LDC-203974 (IMT-1B)

Mitochondrial RNA polymerase (POLRMT)

IMT1B 是一种非竞争性制剂，通过引起 POLRMT 构象变化来消耗体外底物结合和重塑 [1]。在 A2780、A549 和 HeLa 细胞中，IMT1B（0.01 nM–10 μM；72–168 小时）剂量会降低细胞活力 [1]。 IMT1B 会进一步消耗细胞 [1]。 AMP/ATP 比例和磷酸化 AMPK 的水平是由 IMT1B 的单磷酸化和二磷酸化升高产生的。

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抑制细胞mtDNA表达[1]
我们发现IMT1和IMT1B/LDC-203974导致HeLa细胞线粒体转录物水平的剂量依赖性降低（图1f，扩展数据图2a）和mtDNA的逐渐耗尽（图1g，扩展数据图2b）。我们还观察到呼吸链复合物I， III和IV亚基（NDUFB8， UQCRC2和COXI）水平的剂量依赖性降低，其稳定性取决于mtDNA编码的亚基；相比之下，ATP合酶的纯核编码复合体II和F1亚复合体的亚基（SDHB和ATP5A）保持不变（图1，扩展数据图2c）。与这些结果一致，我们对imt1处理的A2780卵巢癌细胞的定量蛋白质组学分析显示复合物I， III和IV的亚基水平快速进行性降低（扩展数据图2d）。IMT1治疗24小时后，胞质核糖体蛋白水平未受影响，而线粒体核糖体蛋白水平严重耗尽，这与mtDNA编码的12S和16S核糖体RNA缺乏一致（扩展数据图2d）。由于mtDNA表达受损，暴露于IMT1的完整HeLa细胞的基础呼吸显著减少（扩展数据图2e）。IMT1和IMT1B在HeLa和A2780细胞中表现出非常相似的mtDNA转录抑制模式，这表明这两种化合物在体外具有相当的效果（扩展数据图2f）。
我们进行了单核苷酸掺入实验，发现IMT1B/LDC-203974不抑制出芽酵母线粒体RNA聚合酶（RPO41）、噬菌体T7 RNA聚合酶、大肠杆菌RNA聚合酶或逆转录酶（扩展数据图3a-d）。此外，IMT1B在体外不抑制人多亚基RNA聚合酶II (RNA Pol II)（扩展数据图3e），并且对细胞系中RNA Pol I-， II-和iii依赖性转录水平没有影响（扩展数据图3f）。此外，IMT1B在体外没有抑制人线粒体DNA聚合酶γ或细胞器中线粒体蛋白的合成（扩展数据图3g， h）。作为独立对照，我们使用SC-6238532 （con IMT）——一种与IMT结构相关的化合物——发现它既不结合POLRMT，也不影响mtDNA的表达（扩展数据图3i-p）。因此，我们得出结论，IMT1和IMT1B是高度特异性的POLRMT抑制剂。

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IMTs选择性靶向细胞中的POLRMT[1]
为了确定IMTs的体内靶点，我们在A2780细胞中进行了无偏倚的全基因组正向遗传筛选19（扩展数据图4a）。我们将细胞暴露于诱变原并用野生型致死剂量的IMT1类似物处理它们。耐药克隆的外显子组测序显示，POLRMT突变是IMT毒性的唯一候选抑制因子。我们一共在POLRMT中发现了6个独立的点突变，导致4个氨基酸替换（L796Q、F813L、L816Q和A821V、A821S或A821Q）聚集在POLRMT蛋白的同一区域（图2a）。我们表达并纯化了POLRMT的重组突变体形式，发现添加10 μM IMT1B/LDC-203974不会导致POLRMT突变体的任何热转移，这与IMT1B结合受损一致（图2b）。此外，IMT1B浓度的增加并没有抑制突变型重组POLRMT的转录（扩展数据图4b-f）。用L796Q或L816Q替换表达POLRMT的A2780细胞（扩展数据图4）在细胞增殖试验中显示出对IMT1的强抗性（图2）。在表达野生型POLRMT的A2780细胞中，IMT1严重损害了mtDNA基因的表达，而表达突变型POLRMT （L796Q或L816Q）的细胞则具有耐药性（扩展数据图4h）。综上所述，这些数据表明POLRMT是IMT1的体内靶点。

