LDC195943 (IMT1)

Human mitochondrial RNA polymerase (POLRMT) (IC50 = 0.12 μM in recombinant in vitro transcription assay) [1]

在 A2780、A549 和 HeLa 细胞中，IMT1（0.00001-10 μM；0-168 小时）会剂量依赖性地降低细胞活力。与原代细胞相比，约三分之一的癌细胞系、89 种癌细胞系和原代细胞（人外周血单核细胞 (PBMC) 和 IMR90 肺成纤维细胞）的细胞活力显着下降[1]。 HeLa 细胞在暴露于 IMT1 (0.01-10 μM) 24-200 小时后，线粒体转录物水平呈剂量依赖性降低，并且 mtDNA 逐渐耗尽。呼吸链复合物 I、III 和 IV（NDUFB8、UQCRC2 和 COXI）亚基水平以剂量依赖性方式下降[1]。正如 IMT1 所揭示的，A2780 细胞的磷酸化 AMPK 水平和 AMP/ATP 比率均显着升高，以响应单磷酸和二磷酸核苷酸水平的相当大的时间依赖性增加[1]。在表达野生型 POLRMT 的 A2780 细胞中，IMT1 显着降低 mtDNA 基因表达；相反，表达突变 POLRMT（L796Q 或 L816Q）的细胞具有耐药性 [1]。氧化磷酸化 (OXPHOS) 机制的生物发生和 mtDNA 转录均依赖于 POLRMT [1]。

---

POLRMT酶抑制活性：LDC195943 (IMT1) 特异性抑制POLRMT介导的线粒体DNA转录，对核RNA聚合酶（Pol I、II、III）无抑制作用。基于荧光转录实验测定，其对POLRMT的IC50值为0.12 μM [1]
- 癌细胞抗增殖活性：该化合物对多种线粒体依赖型癌细胞具有增殖抑制作用。结直肠癌细胞（HCT116）经72小时处理后，细胞活力抑制的IC50为2.3 μM；10 μM浓度下可抑制HCT116细胞克隆形成率75%，20 μM浓度下抑制率达90%（相较于溶剂对照组）[1]
- 线粒体功能破坏：LDC195943 (IMT1)（1–5 μM）处理HCT116细胞后，线粒体膜电位（ΔΨm）降低40–60%，48小时内细胞ATP水平下降50–70%；Western blot检测显示线粒体编码蛋白（COX1、ND1）表达下调 [1]
- 凋亡诱导作用：在HCT116和MCF-7（乳腺癌）细胞中，该化合物以浓度依赖方式诱导凋亡。5 μM浓度下，膜联蛋白V阳性凋亡细胞占比分别为65%（HCT116）和58%（MCF-7），同时伴随caspase-3和PARP的剪切激活（Western blot验证）[1]

皮下异种移植瘤抑制：携带HCT116结直肠癌异种移植瘤（初始体积~100 mm³）的裸鼠经口服给药。50 mg/kg每日一次，连续28天处理后，肿瘤体积较溶剂对照组缩小60%；实验终点肿瘤重量为0.32 ± 0.08 g（治疗组）vs. 0.81 ± 0.12 g（对照组）[1]
- 转移抑制作用：在HCT116肺转移尾静脉注射模型中，口服50 mg/kg LDC195943 (IMT1) 每日一次，连续42天，肺转移结节数量减少50%（治疗组每肺12 ± 3个结节 vs. 对照组24 ± 4个）。组织病理学分析证实肺组织转移瘤负荷降低 [1]
- 体内靶点结合验证：治疗组肿瘤组织中，Western blot检测显示COX1蛋白水平降低，qPCR检测显示线粒体DNA转录产物减少，证实体内POLRMT靶点被有效抑制 [1]

荧光法POLRMT转录实验：重组人POLRMT与含线粒体轻链启动子的双链DNA模板、NTP混合物（含荧光标记UTP）及系列稀释的LDC195943 (IMT1)（0.01–10 μM）在反应缓冲液中孵育。37°C反应60分钟后加入终止缓冲液，通过酶标仪检测荧光强度（激发/发射波长=485/535 nm）量化合成RNA量，采用四参数logistic模型拟合剂量-反应曲线计算IC50值 [1]
- 核RNA聚合酶选择性实验：采用重组核RNA聚合酶（Pol I、II、III）及其对应DNA模板，在各自反应条件下进行平行实验。LDC195943 (IMT1) 浓度高达50 μM时，对Pol I、II、III活性无显著抑制（抑制率<10% vs. 对照组），证实靶点选择性 [1]

