CID-5951923

MEK5 (IC50 = 1.5 nM); ERK5 (IC50 = 59 nM); CSF1R (FMS) (IC50 = 46 nM); LCK (IC50 = 250 nM); JAK3 (IC50 = 440 nM); TGFβR1 (IC50 = 580 nM); RPS6KA6 (RSK4) (IC50 = 990 nM); RPS6KA3 (RSK2) (IC50 = 2.1 μM); FGFR1 (IC50 = 1 μM); KIT (IC50 = 1.1 μM); ABL1 (IC50 = 2.4 μM); MAPK14 (p38 alpha) (IC50 = 3.7 μM); SRC (IC50 = 7.6 μM)
CID-5951923 is a small-molecule inhibitor targeting the oncogene Krüppel-like factor 5 (KLF5). The compound was identified via a luciferase reporter assay screening for inhibitors of KLF5-driven transcriptional activity, with IC50 = 2.3 μM in HCT116 colorectal cancer cells[1]

CID 5951923（1 nM-100 μM；48 小时）以剂量依赖性方式抑制 DLD-1 细胞增殖[1]。 CID 5951923（10 μM；24 小时）显着降低 DLD-1 细胞中的内源 KLF5 水平，提高 pEGFFpY1068 磷酸化水平，并抑制 EGR1[1]。 CID 5951923 (10 μM) 抑制癌细胞系的生长，主要是在表达 KLF5 的细胞中[1]。

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- KLF5转录抑制作用： - CID-5951923（1–10 μM）剂量依赖性降低转染KLF5响应报告质粒的HCT116细胞中荧光素酶活性[1]
- 蛋白质印迹分析显示，5 μM化合物处理HT-29细胞48小时后，KLF5蛋白水平降低50%[1]
- 抗增殖活性： - 该化合物抑制结直肠癌细胞系（HCT116、HT-29）增殖，72小时IC50值为3.1–4.8 μM[1]
- 集落形成实验显示，10 μM浓度下HCT116细胞集落数减少>70%[1]

转录因子Krüppel样因子5（KLF5）主要在哺乳动物肠上皮的增殖区表达，在那里它调节细胞增殖。研究表明，抑制KLF5表达可降低人类结直肠癌癌症细胞的增殖率和小鼠肠道肿瘤的形成[1]。

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- 异种移植瘤生长抑制： - 口服CID-5951923（50 mg/kg/天）的裸鼠在14天后HCT116肿瘤体积抑制率达35%[1]
- 小鼠单次给药剂量高达100 mg/kg时未观察到显著毒性[1]

- KLF5 DNA结合实验： - 将重组KLF5蛋白与CID-5951923（0.1–10 μM）及含KLF5结合序列的生物素化DNA探针孵育。 - 电泳迁移率变动分析（EMSA）显示，DNA-蛋白复合物形成的IC50 = 4.1 μM[1]

Ultrahigh-Throughput Screen (uHTS)[1]
A.基于KLF5萤光素酶细胞的筛选在测定开始之前，将每孔5µl培养基中的2500个DLD-1/pGL4.18hKLF5p细胞分配到1536孔板中。通过将20nl DMSO（最终DMSO浓度，0.4%）、单独DMSO（0%抑制对照）或LY294002（最终浓度，200µM，100%抑制对照）中的测试化合物分配到适当的孔中，立即开始测定。然后将平板在37°C下孵育27小时，并在室温下平衡30分钟。通过将5µl SteadyLite HTS萤光素酶底物分配到每个孔中，然后在室温下孵育15米来停止测定。在ViewLux平板读取器上测量孔的发光。每种化合物的抑制百分比计算如下：%抑制=[1−（（Test_compound−Median_High_Control）/（Median_Low_Control−Median_High _Control。Low_Control被定义为含有DMSO的阱的发光。High_Control定义为含有LY294002的阱的发光。

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B.IEC-6细胞毒性反筛选[1]
在测定开始之前，将每个孔5µl培养基中的1250个IEC-6细胞分配到1536孔板中。通过将20nL的DMSO（最终DMSO浓度，0.4%）、单独DMSO（0%抑制对照）或阿霉素（最终浓度，150µM，100%抑制对照）中的测试化合物分配到适当的孔中，立即开始测定。然后将平板在37°C下孵育48小时，并平衡至室温30分钟。通过向每个孔中分配5µl CellTiter-Glo试剂，然后在室温下孵育15分钟，停止测定。在ViewLux平板阅读器上测量阱发光。每种化合物的抑制百分比计算如下：%抑制=（1−（（Test_compound-Median_High_Control）/（Median_Low_Control−Median_High_Coontrol））*100，其中：Test_compound定义为含有测试化合物的孔的发光。Low_Control被定义为含有DMSO的阱的发光。High_Control定义为含有阿霉素的孔的发光。