IMT1B（100 mg/kg；肺；每日；4 周）可有效降低小鼠异种移植肿瘤的生长 [1]。 IMT1B 降低恶性肿瘤中的线粒体 DNA 水平和呼吸链亚基水平 [1]。在小鼠模型中具有出色的侧壁生物利用度（小鼠中为 101%）和 Cmax（小鼠中为 5149 ng/mL）（小鼠中为 10 mg/kg）[1]。

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抑制肿瘤生长无毒性[1]
由于IMTs影响癌细胞的生长而不影响对照细胞（扩展数据图6c），我们口服了含有人A2780或DLD-1癌细胞异种移植物 / LDC-203974/IMT1B的小鼠（图4，扩展数据图8）。IMT1B体内药代动力学参数（扩展数据图8a， b）允许每天单次口服剂量。重要的是，根据我们的差示扫描荧光法和体外转录测定，小鼠POLRMT对IMT1B处理的敏感程度与人类POLRMT相似（扩展数据图8c， d）。每公斤体重100mg IMT1B的应用导致肿瘤体积明显减少（图4a，扩展数据图8e， f）。处理过的小鼠显示出轻微降低体重的不显著趋势（扩展数据图8g）。我们没有观察到急性或慢性肝或肾毒性的迹象（扩展数据图8），并且在用IMT1B治疗野生型小鼠四周后，没有诱导贫血、血小板减少或白细胞减少（扩展数据图8）。IMT1B的剂量足以减少含有异种移植物的小鼠的肿瘤大小。
在分子水平上，LDC-203974/IMT1B小鼠的处理降低了肿瘤中mtDNA转录水平和呼吸链亚基水平（图4b， c），而分化组织中的线粒体转录物降低程度较小（图4b），肝脏和心脏中的OXPHOS蛋白水平保持正常（图4c）。mtDNA水平在肿瘤中显示出轻微的、不显著的下降，而在肝脏和心脏中没有变化（扩展数据图8j）。观察到用IMT治疗会导致HeLa细胞的mtDNA缺失，但在异种移植物肿瘤中不会，这可能是由组织培养中HeLa细胞的快速细胞分裂所解释的。体内IMT1B治疗引起肿瘤组织中磷酸化AMPK的上调和磷酸化核糖体S6蛋白的减少（扩展数据图8k），尽管这并不像组织培养细胞那样极端（图3e）。
小鼠对LDC-203974/IMT1B治疗长达四周的耐受性非常好，这一发现与先前的遗传实验一致，这些实验表明，在有丝分裂后的组织中，mtDNA表达的缺失可以耐受很长时间，尽管生殖系的破坏会导致胚胎致死。这些发现强调了mtDNA表达对快速分裂细胞中OXPHOS系统生物发生的关键重要性，并解释了为什么mtDNA基因表达的破坏可以在不影响正常组织的情况下具有实质性的抗肿瘤作用。

差示扫描荧光法[1]
荧光染料Sypro Orange (Thermo Fisher Scientific；λex = 490 nm, λem = 570 nm)用于监测载脂蛋白和抑制剂结合的人类和小鼠POLRMT的温度诱导展开。该实验基本如上所述31,0-10 μM LDC-203974/IMT1B, 1.6 μM human POLRMT。

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微尺度热泳[1]
采用微尺度热泳术（MST）分析了抑制剂对人POLRMT与DNA-RNA模板结合亲和力的影响。将28-mer 5 ' alexa488标记的非模板DNA寡核苷酸（5 ' -CATGGGGTAATTATTATTTCGCCAGACG-3 ‘）以1:1:1的摩尔比退火到其互补模板链（5 ’ -CGTCTGGCGTGCGCGCCGCTACCCCATG-3 ‘）和14-mer RNA寡核苷酸（5 ’ - agucugcggcgcgccg -3 '）。用MST缓冲液（25 mM Tris-HCl pH 8.0, 0.1 M NaCl, 10 mM MgCl2, 0.5 mM ATP, 10% （w/v）甘油，1 mM DTT和0.05% (v/v) tweent -20）稀释12倍POLRMT （0.45-1,000 nM）。稀释液用15 nM DNA-RNA模板和0-10 μM IMT1B/LDC-203974在24℃下孵育10 min。使用带有标准毛细管和40% LED和MST电源的Monolith NT.115仪器，在24°C下分析样品30秒，一式三份。结合热泳和温度跳变数据进行基线校正，并在NanoTemper分析软件中计算Kd、结合态和非结合态。

