E260 is a Fer and FerT inhibitor that selectively generates metabolic stress in cancer cells by causing mitochondrial malfunction and deformation and activating energy-consuming autophagy, ultimately lowering cellular ATP levels. To demonstrate that E260 directly targets Fer and FerT, in vitro kinase tests were performed utilizing purified kinase domain (KD)-containing fragments of these enzymes. This investigation indicates a direct inhibitory action of E260 on this domain, demonstrating a substantial drop in the autophosphorylation level of Fer/FerT KD upon incubation with ATP and increasing concentrations of E260. Furthermore, computational examination of E260 docking in the simulated complete Fer protein demonstrated that the highest scoring binding mode of E260 to Fer lay within the ATP-binding pocket of enzyme KD. To estimate the dissociation constant (Kd) of E260 from Fer/FerT KD, microscale thermophoresis (MST) assays were performed using increasing concentrations of E260. This research confirmed the direct binding of E260 to the Fer/FerT KD and established the Kd to be 0.85 µM. To assess the effect of E260 micellar formulations on Fer in cancerous cells, immunoprecipitation of kinases was done in untreated and E260-treated SW620 CC cells. When given to metastatic grade IV SW620 CC cells (serum deprived for 16 hours and treated with 3 mM H2O2 to activate Fer), E260 displayed an inhibitory effect on Fer kinase activity, which was reflected in the suppression of the autophosphorylation activity of the enzyme. To describe the effect of E260 on malignant cells, metastatic SW620 cells were treated with E260 and then analyzed for vitality. The commencement of death was detected in E260-treated cells with EC50 values of 400 nM after 24 hours and 300 nM after 48 hours. E260 exhibits an EC50 of 3.2 µM after 72 hours of treatment of non-metastatic PANC-1 cells originating from primary pancreatic ductal cancer. Furthermore, the maximal amount of death in these cells was around 70% after 72 hours of treatment with 4 µM E260. In contrast, metastatic ductal carcinoma cell line SU.86.86 was demonstrated to be more susceptible to E260, with an EC50 of 1.1 µM after 72 hours of treatment, and 2 µM E260 resulted in 100% fatal levels [1].

In vivo xenograft progression is inhibited by E260. In mice, the pharmacokinetic (PK) profile of E260 was established. E260 is efficiently dispersed in animal tissues, as evidenced by its T1/2 in blood of 175 minutes and distribution volume of 4244 mL/kg. Swine Pancreatic SW620 cells were injected subcutaneously into immunocompromised "nude" mice in order to assess the effect of E260 on tumor growth. During the course of the trial, the administration of E260 significantly slowed the progression of the tumor, and after 22 days of treatment, the mean tumor volume had decreased by ten times. E260's in vivo anticancer effect was further demonstrated by treating mice with xenografts made of SW48 cells, and tracking the development of tumors was done. When compared to control treatment groups, the progression of the tumor was halted five or six times in mice administered E260 [1].

[1]. A novel Fer/FerT targeting compound selectively evokes metabolic stress and necrotic death in malignant cells. Nat Commun. 2017 Oct 16;8(1):940.

C24H34N6S

438.6

438.256

1241537-79-0

46898309

White to off-white solid powder

4.7

0

6

5

31

566

0

CC(C)C1=CC=C(C2=CN3C(SC(N4CCC(CN5CCN(C)CC5)CC4)=N3)=N2)C=C1

RFHSWNJYHRAOMP-UHFFFAOYSA-N

InChI=1S/C24H34N6S/c1-18(2)20-4-6-21(7-5-20)22-17-30-23(25-22)31-24(26-30)29-10-8-19(9-11-29)16-28-14-12-27(3)13-15-28/h4-7,17-19H,8-16H2,1-3H3

2-[4-[(4-methylpiperazin-1-yl)methyl]piperidin-1-yl]-6-(4-propan-2-ylphenyl)imidazo[2,1-b][1,3,4]thiadiazole

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~1 mg/mL (~2.28 mM)

Solubility in Formulation 1: ≥ 0.1 mg/mL (0.23 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 1.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 0.1 mg/mL (0.23 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (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 1.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 0.1 mg/mL (0.23 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 1.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 22 mg/mL (50.16 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)