Approved Drugs Dissolve Mutant Polymerase Aggregates and Restore Motor Function in Mice

Two medications that doctors already prescribe for entirely unrelated conditions can dissolve the protein clumps that clog mitochondria in a mouse model of progressive external ophthalmoplegia, according to a new study published in EMBO Molecular Medicine. Diltiazem hydrochloride, a blood pressure drug, and sulbactam, a beta-lactamase inhibitor used alongside antibiotics, restored the import of a mutant DNA polymerase into mitochondria, revived mitochondrial DNA replication, and significantly improved grip strength and rotarod performance in seven-week-old mice carrying a disease-relevant mutation.

Progressive external ophthalmoplegia, or PEO, is one of the most common manifestations of mitochondrial disorder, affecting roughly twenty percent of diagnosed mitochondrial disease patients. It arises from mutations in POLGA, the catalytic subunit of polymerase gamma, the enzyme responsible for copying and maintaining mitochondrial DNA. Patients typically develop drooping eyelids, progressive weakening of the eye muscles, and ataxia, with enormous variability in symptoms. There is currently no cure; treatment is limited to surgical management of ptosis and supplements such as coenzyme Q10, B vitamins, and antioxidants intended to support mitochondrial function.

The new work builds on the research team’s earlier finding that a subset of PEO-associated POLGA mutants is hyperubiquitylated by MITOL, a mitochondrial E3 ligase that attaches ubiquitin to lysine 1060 of the mutant proteins. This excessive ubiquitination triggers conformational changes, drives the mutants into insoluble aggregates enriched on the mitochondrial surface, and blocks their interaction with the import receptor Tom20. Unable to enter the mitochondrial matrix, the mutant polymerase cannot replicate mitochondrial DNA, and the resulting dysfunction cascades into fragmented mitochondrial networks, elevated fission markers such as MFF and phosphorylated Drp1, and increased mitophagy, the cellular machinery that clears damaged mitochondria.

To test whether this aggregation mechanism was widespread rather than an oddity of one or two mutations, the researchers extended their analysis to three additional PEO mutants, T251I, L304R, and R943H, alongside the previously characterized A467T and F961S variants. All of the newly tested mutants showed enhanced ubiquitination compared with wild-type POLGA, and several formed stable, SDS-resistant aggregates detectable by semi-denaturing detergent agarose gel electrophoresis and dynamic light scattering, which revealed increased hydrodynamic radii for the mutant proteins. Cells expressing these mutants showed reduced BrdU incorporation into mitochondrial DNA, indicating impaired replication.

With the aggregation mechanism confirmed across multiple variants, the team screened the Prestwick library of 1,280 small molecules, 98 percent of which are marketed approved drugs, using a split-GFP complementation assay in which fluorescence returns only when POLGA aggregates are disrupted. Nine compounds broke apart the mutant aggregates by more than 95 percent at low nanomolar concentrations without any detectable cellular toxicity in MTT assays. From these, the investigators selected diltiazem hydrochloride, designated C1, and sulbactam, designated C4, based on their lowest effective concentrations and minimal expected impact on mitochondrial homeostasis.

Biochemical experiments showed that sulbactam binds directly to the mutant polymerase but not to MITOL, and it did not alter the ligase’s activity toward other substrates such as RECQL4. Instead, the compound appears to reshape the mutant protein itself: tryptophan fluorescence spectra of treated mutants reverted to patterns resembling wild-type POLGA, hydrodynamic radii shrank to wild-type dimensions, and aggregate signals on SDD-AGE gels disappeared. Treated mutants regained interaction with Tom20, entered the mitoplast in in vitro import assays, and showed reduced hyperubiquitylation. In vitro primer extension assays demonstrated that both compounds enhanced the replication capability of the A467T mutant, and BrdU incorporation assays confirmed restored mitochondrial DNA synthesis, including faithful amplification of the full 16.6-kilobase mitochondrial genome in treated cells.

