KLF5 (IC50 = 29 nM)
ML264 potently halts DLD-1 viability (IC50 = 29 nM) with high maximal effect (>90%). Human colorectal adenocarcinoma cells are DLD-1 cells. Other cell types, such as HCT116 (human colorectal carcinoma), HT29 (human colorectal adenocarcinoma), and SW620 (human colorectal adenocarcinoma), are also significantly affected by ML264 at submicromolar doses. With inhibition below 50% at the highest dose, the IEC-6 anti-target (a nontransformed rat intestinal epithelial cell line) is largely unaffected[1]. Through changes to the cell cycle profile, this substance effectively prevents the proliferation of CRC cells in vitro[2].

ML264 potently halts DLD-1 viability (IC50 = 29 nM) with high maximal effect (>90%). Human colorectal adenocarcinoma cells are DLD-1 cells. Other cell types, such as HCT116 (human colorectal carcinoma), HT29 (human colorectal adenocarcinoma), and SW620 (human colorectal adenocarcinoma), are also significantly affected by ML264 at submicromolar doses. With inhibition below 50% at the highest dose, the IEC-6 anti-target (a nontransformed rat intestinal epithelial cell line) is largely unaffected[1]. Through changes to the cell cycle profile, this substance effectively prevents the proliferation of CRC cells in vitro[2].

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ML264 (10 µM) significantly inhibited the proliferation of DLD-1 and HCT116 colorectal cancer (CRC) cell lines within 24 hours, with a 15- to 30-fold reduction in live cell numbers compared to vehicle after 72 hours. [2]
MTS assay confirmed reduced metabolic activity/viability in DLD-1 and HCT116 cells treated with 10 µM ML264 over 72 hours. [2]
Treatment with 10 µM ML264 significantly reduced the number of mitotic figures in DLD-1 cells at 24, 48, and 72 hours. [2]
Cell cycle analysis showed that 10 µM ML264 treatment caused a significant decrease in G0/G1 phase population and an increase in S-phase population in both DLD-1 and HCT116 cells, suggesting S-phase arrest. Changes in G2/M population were cell line-dependent. [2]
Annexin V/PI staining indicated a modest but significant increase in apoptotic cells (both early and late apoptotic) in DLD-1 and HCT116 cells treated with 10 µM ML264, with the effect more prominent at 48-72 hours. [2]
Western blot analysis showed that 10 µM ML264 treatment decreased protein levels of KLF5 and EGR1 in both DLD-1 and HCT116 cells over 72 hours. [2]
ML264 treatment (10 µM) modulated components of MAPK, PI3K, and WNT pathways: it decreased p-EGFR and EGFR levels in HCT116 but increased p-EGFR in DLD-1; decreased total ERK but increased p-ERK in both lines; decreased levels of AKT, pAKT, GSK3β, pGSK3β, and β-catenin phosphorylated at Ser552 in both cell lines. In HCT116, total β-catenin and β-catenin phosphorylated at Thr41/Ser45 were also reduced. [2]
ML264 treatment (10 µM) downregulated protein and mRNA levels of cyclins E1, A2, and B1 in both DLD-1 and HCT116 cells over 72 hours. Cyclin D1 protein was downregulated in HCT116 but not significantly in DLD-1, and its mRNA levels were largely unchanged. [2]

In a well-established mouse xenograft model of colon cancer, ML264 effectively inhibits tumor growth five days after administration. Since ML264 potently inhibits the expression of KLF5 and EGR1, a transcriptional activator of KLF5, this effect is brought on by a significant decrease in proliferation[2].

