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

In A2780, A549, and HeLa cells, IMT1 (0.00001-10 μM; 0-168 hours) dose-dependently lowers cell viability. About one-third of cancer cell lines, 89 cancer cell lines, and primary cells (human peripheral blood mononuclear cells (PBMC) and IMR90 lung fibroblasts) displayed a marked decline in cell viability as compared to the primary cells[1]. HeLa cells exhibit a dose-dependent reduction in mitochondrial transcript levels and a progressive depletion of mtDNA upon exposure to IMT1 (0.01-10 μM) for a duration of 24-200 hours. In a dose-dependent manner, the levels of subunits of respiratory chain complexes I, III, and IV (NDUFB8, UQCRC2, and COXI) drop [1]. A2780 cells' phosphorylated AMPK levels and the AMP/ATP ratio both significantly rise in response to a considerable time-dependent increase in mono- and diphosphate nucleotide levels, as revealed by IMT1 [1]. In A2780 cells expressing wild-type POLRMT, IMT1 significantly reduces mtDNA gene expression; in contrast, cells expressing mutant POLRMT (L796Q or L816Q) are resistant [1]. Both the biogenesis of the oxidative phosphorylation (OXPHOS) machinery and mtDNA transcription depend on POLRMT [1].

---

POLRMT enzyme inhibition: LDC195943 (IMT1) specifically suppresses POLRMT-mediated mitochondrial DNA transcription without inhibiting nuclear RNA polymerases (Pol I, II, III). The IC50 value for POLRMT inhibition is 0.12 μM, as determined by fluorescence-based transcription assays [1]
- Cancer cell antiproliferative activity: The compound inhibits the proliferation of various cancer cell lines with different mitochondrial dependency. In colorectal cancer cells (HCT116), the IC50 for cell viability reduction is 2.3 μM after 72-hour treatment. It also suppresses colony formation of HCT116 cells by 75% at 10 μM and 90% at 20 μM compared to the vehicle control [1]
- Mitochondrial function disruption: Treatment with LDC195943 (IMT1) (1–5 μM) reduces mitochondrial membrane potential (ΔΨm) by 40–60% in HCT116 cells, decreases cellular ATP levels by 50–70% within 48 hours, and downregulates the expression of mitochondrial-encoded proteins (COX1, ND1) as detected by western blot [1]
- Apoptosis induction: In HCT116 and MCF-7 (breast cancer) cells, the compound induces apoptosis in a concentration-dependent manner. At 5 μM, Annexin V-positive apoptotic cells account for 65% (HCT116) and 58% (MCF-7) of total cells, accompanied by cleavage of caspase-3 and PARP (western blot verification) [1]

Subcutaneous xenograft tumor inhibition: Nude mice bearing HCT116 colorectal cancer xenografts (initial volume ~100 mm³) were treated with LDC195943 (IMT1) via oral gavage. At a dose of 50 mg/kg once daily for 28 days, the tumor volume was reduced by 60% compared to the vehicle group. Tumor weight at the end of the experiment was 0.32 ± 0.08 g (treatment group) vs. 0.81 ± 0.12 g (control group) [1]
- Metastasis suppression: In a tail vein injection model of HCT116 lung metastasis, oral administration of 50 mg/kg LDC195943 (IMT1) daily for 42 days reduced the number of lung metastatic nodules by 50% (12 ± 3 nodules per lung in treatment group vs. 24 ± 4 in control group). Histopathological analysis confirmed reduced metastatic tumor burden in lung tissues [1]
- Mitochondrial target engagement in vivo: Tumor tissues from treated mice showed decreased levels of mitochondrial-encoded COX1 protein (by western blot) and reduced mitochondrial DNA transcription products (detected by qPCR), confirming on-target inhibition of POLRMT in vivo [1]

