancreatic cancers) with nanomolar IC₅₀ values, regardless of tissue origin or genetic background (e.g., TP53, KRAS, PIK3CA mutations). It was more effective than cetuximab, erlotinib, cisplatin, and BCNU in inhibiting proliferation of colorectal cancer (SW620, COLO-205, HCT-15), NSCLC (H292, H1975), and glioblastoma (U87-MG, SNB-19) cell lines. Treatment with COTI-2 induced apoptosis in cancer cells (e.g., SHP-77, DMS-114, DMS-153) via caspase cascade activation, as evidenced by Annexin V/7AAD staining and abrogation of apoptosis by the pan-caspase inhibitor Z-VAD-FMK. It reduced phosphorylation of AKT (Ser473) and caspase-9 (Thr125) in SHP-77 cells. COTI-2 did not significantly inhibit over 200 kinases or Hsp90 ATPase activity [2] |
| ln Vivo |
At a dose of 10 mg/kg, COTI-2 significantly slowed the growth of the xenografts of the human colorectal tumor HT-29. At doses as low as 3 mg/kg, COTI-2 also markedly slowed the growth of the tumor in the SHP-77 SCLC xenograft model. OVCAR-3, MDA-MB-231, and U87-MG xenograft growth are all delayed by its treatment. A secure toxicity profile is shown in vivo by COTI-2 treatment. As shown by the in vitro data, COTI-2 selectively targets a wide range of human cancer cell lines while having minimal negative effects on normal cells[2].
COTI-2 demonstrated antitumor efficacy in multiple mouse xenograft models. In HT-29 colorectal cancer xenografts (NCr-nu mice), intraperitoneal (IP) administration of 10 mg/kg COTI-2 5 days/week for 7 weeks significantly inhibited tumor growth and delayed tumor progression (control tumors reached 618 mm³ in 32 days vs. 48 days for treated tumors). In SHP-77 SCLC xenografts, 3 mg/kg COTI-2 IP once every 2 days for 38 days was more effective than cisplatin (3 mg/kg/week) and paclitaxel (12.5 mg/kg once every 2 days) in suppressing tumor growth. For U87-MG glioblastoma xenografts (nude mice), 8 mg/kg COTI-2 IP 3 times/week delayed tumor growth (control tumors reached 828 mm³ in 5 days vs. 10 days for treated tumors). Oral (PO) administration of 200 mg/kg COTI-2 5 days/week delayed MDA-MB-231 breast cancer xenograft growth. In OVCAR-3 ovarian cancer xenografts (NIH III nu/nu mice), 20 mg/kg COTI-2 IV 3 times/week induced tumor regression, and 75 mg/kg PO 5 days/week maintained low stable tumor volumes for 60 days. COTI-2 was well-tolerated with no overt toxicity or weight loss in tumor-bearing and non-tumor-bearing mice [2] |
| Enzyme Assay |
The interaction of COTI-2 with 227 kinases is tested using the AMBIT BIOSCIENCES KINOMESCAN assay. In a brief, affinity resins for kinase assays are produced by treating streptavidin-coated magnetic beads with biotinylated small molecule ligands for 30 min at 25°C. The liganded beads are blocked with excess biotin and then washed in blocking buffer (1% BSA, 0.05% Tween 20, 1 mM DTT), which helps to remove unbound ligand and lessen non-specific binding. In one binding buffer (20% SeaBlock, 0.17× PBS, 0.05% Tween 20, 6 mM DTT), phage lysates, liganded affinity beads, and COTI-2 are combined. In a final volume of 0.1 mL, all reactions are conducted in polystyrene 96-well plates that have already been pre-treated with blocking buffer.
ATP-competitive kinase assay: Streptavidin-coated magnetic beads were loaded with biotinylated ligands, blocked, and incubated with phage lysates, beads, and COTI-2 (0.1% DMSO final concentration) at 25°C for 1 h. After washing, bound kinases were eluted and quantified by plaque assay or qPCR to assess binding affinity. Radiometric functional kinase assay: Kinase reactions were performed with γ-³³P-ATP in reaction buffer containing substrates, cofactors, and COTI-2 (3-fold serial dilutions starting at 100 μM). Activity was detected via P81 filter-binding to measure substrate phosphorylation. Hsp90 ATPase inhibition assay: A fluorescence polarization assay was used to test COTI-2 (10-dose IC₅₀, 3-fold serial dilutions starting at 100 μM) for competition with FITC-geldanamycin for binding to Hsp90α and Hsp90β [2] |
| Cell Assay |
For 48 hours, SHP-77 cells were cultured with different COTI-2 concentrations. The cells were then stained with Annexin V and 7AAD after being washed twice with 1X cold PBS. In a nutshell, 1 × 105 cells were treated with 5 l of Annexin V and 7AAD, and they were then left to sit in the dark at room temperature for 15 minutes. A 400 μl dose of the 1X binding buffer was then injected into the cells.