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In a DLD-1 xenograft model in nude mice, intraperitoneal (i.p.) administration of ML264 at 10 mg/kg twice daily or 25 mg/kg twice daily for 10 days significantly inhibited tumor growth compared to vehicle, with effects detectable as early as 2 days post-treatment. A single daily dose of 10 mg/kg was ineffective. [2]
Histological analysis of xenografts from mice treated with ML264 (25 mg/kg twice daily) showed a significant reduction in mitotic figures. [2]
Immunohistochemistry and Western blot analysis of xenograft tumors revealed significant reduction in KLF5 and EGR1 protein levels after ML264 treatment (10 and 25 mg/kg twice daily). [2]
Ki-67 staining (proliferation marker) was significantly reduced in xenografts from ML264-treated mice (25 mg/kg twice daily). [2]
Xenografts from ML264-treated mice showed increased fibrosis (vimentin staining) and inflammation (increased Mac-3 positive mononuclear phagocytes) compared to vehicle-treated controls. [2]

ML264 is highly active (IC50=29 nM is a cell-based assay for proliferation of DLD-1 cells, IC50=81 nM in a cell-based luciferase assay). ML264 lacks cytotoxicity in the IEC-6 control cell line (IC50>50 μM,<50% inhibition is observed at 100 μM). Robust activity is also seen in several other KLF5-expressing cell types as well (e.g., HCT116, IC50=560 nM; HT29, IC50=130 nM; SW620, IC50=430 nM). Western blot analysis shows that ML264 significantly reduces KLF5 expression.

DLD-1 and HCT116 cells are treated with 10 μM ML264 or DMSO in experiments testing cell proliferation. Live cells are collected 24, 48, and 72 hours after treatment, and the number of each are counted using a Coulter counter. DLD-1 and HCT116 cells are treated with 10 μM ML264 or with the control (DMSO) in the MTS assay. Each well receives 20 μL of MTS solution following incubations of 24, 48, and 72 hours. An analysis is then carried out in accordance with the manufacturer's instructions.

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Cell Proliferation and Viability Assay: DLD-1 and HCT116 cells were seeded in plates with medium containing either DMSO (vehicle) or 10 µM ML264. Live cell counts were performed at 24, 48, and 72 hours post-treatment using a cell counter. For MTS assay, cells were treated similarly, and after incubation periods, MTS solution was added to each well and absorbance was measured according to standard protocol. [2]
Cell Cycle Analysis: DLD-1 and HCT116 cells were seeded in plates and treated with DMSO or 10 µM ML264. After 24, 48, and 72 hours, cells were collected, fixed, stained with propidium iodide (PI), and analyzed by flow cytometry to determine cell cycle distribution. [2]
Apoptosis Assay: DLD-1 and HCT116 cells were treated with DMSO or 10 µM ML264. After 24, 48, and 72 hours, cells were stained with Alexa Fluor 488 Annexin V and PI according to the kit instructions and analyzed by flow cytometry to quantify apoptotic cells. [2]
Mitotic Figures Quantification (In Vitro): DLD-1 cells were seeded on slides with medium containing DMSO or 10 µM ML264, fixed after 24, 48, and 72 hours, and stained with Hoechst for DNA. The number of cells undergoing mitosis was counted in five fields (100 cells per field). [2]
Western Blot Analysis: Total protein was extracted from DLD-1 and HCT116 cells treated with DMSO or 10 µM ML264 at various time points using Laemmli buffer. Proteins were separated by SDS-PAGE, transferred to membranes, and probed with specific primary antibodies, followed by appropriate secondary antibodies for detection. [2]
Quantitative PCR (qPCR): Total RNA was extracted from treated DLD-1 and HCT116 cells using TRIzol reagent. cDNA was synthesized, and qPCR was performed using SYBR Green chemistry and gene-specific primers for KLF5, EGR1, CTNNB1, CCND1, CCNE1, CCNA2, CCNB1, and GAPDH. Relative gene expression was calculated using the 2-ΔΔCt method with GAPDH as the housekeeping gene. [2]

Mice: In ventilated, filtered cages with positive pressure, naked mice are kept in a pathogen-free environment. The right flank of 6-7 week old male nude mice is injected subcutaneously with 5×106 DLD-1 human colorectal cells to produce xenograft tumors. By measuring with a caliper and using established formulas, tumor volume is calculated. Mice are treated intraperitoneally (i.p.) with ML264 at doses of 10 mg/kg daily, 10 mg/kg twice daily, and 25 mg/kg twice daily for a total of 10 days when tumor volumes reach approximately 100 mm6. The control treatment is the vehicle solution. Each and every two days, mice are observed and weighed. When the tumor's largest measurement reaches 2 cm, the experiment is over. Tumors are removed and saved for additional research[2].

