Discovery, Synthesis, and Optimization of Diarylisoxazole-3- carboxamides as Potent Inhibitors of the Mitochondrial Permeability Transition Pore

This is the peer reviewed version of the following article: Roy, S., Šileikytė, J., Schiavone, M., Neuenswander, B., Argenton, F., Aubé, J., … Schoenen, F. J. (2015). Discovery, Synthesis, and Optimization of Diarylisoxazole-3-carboxamides as Potent Inhibitors of the Mitochondrial Permeability Transition Pore. ChemMedChem, 10(10), 1655–1671. http://doi.org/10.1002/cmdc.201500284, which has been published in final form at doi.org/10.1002/cmdc.201500284. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.The mitochondrial permeability transition pore (mtPTP) is a Ca2+-requiring mega-channel which, under pathological conditions, leads to the deregulated release of Ca2+ and mitochondrial dysfunction, ultimately resulting in cell death. Although the mtPTP is a potential therapeutic target for many human pathologies, its potential as a drug target is currently unrealized. Herein we describe an optimization effort initiated around hit 1, 5-(3-hydroxyphenyl)-N-(3,4,5-trimethoxyphenyl)isoxazole-3-carboxamide, which was found to possess promising inhibitory activity against mitochondrial swelling (EC50 100 µm). This enabled the construction of a series of picomolar mtPTP inhibitors that also potently increase the calcium retention capacity of the mitochondria. Finally, the therapeutic potential and in vivo efficacy of one of the most potent analogues, N-(3-chloro-2-methylphenyl)-5-(4-fluoro-3-hydroxyphenyl)isoxazole-3-carboxamide (60), was validated in a biologically relevant zebrafish model of collagen VI congenital muscular dystrophies

The Mitochondrial Permeability Transition in Mitochondrial Disorders

Mitochondrial permeability transition pore (PTP), a (patho)physiological phenomenon discovered over 40 years ago, is still not completely understood. PTP activation results in a formation of a nonspecific channel within the inner mitochondrial membrane with an exclusion size of 1.5 kDa. PTP openings can be transient and are thought to serve a physiological role to allow quick Ca2+ release and/or metabolite exchange between mitochondrial matrix and cytosol or long-lasting openings that are associated with pathological conditions. While matrix Ca2+ and oxidative stress are crucial in its activation, the consequence of prolonged PTP opening is dissipation of the inner mitochondrial membrane potential, cessation of ATP synthesis, bioenergetic crisis, and cell death—a primary characteristic of mitochondrial disorders. PTP involvement in mitochondrial and cellular demise in a variety of disease paradigms has been long appreciated, yet the exact molecular entity of the PTP and the development of potent and specific PTP inhibitors remain areas of active investigation. In this review, we will (i) summarize recent advances made in elucidating the molecular nature of the PTP focusing on evidence pointing to mitochondrial FoF1-ATP synthase, (ii) summarize studies aimed at discovering novel PTP inhibitors, and (iii) review data supporting compromised PTP activity in specific mitochondrial diseases

The translocator protein (peripheral benzodiazepine receptor) mediates rat-selective activation of the mitochondrial permeability transition by norbormide

AbstractWe have investigated the mechanism of rat-selective induction of the mitochondrial permeability transition (PT) by norbormide (NRB). We show that the inducing effect of NRB on the PT (i) is inhibited by the selective ligands of the 18kDa outer membrane (OMM) translocator protein (TSPO, formerly peripheral benzodiazepine receptor) protoporphyrin IX, N,N-dihexyl-2-(4-fluorophenyl)indole-3-acetamide and 7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin-2-one; and (ii) is lost in digitonin mitoplasts, which lack an intact OMM. In mitoplasts the PT can still be induced by the NRB cationic derivative OL14, which contrary to NRB is also effective in intact mitochondria from mouse and guinea pig. We conclude that selective NRB transport into rat mitochondria occurs via TSPO in the OMM, which allows its translocation to PT-regulating sites in the inner membrane. Thus, species-specificity of NRB toward the rat PT depends on subtle differences in the structure of TSPO or of TSPO-associated proteins affecting its substrate specificity

Regulation of the Inner Membrane Mitochondrial Permeability Transition by the Outer Membrane Translocator Protein (Peripheral Benzodiazepine Receptor)*

We studied the properties of the permeability transition pore (PTP) in rat liver mitochondria and in mitoplasts retaining inner membrane ultrastructure and energy-linked functions. Like mitochondria, mitoplasts readily underwent a permeability transition following Ca2+ uptake in a process that maintained sensitivity to cyclosporin A. On the other hand, major differences between mitochondria and mitoplasts emerged in PTP regulation by ligands of the outer membrane translocator protein of 18 kDa, TSPO, formerly known as the peripheral benzodiazepine receptor. Indeed, (i) in mitoplasts, the PTP could not be activated by photo-oxidation after treatment with dicarboxylic porphyrins endowed with protoporphyrin IX configuration, which bind TSPO in intact mitochondria; and (ii) mitoplasts became resistant to the PTP-inducing effects of N,N-dihexyl-2-(4-fluorophenyl)indole-3-acetamide and of other selective ligands of TSPO. Thus, the permeability transition is an inner membrane event that is regulated by the outer membrane through specific interactions with TSPO