Zagociguat prevented stressor-induced neuromuscular dysfunction, improved mitochondrial physiology, and increased exercise capacity in diverse mitochondrial respiratory chain disease zebrafish models

Leonard Burg¹Heeyong Yoon¹Min Peng¹Peter Germano²Emily Reesey Gretzmacher¹Vernon Anderson¹Eiko Nakamaru-Ogiso¹Marni Falk^1,3*

• ¹Children's Hospital of Philadelphia, Philadelphia, United States
• ²Cyclerion Therapeutics, Cambridge, Maryland, United States
• ³Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States

BACKGROUND: Zagociguat (zag) is a tricyclic, CNS-penetrant, soluble guanylate cyclase (sGC) stimulator that has been evaluated in phase 2a/b clinical studies of primary mitochondrial disease (PMD) subjects with Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like episodes syndrome (MELAS). To explore utility in a broader array of PMDs and secondary mitochondrial disorders, we performed pre-clinical modeling of zag across larval and adult zebrafish models with biochemical deficiencies in diverse respiratory chain (RC) complexes or dihydrolipoamide dehydrogenase.Zag was evaluated for tissue uptake, gross toxicity, protection from RC toxin-induced brain death, neuromuscular dysfunction, heartbeat loss, and biochemical dysfunction in transgenic or toxin exposed zebrafish with mitochondrial enzyme deficiencies in complex I (ndufs2 -/-or rotenoneexposed wild-type (WT)); complex IV (surf1 -/-or azide exposed WT); multiple RC complexes (fbxl4 - /-); or pyruvate dehydrogenase complex (dldh -/-). Zag effects were also studied on whole body oxygen consumption capacity (MO2) and swimming activity of WT and complex IV disease adult zebrafish.RESULTS: Similar zag levels were observed in adult brains and tail muscle. No morphological or functional toxic effects of zag were observed on larvae viability. Zag provided neuromuscular protection in complex I deficient genetic and pharmacologic inhibitor models. In complex IV deficient models, prevention from brain death occurred at 100 nM zag in high dose azide-exposed WT larvae, although no rescue of swimming or neuromuscular phenotypes in low dose azideexposed surf1 -/-larvae was observed. 100 nM zag rescued MO2 and maximum swimming speed in adult surf1 -/-zebrafish. Larval swimming activity was also preserved with 10 nM zag treatment in azide-stressed fbxl4 -/-larvae, but not at 10 nM, 100 nM, or 1 µM zag in dldh -/-larvae. 10 nM zag enhanced complex I enzyme activity suggestive of mitochondrial biogenesis and key aspects of mitochondrial physiology in azide-exposed surf1 -/-and fbxl4 -/-larvae.Pre-clinical evaluation of zag demonstrated its safety, significant protection of neuromuscular dysfunction and/or acute RC stressor induced decompensation, and improved mitochondrial physiology across multiple different genetic and/or pharmacologic models of RC deficient PMD. Zag may yield therapeutic potential for an array of diseases with mitochondrial dysfunction beyond MELAS, potentially including Leigh syndrome spectrum disorder and primary mitochondrial myopathies.