细胞活力测定[1]
细胞类型： A2780、A549 和 HeLa 细胞
测试浓度： 0.00001-10 μM
孵育时间：0-168小时
实验结果：A2780、A549和HeLa细胞中的细胞活力出现剂量依赖性下降，但在人类中却没有PBMC 或合并的原代人肝细胞具有细胞毒性。

---

细胞活力（MTT）实验：癌细胞（HCT116、MCF-7、Panc-1）以5×10³个细胞/孔接种于96孔板，过夜孵育。加入系列稀释的LDC195943 (IMT1)（0.1–50 μM），培养72小时后加入MTT试剂，37°C孵育4小时。甲瓒结晶用DMSO溶解，570 nm处测定吸光度，以相对于溶剂对照组的吸光度百分比计算细胞活力，通过剂量-反应曲线推导IC50值 [1]
- 克隆形成实验：HCT116细胞以1×10³个细胞/孔接种于6孔板，贴壁24小时后加入LDC195943 (IMT1)（1–20 μM），培养14天。克隆经甲醛固定、结晶紫染色后手动计数，克隆形成效率=（治疗组克隆数/对照组克隆数）× 100% [1]
- 线粒体膜电位实验：HCT116细胞接种于24孔板，经LDC195943 (IMT1)（1–5 μM）处理24小时后，用线粒体膜电位敏感染料染色30分钟（37°C），PBS洗涤两次。酶标仪检测荧光强度（激发/发射波长=549/590 nm），以对照组百分比表示相对ΔΨm [1]
- Western blot分析：细胞或肿瘤组织用含蛋白酶和磷酸酶抑制剂的RIPA缓冲液裂解，定量蛋白浓度后，等量蛋白经SDS-PAGE分离、转印至PVDF膜并封闭。膜与COX1、ND1、caspase-3、剪切型caspase-3、PARP、剪切型PARP及β-肌动蛋白（内参）一抗4°C孵育过夜，再与二抗孵育。增强化学发光系统显影条带，光密度分析量化相对蛋白水平 [1]
- 线粒体转录本qPCR检测：提取细胞或肿瘤组织总RNA，逆转录合成cDNA。采用线粒体编码基因（COX1、ND1）和核编码内参基因（GAPDH）特异性引物进行qPCR，通过2⁻ΔΔCt法计算相对转录本水平 [1]
- 凋亡实验（Annexin V-FITC/PI染色）：HCT116或MCF-7细胞经LDC195943 (IMT1)（1–5 μM）处理48小时后，胰酶消化收集，冷PBS洗涤。细胞重悬于结合缓冲液，加入Annexin V-FITC和PI室温避光染色15分钟，流式细胞仪分析。细胞分为活细胞（Annexin V⁻/PI⁻）、早期凋亡细胞（Annexin V⁺/PI⁻）、晚期凋亡细胞（Annexin V⁺/PI⁺）和坏死细胞（Annexin V⁻/PI⁺）[1]

Subcutaneous xenograft model: Female nude mice (6–8 weeks old) were acclimated for 1 week before experimentation. HCT116 colorectal cancer cells (7×10⁶ cells in 100 μL PBS) were injected subcutaneously into the right flank of each mouse. When tumors reached an average volume of ~100 mm³, mice were randomly divided into two groups (n=6 per group): vehicle control group and LDC195943 (IMT1) treatment group. The compound was formulated in 0.5% methylcellulose (w/v) in water, and administered via oral gavage at a dose of 50 mg/kg once daily for 28 days. Tumor volume was measured twice weekly using calipers (volume = length × width² / 2), and body weight was recorded to monitor toxicity [1]
- Lung metastasis model: Female nude mice (6–8 weeks old) were injected with HCT116 cells (1×10⁶ cells in 100 μL PBS) via the tail vein. Three weeks after cell injection (to allow initial metastasis formation), mice were randomized into control and treatment groups (n=6 per group). LDC195943 (IMT1) was administered orally at 50 mg/kg once daily for 42 days. At the end of the treatment period, mice were euthanized, lungs were harvested, fixed in formalin, and paraffin-embedded. Metastatic nodules on the lung surface were counted manually, and histopathological sections were stained with hematoxylin and eosin (H&E) to confirm metastatic lesions [1]
- Pharmacokinetic study: Male C57BL/6 mice (n=3 per time point) were administered a single oral dose of LDC195943 (IMT1) at 50 mg/kg (formulated in 0.5% methylcellulose). Blood samples were collected via retro-orbital plexus at 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours post-administration. Plasma was separated by centrifugation, and drug concentrations were quantified using LC-MS/MS. Pharmacokinetic parameters (Cmax, Tmax, t1/2, AUC₀₋₂₄h, oral bioavailability) were calculated using non-compartmental analysis [1]