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体外转录测定[1]
通过对含有线粒体LSP的线性和超卷曲模板进行体外转录试验，探讨了抑制剂对线粒体转录的影响。转录反应（25 μl）在0 ~ 10 μM LDC-203974/IMT1B、500 fmol POLRMT、1.5 pmol TFB2M、2.5 pmol TFAM和500 fmol TEFM存在下进行。转录产物用乙醇沉淀法纯化，并在先前发表的8%变性聚丙烯酰胺凝胶上进行分析。以同样的方法对小鼠转录系统进行了实验和分析。

细胞活力测定[1]
细胞类型： A2780 细胞
测试浓度： 0.01 nM、1 nM、10 nM、100 nM、1 μM、10 μM
孵育时间：72 hrs（小时）、96 hrs（小时）、168 hrs（小时）
实验结果：明显增加减少[1]。细胞活力呈剂量依赖性。

Animal/Disease Models: 7-9 weeks old female BALB/c nude mice, A2780 cell xenografts [1]
Doses: 100 mg/kg
Route of Administration: Orally, one time/day, 4 times a week.
Experimental Results: The tumor volume was Dramatically diminished.

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Animal/Disease Models: Mouse[1]
Doses: 1 mg/kg, intravenous (iv) (iv)injection; oral 10 mg/kg (pharmacokinetic/PK/PK analysis)
Route of Administration: intravenous (iv) (iv)administration and oral administration
Experimental Results: Oral bioavailability (101% ), Cmax (5149 ng/mL), T1/2 (1.88 h).

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The DLD-1 xenograft model was conducted at Crown Bioscience, using female BALB/c nude mice (7–9 weeks of age). Mice were housed in standard individually ventilated cages (maximum four mice per cage) in a 12 h light/dark cycle in controlled environmental conditions (22.3 ± 2 °C, 44–58% relative humidity). Mice were fed a normal chow diet and water ad libitum. Group assignment was based on a stratified random sampling procedure. All experimental procedures were approved by and conducted in accordance with the regulations of the local Animal Welfare Authorities. Human ovary carcinoma A2780 (ECACC 93112519) (5 × 107 cells in 100 μl of PBS and Matrigel) or DLD-1 colon carcinoma cells (5 × 106 cells in 100 μl of PBS and Matrigel), isolated during exponential growth phase from in vitro cell culture, were transplanted subcutaneously into each female nude mice at day 0. Mice were stratified (n = 8 per group each) when the mean tumour volume had reached approximately 0.1 cm3. Mice were orally treated with either vehicle or 100 mg per kg body weight LDC-203974/IMT1B. IMT1B/LDC-203974 was administered using 25% PEG400 in 30% aqueous hydroxypropyl-β-cyclodextrin as vehicle. Measurement of body weight and tumour volume was performed three times per week. For A2780 xenografts, individual mice were killed if tumour volumes progressed to >1.5 cm3; if the mean tumour volume of a group was >1 cm3; or if ulceration was observed. For DLD-1 xenografts, individual mice were killed if tumour volumes progressed to 3 cm3; if the mean tumour volume of a group was >2 cm3; or if ulceration was observed. A gross autopsy was performed from all mice upon study termination and tissues samples were isolated.

Toxicity panels [1]
Toxicity panels, blood parameters and pharmacokinetic analyses were carried out at Synovo. Mice (4 per cage) were housed in standard individually ventilated cages in a 12 h light/dark cycle in controlled environmental conditions (22 ± 1 °C, 55–65% relative humidity). Mice were fed a normal chow diet and water ad libitum
For toxicity and blood parameters, female NMRI mice obtained from Janvier Labs were used. Before allocation, mice were weighed and groups were assigned according to a randomized block design such that mean starting weight was similar in all groups. Mice received either vehicle (25% PEG400 in 30% aqueous hydroxypropyl-β-cyclodextrin) or 100 mg per kg body weight IMT1B/LDC-203974 once daily for four weeks followed by a wash-out period. Body weight was measured once daily. Blood samples were taken for analysis at day 0 and day 28.
To establish a suitable dosing regimen for an in vivo efficacy study, the pharmacokinetic profiles of the test compounds after administration of single doses of 1 mg per kg intravenously and 10 mg per kg orally to male CD1 mice were determined. Plasma samples were obtained over a 24-h interval and test compound concentrations were analysed by LC–MS. To this end, test compounds were extracted from plasma by protein precipitation using ACN. Samples were analysed by liquid chromatography–tandem mass spectrometry using a Prominence UFLC system coupled to a Qtrap 5500 instrument. Test compounds were separated on a C18 column with an ACN and water mixture containing 0.1% formic acid as solvent. Chromatographic conditions and mass spectrometer parameters were optimized for each test compound before sample analysis. Concentrations of each test compound were calculated by means of a standard curve. Pharmacokinetic parameters were calculated by non-compartmental analysis using PKSolver Plug-in for Microsoft Excel (v.2.0)