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Xenograft Efficacy Study: Nude mice were subcutaneously injected with 5×10^6 DLD-1 human colorectal cancer cells into the right flank. When tumors reached approximately 100 mm³, mice were randomized and treated intraperitoneally (i.p.) for 10 days with varying regimens: vehicle (control), ML264 at 10 mg/kg once daily, 10 mg/kg twice daily, or 25 mg/kg twice daily. The vehicle solution consisted of 80% dH2O, 10% DMSO, and 10% Tween 80. Tumor dimensions were measured regularly with calipers, and volume was calculated. Mice were weighed every two days. The study was terminated when tumors reached 2 cm in greatest dimension, at which point tumors were excised for analysis. [2]
Tissue Analysis: Excised tumors were fixed, paraffin-embedded, and sectioned. Sections were used for Hematoxylin and Eosin (H&E) staining, immunohistochemistry (IHC) for KLF5, EGR1, vimentin, and Mac-3, and immunofluorescence for Ki-67. Mitotic figures were counted in H&E-stained sections. Protein was also extracted from tumor tissues for Western blot analysis. [2]

In vitro drug metabolism and pharmacokinetics (DMPK) studies showed that ML264 has high stability in liver microsomes, suggesting high stability against first-pass metabolism. [2] ML264 does not inhibit the activity of cytochrome P450 isoenzymes. [2] In vivo DMPK studies in mice showed that the plasma half-life of ML264 is approximately 2 hours. [2] The bioavailability of ML264 orally in mice is 47%. [2]

ML264 has been reported to be inactive against 47 kinases and 67 protein targets of therapeutic/toxicological significance, suggesting that it may be selective. [2] ML264 does not inhibit cytochrome P450 isoenzymes, indicating that it has a low risk of drug interactions in this regard. [2] Compared with the vector control group, nude mice treated with ML264 twice daily at doses up to 25 mg/kg for 10 days did not show significant changes in body weight. [2]

[1]. ML264: An Antitumor Agent that Potently and Selectively Inhibits Krüppel-like Factor Five (KLF5) Expression: A Probe for Studying Colon Cancer Development and Progression.

[2]. ML264, A Novel Small-Molecule Compound That Potently Inhibits Growth of Colorectal Cancer. Mol Cancer Ther. 2016 Jan;15(1):72-83.

ML264 is a third-generation small molecule compound discovered through ultra-high-throughput screening (uHTS) to find KLF5 expression inhibitors for the treatment of colorectal cancer. [2] KLF5 is a transcription factor overexpressed in intestinal tumors and a mediator of the RAS/MAPK and WNT signaling pathways. ML264 is thought to exert its antitumor effect by downregulating KLF5 and its activator EGR1. [2] The mechanism by which ML264 inhibits KLF5 expression is not fully elucidated; it may affect transcription directly or indirectly. [2] Current research is underway to identify the direct molecular targets of ML264 and to test its efficacy in a genetic mouse model of intestinal adenoma. [2]

C17H21CLN2O4S

384.88

384.091

C, 53.05; H, 5.50; Cl, 9.21; N, 7.28; O, 16.63; S, 8.33

1550008-55-3

51003603

White to off-white solid powder

1.4±0.1 g/cm3

701.0±60.0 °C at 760 mmHg

377.7±32.9 °C

0.0±2.2 mmHg at 25°C

1.599

0.46

1

4

5

25

601

0

ClC1=CC=CC(=C1)/C=C/C(NCC(N(C)C1CCS(CC1)(=O)=O)=O)=O

AJCDZIDKYKCOMZ-AATRIKPKSA-N

InChI=1S/C17H21ClN2O4S/c1-20(15-7-9-25(23,24)10-8-15)17(22)12-19-16(21)6-5-13-3-2-4-14(18)11-13/h2-6,11,15H,7-10,12H2,1H3,(H,19,21)/b6-5+

(E)-3-(3-chlorophenyl)-N-[2-[(1,1-dioxothian-4-yl)-methylamino]-2-oxoethyl]prop-2-enamide

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)

Solubility in Formulation 1: ≥ 3.25 mg/mL (8.44 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 32.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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