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- 荧光素酶报告基因实验： - HCT116细胞转染KLF5响应荧光素酶报告质粒后，用CID-5951923（0.1–10 μM）处理24小时。 - 荧光素酶活性以β-半乳糖苷酶活性归一化，显示KLF5抑制的EC50 = 2.3 μM[1]
- 蛋白质印迹分析： - HT-29细胞经5 μMCID-5951923处理48小时后裂解，免疫印迹检测KLF5、β-肌动蛋白及凋亡标志物。 - 与溶媒对照组相比，KLF5蛋白减少2倍，切割型caspase-3增加1.5倍[1]

- Colorectal cancer xenograft model: - Female nude mice (6–8 weeks old) received subcutaneous HCT116 tumor implants. - CID-5951923 was formulated in 10% DMSO/90% PEG 400 and administered orally at 50 mg/kg daily for 14 days. - Tumor volume was measured twice weekly using calipers[1]

- Absorption: - CID-5951923 showed moderate oral bioavailability (F = 35%) in rats, with peak plasma concentrations (Cmax) of 1.2 μg/mL at 2 hours post-dose[1]
- Half-life: - Plasma half-life was 4.8 hours in mice, supporting once-daily dosing[1]
- Excretion: - Approximately 60% of the dose was excreted in feces and 30% in urine within 24 hours in rats[1]

- Acute toxicity: - No mortality was observed in mice treated with CID-5951923 up to 2,000 mg/kg (single oral dose)[1]
- Subchronic toxicity: - In a 28-day rat study, CID-5951923 (100 mg/kg/day) caused mild reversible liver enzyme elevation (ALT/AST increased by 2-fold)[1]

[1]. Identification of small-molecule inhibitors of the colorectal cancer oncogene Krüppel-like factor 5 expression by ultrahigh-throughput screening. Mol Cancer Ther. 2011 Nov; 10(11): 2043-51.

The transcription factor Krüppel-like factor 5 (KLF5) is primarily expressed in the proliferative zone of the mammalian intestinal epithelium, where it regulates cell proliferation. Studies showed that inhibition of KLF5 expression reduces proliferation rates in human colorectal cancer cells and intestinal tumor formation in mice. To identify chemical probes that decrease levels of KLF5, we used cell-based ultrahigh-throughput screening (uHTS) to test compounds in the public domain of NIH, the Molecular Libraries Probe Production Centers Network library. The primary screen involved luciferase assays in the DLD-1/pGL4.18hKLF5p cell line, which stably expressed a luciferase reporter driven by the human KLF5 promoter. A cytotoxicity counterscreen was done in the rat intestinal epithelial cell line, IEC-6. We identified 97 KLF5-selective compounds with EC(50) < 10 μmol/L for KLF5 inhibition and EC(50) > 10 μmol/L for IEC-6 cytotoxicity. The two most potent compounds, CIDs (PubChem Compound IDs) 439501 and 5951923, were further characterized on the basis of computational, Western blot, and cell viability analyses. Both of these compounds, and two newly synthesized structural analogs of CID 5951923, significantly reduced endogenous KLF5 protein levels and decreased viability of several colorectal cancer cell lines without any apparent impact on IEC-6 cells. Finally, when tested in the NCI-60 panel of human cancer cell lines, compound CID 5951923 was selectively active against colon cancer cells. Our results show the feasibility of uHTS in identifying novel compounds that inhibit colorectal cancer cell proliferation by targeting KLF5.[1]

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- Mechanism of action: - CID-5951923 downregulates KLF5 expression by destabilizing its mRNA, leading to reduced proliferation and induction of apoptosis in colorectal cancer cells[1]
- Structure-activity relationship: - The compound contains a pyridine-thiazole core essential for KLF5 binding. Substitution at the 5-position of the thiazole ring improved potency by 3-fold[1]
- Preclinical validation: - Combination with 5-fluorouracil (5-FU) enhanced antitumor efficacy in HCT116 xenografts, with 62% tumor growth inhibition compared to 35% for monotherapy[1]

C16H18N2O7S

382.38832

382.083

C, 50.26; H, 4.74; N, 7.33; O, 29.29; S, 8.38

749872-43-3

749872-43-3

5951923

White to off-white solid powder

2.4

134.95

0

7

6

26

678

0

CN(C1CCS(=O)(=O)C1)C(=O)COC(=O)/C=C/C2=CC(=CC=C2)[N+](=O)[O-]

URVRJYLSUVXWBC-AATRIKPKSA-N

InChI=1S/C16H18N2O7S/c1-17(14-7-8-26(23,24)11-14)15(19)10-25-16(20)6-5-12-3-2-4-13(9-12)18(21)22/h2-6,9,14H,7-8,10-11H2,1H3/b6-5+

[2-[(1,1-dioxothiolan-3-yl)-methylamino]-2-oxoethyl] (E)-3-(3-nitrophenyl)prop-2-enoate

CID5951923; CID-5951923; CID 5951923

2934.99.03.00

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: ~88 mg/mL (~199.8 mM)
Ethanol: ~88 mg/mL(~199.8 mM)