Proliferation/viability assays: Cancer cells were seeded in 96-well plates, treated with serial concentrations of COTI-2 (or comparator drugs), and incubated for 4-7 days. Viability was measured via alamarBlue (metabolic activity) or CellTiter-Blue assays, and IC₅₀ values were calculated from 3-4 independent experiments. Apoptosis assay: SHP-77, DMS-114, and DMS-153 cells were treated with COTI-2 (IC₅₀ concentrations) with/without Z-VAD-FMK (20 μM pre-treatment), stained with Annexin V-FITC/PI or Annexin V/7AAD, and analyzed by flow cytometry. Western blot: SHP-77 cells were treated with 250 nM COTI-2 for 3-6 h, lysed, and proteins separated by electrophoresis. Membranes were probed with antibodies against phospho-AKT, phospho-caspase-9, cleaved caspase-9, and β-tubulin [2] |
| Animal Protocol |
SHP-77 and HT-29 cells are injected into the flanks of NCr-nu mice (2×106 cells per injection site) using 50% matrigel (n = 5 mice per group). With regard to SHP-77 xenografts, COTI-2 therapy is started before palpable tumors appear. Animals are given 3 mg/kg of COTI-2 (once every two days, up to 38 days) a day after SHP-77 cells are injected into them. Standard caliper measurements are used to estimate the size of tumors at 5, 10, 17, 24, and 38 days. The ability of COTI-2 to inhibit the growth of established tumors is evaluated in the context of HT-29 xenografts. Before beginning IP treatment with COTI-2 (10 mg/kg, 5 days a week for 7 weeks) or saline IP, HT-29 xenografts are allowed to reach a size of 200 mm3. Every 4 days, caliper measurements are used to measure tumor growth.
Xenograft models: HT-29 (2×10⁶) or SHP-77 (2×10⁶) cells in 50% matrigel were injected subcutaneously (SC) into NCr-nu mice flanks. For HT-29, treatment started when tumors reached ~200 mm³: 10 mg/kg COTI-2 IP 5 days/week for 7 weeks. For SHP-77, treatment started 1 day post-injection: 3 mg/kg COTI-2 IP once every 2 days for 38 days. U87-MG cells (2×10⁶) in 50% matrigel were injected SC into nude mice hind legs; 8 mg/kg COTI-2 IP 3 times/week was administered when tumors reached 200-300 mm³. MDA-MB-231 cells (2×10⁶) were injected into SCID mice flanks; 200 mg/kg COTI-2 (dissolved in phosphate-citrate buffer, pH 2.3) was given PO 5 days/week when tumors reached 100-200 mm³. OVCAR-3 cells (7×10⁶) were injected SC into NIH III nu/nu mice thighs; 20 mg/kg COTI-2 IV 3 times/week or 75 mg/kg PO 5 days/week was administered when tumors reached 75-100 mm³. Tumor volume was calculated as π/6 × longest diameter × (shortest diameter)². Toxicity study: BALB/c mice were given 4, 8, or 16 mg/kg COTI-2 IP 3 times/week for 28 days; weight and toxicity signs were monitored [2] |
| Toxicity/Toxicokinetics |
COTI-2 showed good safety in vivo. In immunocompetent BALB/c mice, intraperitoneal injection of 4-16 mg/kg COTI-2 for 28 days did not result in weight loss, hair loss, bruising at the injection site, or limited activity. In tumor-bearing mice (OVCAR-3, HT-29, U87-MG models), intraperitoneal, intravenous, or oral treatment with COTI-2 for up to 60 days did not result in significant toxicity or weight loss.[2]
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| References | |
| Additional Infomation |
COTI-2, a mutant p53 activator, is an orally administered third-generation thioaminourea compound that activates mutant p53 protein and has potential antitumor activity. After oral administration, COTI-2 targets and binds to misfolded mutant p53 protein, inducing a conformational change that restores p53 protein to normal and reactivates it. This can induce apoptosis in tumor cells with mutated p53 protein. In addition, COTI-2 inhibits Akt2 activation and prevents activation of the PI3K/AKT/mTOR pathway, thereby inducing apoptosis in cancer cells overexpressing this pathway. p53 is a tumor suppressor protein that plays a key role in controlling cell proliferation and survival. High levels of mutant p53 are present in many cancers, which are associated with uncontrolled cell growth. COTI-2 is a third-generation thioaminourea compound that was identified by the CHEMSAS® computational platform (which integrates pharmacology, statistical modeling, medicinal chemistry and machine learning) [2].