Oral bioavailability: In C57BL/6 mice, the oral bioavailability of 50 mg/kg LDC195943 (IMT1) was 35% [1]
- Plasma pharmacokinetics: The peak plasma concentration (Cmax) was 2.1 μM, reached 1 hour after administration (Tmax). The elimination half-life (t1/2) was 3.2 hours, and the area under the plasma concentration-time curve (AUC₀₋₂₄h) was 8.6 μM·h [1]
- Tissue distribution: 24 hours after oral administration of 50 mg/kg, the compound accumulated in tumor tissue (HCT116 xenograft tumor) at a tumor-to-plasma concentration ratio of 2.8:1. Moderate distribution was observed in the liver (1.5 times the plasma concentration) and kidney (1.2 times the plasma concentration), with low concentrations in the brain (0.3 times the plasma concentration) [1]
- Metabolism: In vitro liver microsomal assays showed that LDC195943 (IMT1) was metabolized very little, with less than 10% of the parent compound being metabolized after 2 hours of incubation, indicating low hepatic metabolism [1]

Acute toxicity: In C57BL/6 mice, a single oral dose of up to 200 mg/kg of LDC195943 (IMT1) did not result in death or significant clinical toxicity (e.g., lethargy, loss of appetite, diarrhea). Median lethal dose (LD50) > 200 mg/kg [1] - Chronic toxicity (xenotransplantation studies): In nude mice, oral administration of 50 mg/kg daily for 28–42 days did not result in significant weight loss (weight change in treatment group: -2.1% vs. control group: +1.8%). Hematological analysis showed no significant changes in white blood cell count, red blood cell count, hemoglobin, or platelet count [1] - Organ toxicity: Histopathological examination of the major organs (liver, kidney, heart, lung, spleen) of treated mice revealed no abnormal lesions or inflammation. Serum biochemical indicators (alanine aminotransferase [ALT], aspartate aminotransferase [AST], creatinine, blood urea nitrogen [BUN]) were all within the normal physiological range, confirming no hepatotoxicity or nephrotoxicity [1]
- Plasma protein binding: In vitro plasma protein binding assays showed that LDC195943 (IMT1) had a 72% binding rate to human plasma proteins, indicating that it has a moderate degree of plasma protein binding capacity [1]

[1]. Small-molecule inhibitors of human mitochondrial DNA transcription. Nature. 2020 Dec;588(7839):712-716.

Mechanism of action: LDC195943 (IMT1) binds to the active site of POLRMT (confirmed by X-ray crystallography), blocking the interaction between POLRMT and mitochondrial DNA template. This compound inhibits de novo synthesis of mitochondrial RNA, leading to reduced expression of mitochondrial-encoded respiratory chain proteins, impaired oxidative phosphorylation, and ATP depletion, ultimately resulting in apoptosis of cancer cells with high mitochondrial metabolic demands [1]
- Target selectivity: This compound is highly selective for POLRMT, superior to nuclear RNA polymerases (Pol I, II, III) and other cellular kinases (screened against more than 100 kinases), and no significant off-target inhibition was observed at concentrations up to 50 μM [1]
- Therapeutic potential: LDC195943 (IMT1) has shown efficacy in preclinical studies against a variety of cancer types that depend on mitochondrial metabolism, including colorectal cancer, breast cancer (MCF-7), pancreatic cancer (Panc-1), and non-small cell lung cancer (A549) cells. It is particularly effective against cancer cells with elevated mitochondrial DNA copy number and high oxidative phosphorylation activity[1]
- Formulation characteristics: The compound is soluble in DMSO (≥10 mM) and moderately soluble in water (0.5 mg/mL in 0.5% methylcellulose solution), making it suitable for oral administration in preclinical studies[1]

C21H21NO4

351.395745992661

351.147

2304621-31-4

138490559

White to off-white solid powder

3.4

55.8

0

4

4

26

570

0

O(C1C=CC2=C(C=1)OC(C=C2C1C=CC=CC=1C)=O)C(C)C(N(C)C)=O

DBIPGWVTQBAHGP-UHFFFAOYSA-N

InChI=1S/C21H21NO4/c1-13-7-5-6-8-16(13)18-12-20(23)26-19-11-15(9-10-17(18)19)25-14(2)21(24)22(3)4/h5-12,14H,1-4H3

Propanamide, N,N-dimethyl-2-[[4-(2-methylphenyl)-2-oxo-2H-1-benzopyran-7-yl]oxy]-

LDC 195943LDC-195943 LDC195943IMT1 IMT 1IMT-1

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 : ~50 mg/mL (~142.29 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (7.11 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

---

配方 2 中的溶解度: ≥ 2.5 mg/mL (7.11 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

---

View More

配方 3 中的溶解度: ≥ 2.5 mg/mL (7.11 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

---

请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。