Fluorescence-based POLRMT transcription assay: Recombinant human POLRMT was incubated with a double-stranded DNA template containing the mitochondrial light-strand promoter, a mix of NTPs (including a fluorescently labeled UTP), and serial dilutions of LDC195943 (IMT1) (0.01–10 μM) in reaction buffer. The reaction was carried out at 37°C for 60 minutes, then terminated by adding stop buffer. Fluorescence intensity (excitation/emission = 485/535 nm) was measured using a microplate reader to quantify the amount of synthesized RNA. The IC50 value was calculated by fitting the dose-response curve with a four-parameter logistic model [1]
- Nuclear RNA polymerase selectivity assay: Parallel assays were performed using recombinant nuclear RNA polymerases (Pol I, II, III) with their respective DNA templates and reaction conditions. LDC195943 (IMT1) at concentrations up to 50 μM showed no significant inhibition of Pol I, II, or III activity (inhibition rate < 10% vs. control), confirming target selectivity [1]

Cell Viability Assay[1]
Cell Types: A2780, A549 and HeLa Cell
Tested Concentrations: 0.00001-10 μM
Incubation Duration: 0-168 hrs (hours)
Experimental Results: There was a dose-dependent decrease in cell viability in A2780, A549 and HeLa cells, but not in human PBMC or pooled primary human hepatocytes were cytotoxic.

---

Cell viability (MTT) assay: Cancer cells (HCT116, MCF-7, Panc-1) were seeded in 96-well plates at a density of 5×10³ cells/well and incubated overnight. Serial dilutions of LDC195943 (IMT1) (0.1–50 μM) were added, and cells were cultured for 72 hours. MTT reagent was added to each well and incubated for 4 hours at 37°C. The resulting formazan crystals were dissolved in DMSO, and absorbance was measured at 570 nm. Cell viability was calculated as the percentage of absorbance relative to the vehicle control, and IC50 values were derived from dose-response curves [1]
- Colony formation assay: HCT116 cells were seeded in 6-well plates at 1×10³ cells/well and allowed to attach for 24 hours. LDC195943 (IMT1) (1–20 μM) was added, and cells were cultured for 14 days. Colonies were fixed with formaldehyde, stained with crystal violet, and counted manually. Colony formation efficiency was calculated as (number of colonies in treatment group / number in control group) × 100% [1]
- Mitochondrial membrane potential assay: HCT116 cells were seeded in 24-well plates and treated with LDC195943 (IMT1) (1–5 μM) for 24 hours. Cells were stained with a mitochondrial membrane potential-sensitive dye for 30 minutes at 37°C, then washed twice with PBS. Fluorescence intensity was measured using a microplate reader (excitation/emission = 549/590 nm), and relative ΔΨm was expressed as a percentage of the control group [1]
- Western blot analysis: Cells or tumor tissues were lysed in RIPA buffer containing protease and phosphatase inhibitors. Protein concentrations were quantified, and equal amounts of protein were separated by SDS-PAGE, transferred to PVDF membranes, and blocked. Membranes were incubated with primary antibodies against COX1, ND1, caspase-3, cleaved caspase-3, PARP, cleaved PARP, and β-actin (loading control) overnight at 4°C, followed by secondary antibody incubation. Bands were visualized using an enhanced chemiluminescence system, and densitometric analysis was performed to quantify relative protein levels [1]
- qPCR for mitochondrial transcripts: Total RNA was extracted from cells or tumor tissues, and cDNA was synthesized by reverse transcription. qPCR was performed using primers specific for mitochondrial-encoded genes (COX1, ND1) and nuclear-encoded reference gene (GAPDH). Relative transcript levels were calculated using the 2⁻ΔΔCt method [1]
- Apoptosis assay (Annexin V-FITC/PI): HCT116 or MCF-7 cells were treated with LDC195943 (IMT1) (1–5 μM) for 48 hours, harvested by trypsinization, and washed with cold PBS. Cells were resuspended in binding buffer, stained with Annexin V-FITC and PI for 15 minutes at room temperature in the dark, and analyzed by flow cytometry. Cells were categorized into viable (Annexin V⁻/PI⁻), early apoptotic (Annexin V⁺/PI⁻), late apoptotic (Annexin V⁺/PI⁺), and necrotic (Annexin V⁻/PI⁺) populations [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

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.11 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 25.0 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.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (7.11 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 25.0 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.

---

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (7.11 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

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