Alterations in mitochondrial DNA (mtDNA) expression occur in ageing and a range of human diseases, including inborn errors of metabolism, cancer and neurodegeneration. Writing in Nature, Bonekamp et al. now report the identification of first-in-class inhibitors of human mtDNA transcription (IMTs). In mouse xenograft models, the IMTs reduced mitochondrial gene expression in tumours and potently suppressed tumour growth.

With the aim of aiding understanding of the role of altered mtDNA expression in disease, Bonekamp et al. set out to identify IMTs. Using a high-throughput recombinant in vitro transcription assay system, the authors screened a diverse set of 430,000 small molecules for inhibitory effects on the mitochondrial transcription machinery. This screen identified LDC195943 (IMT1), which, following optimization for in vivo use in animals, generated the lead compound LDC203974 (IMT1B).

A series of assays revealed that the IMTs target human mitochondrial RNA polymerase (POLRMT; essential for mtDNA transcription and biogenesis of the oxidative phosphorylation (OXPHOS) system), affecting enzyme stability, substrate binding and activity. Consequently, in HeLa cells, the IMTs decreased levels of mitochondrial transcripts, gradually depleted mtDNA and reduced OXPHOS protein levels.

To confirm the target and specificity of IMTs, the authors next performed an in vitro unbiased genome-wide forward genetic screen in an ovarian cancer cell line. Exome sequencing of cells exposed to a mutagen identified POLRMT mutations as the only suppressors of IMT toxicity. Determining the cryo-EM structure of POLRMT bound with IMT1B provided further insight, revealing that IMT1B acts as an allosteric inhibitor of POLRMT and that binding displaces the palm loop, which then sterically impairs binding of the DNA–RNA hybrid and translocation of the nascent RNA.

Given that fast-growing tumour cells require mitochondrial metabolism, and that previous studies have reported that the growth of cancer cells and the persistence of therapy-resistant cancer stem cells depend on OXPHOS, the authors next investigated the effect of the IMTs on cancer cell proliferation. A large-scale cell viability assay encompassing 89 cancer cell lines as well as primary cells showed a strong decrease in cell viability after IMT1 treatment in about one third of the cancer cell lines, whereas primary human cells remained unresponsive.

Investigation of the basis of the observed cancer cell toxicity revealed that IMT1 treatment results in depletion of cellular metabolites. Treatment of an ovarian cancer cell line with IMT1 increased the AMP–ATP ratio and levels of phosphorylated AMPK, indicative of a cellular energy crisis as expected from a nonfunctional OXPHOS system. In addition, IMT1 reduced deoxynucleoside triphosphate levels and citric acid cycle intermediates, resulting in a marked depletion of cellular amino acid levels.

In human cancer cell xenograft mouse models, a single daily oral dose of IMT1B for 4 weeks significantly reduced tumour growth, was well tolerated and did not cause dysfunction of the OXPHOS system or toxicity in normal tissues. Levels of mtDNA transcripts and respiratory-chain subunits were reduced in tumours, whereas mitochondrial transcripts were reduced to a lesser extent and OXPHOS protein levels remained normal in differentiated tissues such as the liver and heart.

In summary, IMTs are first-in-class, potent and highly specific allosteric inhibitors of mtDNA transcription, and they represent a potent chemical biology tool to study the role of mtDNA expression in physiology and disease. The authors are continuing to investigate the potential of IMTs as a treatment for cancer alone and in combination with other therapies.