The functional consequences extended throughout mitochondrial physiology. Sulbactam treatment lowered fission markers, raised the fusion marker MFN2, restored mitochondrial membrane potential measured by TMRM staining, and reversed the depressed basal respiration and ATP production recorded in Seahorse metabolic analyses of mutant cells. Mitophagy markers including LC3B, PINK1, and Parkin declined after treatment, and live-cell mtKeima imaging confirmed reduced mitochondrial clearance. Notably, when cells were treated with bafilomycin to block autophagic degradation, sulbactam co-treatment significantly reduced LC3B accumulation in A467T-expressing cells, suggesting the compound prevents the elevated mitophagy rather than merely masking it.

The decisive test came in knock-in mice carrying the A449T mutation, the mouse equivalent of the human A467T variant. In skeletal muscle of both heterozygous and homozygous animals, the mutant polymerase was hyperubiquitylated and formed aggregates; four intravenous treatments with either compound, spaced five days apart, reversed both defects. BrdU incorporation into muscle mitochondrial DNA rose significantly in homozygous mice after sulbactam treatment, and muscle fiber area, which had shrunk in untreated mutants, recovered following the fourth dose. Most strikingly, seven-week-old mice treated with sulbactam showed improvements in body weight, grip strength, and time spent on the accelerating rotarod, while diltiazem significantly improved grip strength and rotarod performance without significantly changing body weight. Treated animals regained motor function to a level approaching their wild-type littermates.

The compounds showed encouraging specificity. In an alpha-synuclein aggregation model using SH-SY5Y cells seeded with pre-formed fibrils, neither diltiazem nor sulbactam changed the number of alpha-synuclein puncta per cell, indicating their aggregate-disrupting activity is not a generic protein anti-aggregation effect but something particular to the mutant polymerase conformation. The authors also note that sulbactam and newer formulations such as sulbactam-durlobactam have already been through numerous clinical trials for other indications, which could ease the path toward repurposing, although the doses, pharmacokinetics, and long-term safety profiles relevant to mitochondrial disease remain unestablished.

Important caveats temper the excitement. The study cannot yet say whether the rescued polymerase replicates mitochondrial DNA with normal fidelity; the compounds restored polymerization activity and mtDNA content, but whether exonuclease proofreading and error rates also recover is an open question the authors explicitly flag. The mouse phenotypes at seven weeks differ in some respects from an earlier study that found no body weight or rotarod differences in homozygous animals at around three months, discrepancies the authors attribute plausibly to age, genetic background, housing conditions, and behavioral protocol differences. The heterozygous aggregate accumulation observed here is interpreted as an early biochemical vulnerability rather than a dominant disease phenotype, given the strictly recessive inheritance of A467T in humans. The authors position the work firmly as preclinical evidence: promising, mechanistically grounded, and validated in vivo, but requiring further studies of pharmacokinetics, long-term safety, and response across different POLG variants before any clinical translation can be considered.

Subject of Research: Repurposing approved small molecules to disrupt mutant POLGA aggregation and restore mitochondrial function in progressive external ophthalmoplegia

Article Title: Small molecules rescue pathogenic POLGA aggregation and restore its functions in a mouse model of PEO

Article References: Mohammed, A., Kaur, T., Panwar, P., Ghosh, S., Ahmad, H., Kumbhakar, S., Muftuoglu, M., Viscomi, C., & Sengupta, S. (2026). Small molecules rescue pathogenic POLGA aggregation and restore its functions in a mouse model of PEO. EMBO Molecular Medicine. https://doi.org/10.1038/s44321-026-00532-3

Image Credits: AI Generated

DOI: 10.1038/s44321-026-00532-3

Keywords: POLGA, progressive external ophthalmoplegia, mitochondrial DNA, MITOL, protein aggregation, drug repurposing, sulbactam, diltiazem, mitophagy, knock-in mice, mitochondrial disease, polymerase gamma