Mitochondrial RNA polymerase (POLRMT)

IMT1B is a non-competitive formulation that depletes substrate binding and remodeling in vitro by causing conformational changes in POLRMT [1]. In A2780, A549, and HeLa cells, IMT1B (0.01 nM–10 μM; 72–168 hours) dose-reduced cell viability [1]. IMT1B depletes cells even more [1]. The AMP/ATP proportion and the levels of phosphorylated AMPK are produced by IMT1B's elevation of mono- and di-phosphorylation.

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Inhibition of cellular mtDNA expression [1]
We found that IMT1 and IMT1B/LDC-203974 caused a dose-dependent decrease in the levels of mitochondrial transcripts (Fig. 1f, Extended Data Fig. 2a) and gradual depletion of mtDNA (Fig. 1g, Extended Data Fig. 2b) in HeLa cells. We also observed a dose-dependent decrease in the levels of subunits (NDUFB8, UQCRC2 and COXI) of respiratory chain complexes I, III and IV, the stability of which depends on subunits encoded by mtDNA; by contrast, subunits (SDHB and ATP5A) of the exclusively nucleus-encoded complex II and F1 subcomplex of ATP synthase remained unchanged (Fig. 1h, Extended Data Fig. 2c). Consistent with these results, our quantitative proteomics analysis of IMT1-treated A2780 ovarian carcinoma cells revealed a rapid progressive decrease in the levels of subunits of complexes I, III and IV (Extended Data Fig. 2d). Levels of cytosolic ribosomal proteins remained unaffected after 24 h of treatment with IMT1, whereas the levels of mitoribosomal proteins were severely depleted—which is consistent with the lack of 12S and 16S ribosomal RNA encoded by mtDNA (Extended Data Fig. 2d). As a consequence of the impaired mtDNA expression, basal respiration was significantly decreased in intact HeLa cells exposed to IMT1 (Extended Data Fig. 2e). IMT1 and IMT1B showed very similar patterns of mtDNA transcription inhibition in HeLa and A2780 cells, which demonstrates that the compounds have comparable effects in vitro (Extended Data Fig. 2f).
We performed single-nucleotide incorporation assays and found that IMT1B/LDC-203974 did not inhibit budding yeast mitochondrial RNA polymerase (RPO41), bacteriophage T7 RNA polymerase, Escherichia coli RNA polymerase or a reverse transcriptase (Extended Data Fig. 3a–d). In addition, IMT1B did not inhibit the human multi-subunit RNA polymerase II (RNA Pol II) in vitro (Extended Data Fig. 3e) and had no effect on RNA Pol I-, II- and III-dependent transcript levels in cell lines (Extended Data Fig. 3f). Furthermore, IMT1B did not inhibit human mitochondrial DNA polymerase γ in vitro or mitochondrial protein synthesis in organello (Extended Data Fig. 3g, h). As an independent control, we used SC-6238532 (con IMT)—a compound that is structurally related to IMTs—and found that it neither binds POLRMT nor affects mtDNA expression (Extended Data Fig. 3i–p). We thus conclude that IMT1 and IMT1B are highly specific inhibitors of POLRMT.

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IMTs selectively target POLRMT in cells [1]
To determine the in vivo target of IMTs, we performed an unbiased genome-wide forward genetic screen19 in A2780 cells (Extended Data Fig. 4a). We exposed the cells to a mutagen and treated them with a wild-type lethal dose of an IMT1 analogue. Exome sequencing of resistant clones revealed POLRMT mutations as the only candidate suppressors of IMT toxicity. In total, we identified six independent point mutations in POLRMT, causing four amino acid substitutions (L796Q, F813L, L816Q and A821V, A821S or A821Q) clustered in the same region of the POLRMT protein (Fig. 2a). We expressed and purified recombinant mutant forms of POLRMT and found that addition of 10 μM IMT1B/LDC-203974 did not result in any thermal shift of POLRMT mutants, consistent with impaired IMT1B binding (Fig. 2b). Furthermore, increasing concentrations of IMT1B did not inhibit transcription with the mutant recombinant versions of POLRMT (Extended Data Fig. 4b–f). A2780 cells genetically engineered to express POLRMT with the L796Q or L816Q substitutions (Extended Data Fig. 4g) displayed strong resistance to IMT1 in cell proliferation assays (Fig. 2c). Treatment with IMT1 severely impaired mtDNA gene expression in A2780 cells that express wild-type POLRMT, whereas cells that express mutant POLRMT (L796Q or L816Q) were resistant (Extended Data Fig. 4h). Together, these data demonstrate that POLRMT is the in vivo target of IMT1.

IMT1B/LDC-203974 (100 mg/kg; lung; daily; 4 weeks) effectively lowers xenograft tumor growth in mice [1]. IMT1B decreases mtDNA levels and respiratory chain subunit levels in malignancies [1]. Excellent side wall bioavailability (101% in mice) and Cmax (5149 ng/mL in mice) in mouse models (10 mg/kg in mice)[1].

