RESUMO
The fast skeletal troponin activators (FSTAs) Reldesemtiv and Tirasemtiv were developed for patients suffering from neuro-degenerative diseases of the motor nervous system, e.g. amyotrophic lateral sclerosis (ALS). The drug candidates can increase the sensitivity of troponin C to calcium by selectively activating the troponin complex resulting in increased skeletal muscle contraction. Although the development of the drug candidates is currently discontinued because of missed end points in phase III clinical studies with patients with ALS, phase I clinical trials showed an increase in muscle contraction force in healthy humans. This effect could be abused by athletes to enhance performance in sports. As the substances are listed on the 2024 edition of the World Anti-Doping Agency's Prohibited List, the aim of this study was to identify and characterize metabolites of Reldesemtiv and Tirasemtiv to ensure their reliable identification in doping control analyses. The biotransformation of the drug candidates was studied in vitro using pooled human liver microsomes and 3D cultivated human hepatic cells of the cell line HepaRG, yielding a total of 11 metabolites of Reldesemtiv and eight of Tirasemtiv. In addition, a human elimination study was conducted to investigate the metabolism and elimination profile of Tirasemtiv and Reldesemtiv in vivo, suggesting the N-glucuronide of Tirasemtiv and hydroxylated 3-fluoro-2-(3-fluoro-1-methylcyclobutyl)pyridine as well as its glucuronide as suitable target analytes for routine doping controls. Applying a validating HPLC-MS/MS method, optimized to detect Reldesemtiv and Tirasemtiv in human urine, microdosing (50 µg) of each substance was traceable for 24-72 h.
RESUMO
Mitochondrial ultrastructure is highly adaptable and undergoes dynamic changes upon physiological and energetic cues. MICOS (mitochondrial contact site and cristae organizing system), a large oligomeric protein complex, maintains mitochondrial ultrastructure as it is required for formation of crista junctions (CJs) and contact sites. MIC13 acts as a critical bridge between two MICOS subcomplexes. Deletion of MIC13 causes loss of CJs resulting in cristae accumulating as concentric rings and specific destabilization of the MIC10-subcomplex. Mutations in MIC13 are associated with infantile lethal mitochondrial hepato-encephalopathy, yet functional regions within MIC13 were not known. To identify and characterize such regions, we systemically generated 20 amino-acids deletion variants across the length of MIC13. While deletion of many of these regions of MIC13 is dispensable for its stability, the N-terminal region and a stretch between amino acid residues 84 and 103 are necessary for the stability and functionality of MIC13. We could further locate conserved motifs within these regions and found that a GxxxG motif in the N-terminal transmembrane segment and an internal WN motif are essential for stability of MIC13, formation of the MIC10-subcomplex, interaction with MIC10- and MIC60-subcomplexes and maintenance of cristae morphology. The GxxxG motif is required for membrane insertion of MIC13. Overall, we systematically found important conserved residues of MIC13 that are required to perform the bridging between the two MICOS subcomplexes. The study improves our understanding of the basic molecular function of MIC13 and has implications for its role in the pathogenesis of a severe mitochondrial disease.