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

Altered expression of mitochondrial DNA (mtDNA) occurs in ageing and a range of human pathologies (for example, inborn errors of metabolism, neurodegeneration and cancer). Here we describe first-in-class specific inhibitors of mitochondrial transcription (IMTs) that target the human mitochondrial RNA polymerase (POLRMT), which is essential for biogenesis of the oxidative phosphorylation (OXPHOS) system. The IMTs efficiently impair mtDNA transcription in a reconstituted recombinant system and cause a dose-dependent inhibition of mtDNA expression and OXPHOS in cell lines. To verify the cellular target, we performed exome sequencing of mutagenized cells and identified a cluster of amino acid substitutions in POLRMT that cause resistance to IMTs. We obtained a cryo-electron microscopy (cryo-EM) structure of POLRMT bound to an IMT, which further defined the allosteric binding site near the active centre cleft of POLRMT. The growth of cancer cells and the persistence of therapy-resistant cancer stem cells has previously been reported to depend on OXPHOS, and we therefore investigated whether IMTs have anti-tumour effects. Four weeks of oral treatment with an IMT is well-tolerated in mice and does not cause OXPHOS dysfunction or toxicity in normal tissues, despite inducing a strong anti-tumour response in xenografts of human cancer cells. In summary, IMTs provide a potent and specific chemical biology tool to study the role of mtDNA expression in physiology and disease.[1]

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In conclusion, IMTs are first-in-class, potent and highly specific allosteric inhibitors of POLRMT, which is essential for mtDNA transcription. The IMTs show potent effects for the treatment of cancer in preclinical mouse models and have the potential to be further developed for clinical applications in human cancer. In addition, IMTs represent a potent chemical biology tool for manipulating mtDNA transcription—and thereby OXPHOS capacity—in a dose-dependent way in animal models to assess how the downregulation of OXPHOS affects a variety of physiological and disease processes relevant for human health. [1]

C24H21CLFNO6

473.878049612045

473.104

C, 60.83; H, 4.47; Cl, 7.48; F, 4.01; N, 2.96; O, 20.26

2304621-06-3

138490769

White to off-white solid powder

3.7

93.1

1

7

5

33

806

2

ClC1C=C(C=CC=1C1=CC(=O)OC2C=C(C=CC=21)O[C@H](C)C(N1CCC[C@H](C(=O)O)C1)=O)F

PFEKWBKJUBCXDT-KGLIPLIRSA-N

InChI=1S/C24H21ClFNO6/c1-13(23(29)27-8-2-3-14(12-27)24(30)31)32-16-5-7-18-19(11-22(28)33-21(18)10-16)17-6-4-15(26)9-20(17)25/h4-7,9-11,13-14H,2-3,8,12H2,1H3,(H,30,31)/t13-,14+/m1/s1

(S)-1-((R)-2-((4-(2-chloro-4-fluorophenyl)-2-oxo-2H-chromen-7-yl)oxy)propanoyl)piperidine-3-carboxylic acid

LDC 203974; IMT1B; LDC203974; IMT-1B; 2304621-06-3; LDC203974; (S)-1-((R)-2-((4-(2-Chloro-4-fluorophenyl)-2-oxo-2H-chromen-7-yl)oxy)propanoyl)piperidine-3-carboxylic acid; IMT1B(3-Piperidinecarboxylicacid,1-[(2R)-2-[[4-(2-chloro-4-fluorophenyl)-2-oxo-2H-1-benzopyran-7-yl]oxy]-1-oxopropyl]-,(3S)-;LDC203974); (3S)-1-[(2R)-2-[4-(2-chloro-4-fluorophenyl)-2-oxochromen-7-yl]oxypropanoyl]piperidine-3-carboxylic acid; CHEMBL5440629; SCHEMBL20839363; IMT-1B; LDC-203974

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 : ~250 mg/mL (~527.56 mM)

配方 1 中的溶解度: ≥ 2.08 mg/mL (4.39 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 20.8 mg/mL澄清DMSO储备液加入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。

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配方 2 中的溶解度: ≥ 2.08 mg/mL (4.39 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 20.8 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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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；
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