COTI-2 is not a traditional kinase or Hsp90 inhibitor, and its exact mechanism of action is still under investigation [2]. Currently, COTI-2 is being clinically studied as an anticancer drug targeting mutant p53-driven tumors [1]. |
| Molecular Formula |
C19H22N6S
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|---|---|---|
| Molecular Weight |
366.48
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| Exact Mass |
366.162
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| Elemental Analysis |
C, 62.27; H, 6.05; N, 22.93; S, 8.75
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| CAS # |
1039455-84-9
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| Related CAS # |
1204956-74-0 (HCl);1039455-84-9;
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| PubChem CID |
91810660
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| Appearance |
Light yellow to yellow solid powder
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| Density |
1.3±0.1 g/cm3
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| Boiling Point |
574.1±60.0 °C at 760 mmHg
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| Flash Point |
301.0±32.9 °C
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| Vapour Pressure |
0.0±1.6 mmHg at 25°C
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| Index of Refraction |
1.715
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| LogP |
0.79
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
2
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| Heavy Atom Count |
26
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| Complexity |
516
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| Defined Atom Stereocenter Count |
0
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| SMILES |
S=C(N/N=C1\C2C(=CC=CN=2)CCC\1)N1CCN(C2C=CC=CN=2)CC1
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| InChi Key |
UTDAKQMBNSHJJB-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C19H22N6S/c26-19(23-22-16-7-3-5-15-6-4-10-21-18(15)16)25-13-11-24(12-14-25)17-8-1-2-9-20-17/h1-2,4,6,8-10H,3,5,7,11-14H2,(H,23,26)
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| Chemical Name |
N-(6,7-dihydro-5H-quinolin-8-ylideneamino)-4-pyridin-2-ylpiperazine-1-carbothioamide
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| Synonyms |
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 0.67 mg/mL (1.83 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 6.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.7287 mL | 13.6433 mL | 27.2866 mL | |
| 5 mM | 0.5457 mL | 2.7287 mL | 5.4573 mL | |
| 10 mM | 0.2729 mL | 1.3643 mL | 2.7287 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
| NCT Number | Recruitment | interventions | Conditions | Sponsor/Collaborators | Start Date | Phases |
| NCT02433626 | Unknown | Drug: COTI2 Drug: Cisplatin |
Ovarian Cancer HNSCC |
Critical Outcome Technologies Inc. | December 2015 | Phase 1 |
A.COTI-2, a third generation thiosemicarbazone, was designed using the CHEMSAS computational platform.B.Human cancer cell lines were treated with COTI-2.Tumor cell proliferation was examined 72 h after treatment with COTI-2. The IC50values were calculated from four independent experiments. Error bars indicate SEM.Oncotarget. 2016 Jul 5; 7(27): 41363–41379. |
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COTI-2 is significantly more effective than cetuximab or erlotinib in inhibiting colorectal cancer cell line proliferation.Oncotarget. 2016 Jul 5; 7(27): 41363–41379. |
Human glioblastoma cell lines are sensitive to COTI-2 treatment.Oncotarget. 2016 Jul 5; 7(27): 41363–41379. |
COTI-2 induces apoptosis in human cancer cells.Oncotarget. 2016 Jul 5; 7(27): 41363–41379. |
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COTI-2 treatment inhibits human HT-29 and SHP-77 xenograft growth.Oncotarget. 2016 Jul 5; 7(27): 41363–41379. |
COTI-2 treatment delays U87-MG xenograft growth.Oncotarget. 2016 Jul 5; 7(27): 41363–41379. |
COTI-2 treatment inhibits OVCAR-3 xenograft growth.Oncotarget. 2016 Jul 5; 7(27): 41363–41379. |
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COTI-2 demonstrates a safe toxicity profilein vivo.Oncotarget. 2016 Jul 5; 7(27): 41363–41379. |
Refolding and reactivation of missense-mutant p53.Nat Rev Cancer.2018 Feb;18(2):89-102. |
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