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Inhibition of tumour growth without toxicity [1]
Because IMTs affect the growth of cancer cells without affecting control cells (Extended Data Fig. 6c), we orally treated mice containing human A2780 or DLD-1 cancer cell xenografts with LDC-203974/IMT1B (Fig. 4, Extended Data Fig. 8). The IMT1B in vivo pharmacokinetic parameters (Extended Data Fig. 8a, b) allowed a single oral dose per day. Importantly, mouse POLRMT is sensitive to IMT1B treatment to a similar degree to human POLRMT, according to our differential scanning fluorimetry and in vitro transcription assays (Extended Data Fig. 8c, d). The application of 100 mg of IMT1B per kg body weight led to a clear reduction of tumour volume (Fig. 4a, Extended Data Fig. 8e, f). Treated mice showed a nonsignificant tendency towards a slightly lower body weight (Extended Data Fig. 8g). We observed no signs of acute or chronic liver or kidney toxicity (Extended Data Fig. 8h) and no induction of anaemia, thrombocytopenia or leukopenia (Extended Data Fig. 8i) after the treatment of wild-type mice with IMT1B for four weeks at a dose sufficient to reduce tumour size in mice containing xenografts.
On a molecular level, the treatment of mice with LDC-203974/IMT1B reduced mtDNA transcript levels and respiratory-chain subunit levels in tumours (Fig. 4b, c), whereas mitochondrial transcripts in differentiated tissues were reduced to a lesser extent (Fig. 4b) and OXPHOS protein levels remained normal in liver and heart (Fig. 4c). The levels of mtDNA showed a slight, nonsignificant decrease in tumours, whereas there were no changes in liver and heart (Extended Data Fig. 8j). The observation that treatment with an IMT causes mtDNA depletion in HeLa cells but not in xenograft tumours is probably explained by the very fast cell division of HeLa cells in tissue culture. Treatment with IMT1B in vivo caused an upregulation of phosphorylated AMPK and a decrease in phosphorylated ribosomal S6 protein in tumour tissue (Extended Data Fig. 8k), although this was not as extreme as in tissue-culture cells (Fig. 3e).
The finding that treatment with LDC-203974/IMT1B for up to four weeks is very well-tolerated in the mouse is consistent with previous genetic experiments that have shown that loss of mtDNA expression can be tolerated for a very long time in post-mitotic tissues, although disruption in the germ line results in embryonic lethality. These findings underscore the critical importance of mtDNA expression for biogenesis of the OXPHOS system in rapidly dividing cells, and explain why disruption of mtDNA gene expression can have substantial anti-tumour effects without affecting normal tissues.

Differential scanning fluorimetry [1]
The fluorescent dye Sypro Orange (Thermo Fisher Scientific; λex = 490 nm, λem = 570 nm) was used to monitor the temperature-induced unfolding of apo- and inhibitor-bound human and mouse POLRMT. The assay was set up essentially as previously described31, with 0–10 μM LDC-203974/IMT1B and 1.6 μM human POLRMT.

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Microscale thermophoresis [1]
The effect of inhibitors on the binding affinity of human POLRMT to a DNA–RNA template was analysed using microscale thermophoresis (MST). A 28-mer 5′ Alexa488-labelled non-template DNA oligonucleotide (5′-CATGGGGTAATTATTATTTCGCCAGACG-3′) was annealed to its complementary template strand (5′-CGTCTGGCGTGCGCGCCGCTACCCCATG-3′) and a 14-mer RNA oligonucleotide (5′-AGUCUGCGGCGCGC-3′) in a 1:1:1 molar ratio. Twelve 2-fold dilutions of POLRMT (0.45–1,000 nM) were made in MST buffer (25 mM Tris-HCl pH 8.0, 0.1 M NaCl, 10 mM MgCl2, 0.5 mM ATP, 10% (w/v) glycerol, 1 mM DTT and 0.05% (v/v) Tween-20). Dilutions were incubated with 15 nM DNA–RNA template and 0–10 μM IMT1B/LDC-203974 for 10 min at 24 °C. Samples were analysed in triplicate using a Monolith NT.115 instrument with standard capillaries and 40% LED and MST power for 30 s at 24 °C. Combined thermophoresis and temperature jump data were baseline-corrected and used to calculate Kd, bound state and unbound state in the NanoTemper analysis software.

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In vitro transcription assays [1]
The effect of inhibitors on mitochondrial transcription was probed using an in vitro transcription assay on linear and supercoiled templates containing the mitochondrial LSP. Transcription reactions (25 μl) were set up as previously described in the presence of 0–10 μM LDC-203974/IMT1B, 500 fmol POLRMT, 1.5 pmol TFB2M, 2.5 pmol TFAM and 500 fmol TEFM. Transcription products were purified by ethanol precipitation and analysed on 8% denaturing polyacrylamide gels as previously published4. Experiments with the mouse transcription system were performed and analysed in the same way.

Cell Viability Assay[1]
Cell Types: A2780 Cell
Tested Concentrations: 0.01 nM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM
Incubation Duration: 72 hrs (hours), 96 hrs (hours), 168 hrs (hours)
Experimental Results: significant increase in decrease [1]. Cell viability was dose-dependent.

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

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)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.39 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 20.8 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.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.39 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 20.8 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.)