Inhibiting Long-Chain 3-Ketoacyl-CoA Thiolase: A Novel Strategy for Treating Mitochondrial Fatty Acids Oxidation Disorders

Al-Walid Mohsen, PhD1*, Anuradha Karunanidhi, MS1, Bianca Seminotti, PhD1, Guilhian Leipnitz, PhD1,2, Catherine Kochersperger, MS1, Lina Ghaloul-Gonzalez1, MD, Shrabani Basu,

Mike Bennet, PhD4, Jerry Vockley, MD, PhD1,4

1Division Medical Genetics, Department Pediatrics, University of Pittsburgh, Pittsburgh, PA, USA

2PPG Ciências Biológicas: Bioquímica, Departamento de Bioquímica, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil

3Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA; Philadelphia, PA, USA

4Department of Human Genetics, University of Pittsburgh, Pittsburgh, PA, USA 

*e-mail: aam27@pitt.edu, Phone: +1 412-692-6498

Background: Mitochondrial fatty acid oxidation (MFAO) has two major components, a transport component comprised of CPTI, CACT, and CPTII, and a catabolic β-oxidation pathway-spiral component, comprised of the acyl-CoA dehydrogenases (SCAD, MCAD and VLCAD) and the mitochondrial trifunctional protein (MTFP). MTFP is an a4b4-octamer containing the enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and 3-ketoacyl-CoA thiolase functions for long-chain substrates. Channeling of intermediates in such structural arrangement for these two major components is expected. Binding of substrates/products to the catalytic sites of proteins often provides stability as has been reported for MCAD. Missense unstable variants have been reported for these MFAO proteins in patients with clinical deficiencies. In this study, trimetazidine, an inhibitor of LCKAT approved as ischemic heart disease therapy in >90 countries, was used to induce accumulation of MFAO intermediates and investigate their effect on upstream MFAO proteins stability.

Methods: Fibroblasts from patients with VLCAD, MCAD, LCHAD, and TFP deficiencies were treated with trimetazidine up to 10 µM in culture. Protein variants presence was monitored using in situ immunostaining, western blotting and/or enzyme assay. Acylcarnitines were monitored in one VLCAD deficient cell line.

Results: In all deficient fibroblasts the level of the mutant protein increased significantly in a dose dependent fashion. The increase varied with mutation and correlated with the relative stability of the protein. Acylcarnitines in one culture VLCAD deficient cell line decreased with up to 0.25 µM trimetazidine supplementation, then increased in the presence of higher concentrations of drug.

Conclusion: Trimetazidine could provide therapeutic benefit for patients with MFAO disorders.

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