A Yeast-Based Repurposing Approach for the Treatment of Mitochondrial DNA Depletion Syndromes Led to the Identification of Molecules Able to Modulate the dNTP Pool

Abstract

Mitochondrial DNA depletion syndromes (MDS) are clinically heterogenous and often severe diseases, characterized by a reduction of the number of copies of mitochondrial DNA (mtDNA) in affected tissues. In the context of MDS, yeast has proved to be both an excellent model for the study of the mechanisms underlying mitochondrial pathologies and for the discovery of new therapies via high-throughput assays. Among the several genes involved in MDS, it has been shown that recessive mutations in MPV17 cause a hepatocerebral form of MDS and Navajo neurohepatopathy. MPV17 encodes a non selective channel in the inner mitochondrial membrane, but its physiological role and the nature of its cargo remains elusive. In this study we identify ten drugs active against MPV17 disorder, modelled in yeast using the homologous gene SYM1. All ten of the identified molecules cause a concomitant increase of both the mitochondrial deoxyribonucleoside triphosphate (mtdNTP) pool and mtDNA stability, which suggests that the reduced availability of DNA synthesis precursors is the cause for the mtDNA deletion and depletion associated with Sym1 deficiency. We finally evaluated the effect of these molecules on mtDNA stability in two other MDS yeast models, extending the potential use of these drugs to a wider range of MDS patients.

Mitochondrial Membranes and Mitochondrial Genome: Interactions and Clinical Syndromes

“…In a yeast model with SYM1 mutation, the ortholog of the human MPV17, several molecules were identified that could increase the dTTP pool and mtDNA stability. Interestingly, some of these molecules affect ergosterol levels and therefore could work through increasing the permeability of mitochondrial membranes [110]. There is an ongoing phase 2 clinical trial to evaluate supplementation of deoxycytidine and deoxythymidine in children with mtDNA depletion syndromes (including MPV17 deficiency) (https://clinicaltrials.gov/ct2/show/NCT04802 707, accessed on 8 May 2022).…”

Saccharomyces cerevisiae as a Tool for Studying Mutations in Nuclear Genes Involved in Diseases Caused by Mitochondrial DNA Instability

“…Very recently, it was shown that the yeast mitochondria of sym1Δ mutant displayed a significant decrease in the levels of all the dNTPs, suggesting that, as in the Mpv17 −/− mouse and in MPV17 -deficient human fibroblasts, the instability of mtDNA in sym1∆ yeast mitochondria is associated with a decrease in dNTPs levels, providing strong evidence that the cause of the mtDNA deletion/depletion in Sym1 deficient cells is a shortage of precursors for DNA synthesis [ 40 ]. Furthermore, it was demonstrated that the nucleotide reduction is not limited to the mitochondrial compartment but is instead extended to the entire cell compartment.…”

“…The whole-cell dNTP pool decrease could be due to a reduced ATP production caused by the OXPHOS impairment; however, OXPHOS defect and mtDNA instability occur independently in sym1Δ yeast cells [ 110 ]. Another tempting explanation links this dNTP shortage to the Sym1 proposed function, as TCA cycle intermediates homeostatic control [ 40 ]. Indeed, it was previously reported that the OXPHOS defect of sym1Δ strain was rescued by the overexpression of mitochondrial transporters of TCA intermediates ( YMC1 and ODC1 ), or by the supplementation of different amino acids (glutamate, aspartate, glutamine, or asparagine) produced from TCA intermediates and all precursors of nucleotides synthesis [ 110 ].…”

“…The most detrimental mutation was L362V, equivalent to human L317V, which increased the petite frequency to ~60% [ 40 ]. The mutant strain harboring this mutation was used to evaluate the capability of some drugs to reduce the mtDNA instability, as reported in Section 4 .…”

“…Further analysis showed that the decrease in mtDNA instability obtained with all ten drugs was associated with an increase in mitochondrial dNTP pools, especially of dTTP, providing evidence that the reduced availability of DNA synthesis precursors is the cause of the mtDNA maintenance defect in Sym1 deficiency. Some of these molecules were also able to reduce mtDNA instability on another MDDS yeast model, characterized by mutations in MIP1 or RNR2 , likely increasing the levels of dNTPs [ 40 ]. Although it is necessary to test these molecules on mammalian cells and model organisms, the identification of molecules capable of reducing the mtDNA instability in different MDDS yeast models makes them a starting point for developing drugs for the treatment of diseases caused by mutations in different genes but all resulting in mtDNA synthesis defects.…”