The mitochondrial rhomboid protease PARL regulates mitophagy by balancing intramembrane proteolysis of PINK1 and PGAM5. It has been implicated in the pathogenesis of Parkinson's disease, but its investigation as a possible therapeutic target is challenging in this context because genetic deficiency of PARL may result in compensatory mechanisms. To address this problem, we undertook a hitherto unavailable chemical biology strategy. We developed potent PARL-targeting ketoamide inhibitors and investigated the effects of acute PARL suppression on the processing status of PINK1 intermediates and on Parkin activation. This approach revealed that PARL inhibition leads to a robust activation of the PINK1/Parkin pathway without major secondary effects on mitochondrial properties, which demonstrates that the pharmacological blockage of PARL to boost PINK1/Parkin-dependent mitophagy is a feasible approach to examine novel therapeutic strategies for Parkinson's disease. More generally, this study showcases the power of ketoamide inhibitors for cell biological studies of rhomboid proteases.

1st Faculty of Medicine Charles University Kateřinská 32 Prague 121 08 Czech Republic

Center for Biochemistry and Cologne Excellence Cluster on Cellular Stress Responses in Aging Associated Diseases Medical Faculty University of Cologne Joseph Stelzmann Strasse 52 Cologne 50931 Germany

Center for Molecular Biology of Heidelberg University DKFZ ZMBH Alliance Im Neuenheimer Feld 282 Heidelberg 69120 Germany

Center for Molecular Medicine Medical Faculty University of Cologne Joseph Stelzmann Strasse 52 Cologne 50931 Germany

Department of Molecular Genetics Faculty of Science Charles University Viničná 5 Prague 128 44 Czech Republic

Institute for Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging Associated Diseases Medical Faculty University of Cologne Joseph Stelzmann Strasse 52 Cologne 50931 Germany

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Science Flemingovo n 2 Prague 160 00 Czech Republic

Max Planck Institute for Biology of Ageing Joseph Stelzmann Str 9b Cologne 50931 Germany

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