U955 - IMRB, Inserm, UPEC, Ecole Nationale Vétérinaire d'Alfort, Créteil, France (M.P., B.G., D.M.);Université Paris-Est, UMR S955, UPEC, Créteil, France (A.A.B., I.R., J.M.P.); INSERM U955, Team « Viruses, Hepatology, Cancer », Hôpital Henri Mondor, Créteil, France (A.A.B., J.M.P., I.R.); Centre de Biochimie Structurale (CBS), INSERM U1054, CNRS UMR5048, Université de Montpellier, Montpellier, France (J.F.G.); and Department of Virology, Hôpital Henri Mondor, Créteil, France (J.M.P.).
U955 - IMRB, Inserm, UPEC, Ecole Nationale Vétérinaire d'Alfort, Créteil, France (M.P., B.G., D.M.);Université Paris-Est, UMR S955, UPEC, Créteil, France (A.A.B., I.R., J.M.P.); INSERM U955, Team « Viruses, Hepatology, Cancer », Hôpital Henri Mondor, Créteil, France (A.A.B., J.M.P., I.R.); Centre de Biochimie Structurale (CBS), INSERM U1054, CNRS UMR5048, Université de Montpellier, Montpellier, France (J.F.G.); and Department of Virology, Hôpital Henri Mondor, Créteil, France (J.M.P.)
J Pharmacol Exp Ther. 2021 Mar;376(3):348-357. doi: 10.1124/jpet.120.000359. Epub 2020 Dec 10.
Mitochondrial permeability transition pore (mPTP) opening is a key event in cell death during myocardial ischemia reperfusion. Inhibition of its modulator cyclophilin D (CypD) by cyclosporine A (CsA) reduces ischemia-reperfusion injury. The use of cyclosporine A in this indication is debated; however, targeting mPTP remains a major goal to achieve. We investigated the protective effects of a new original small-molecule cyclophilin inhibitor C31, which was specifically designed to target CypD. CypD peptidylprolyl isomerase (PPIase) activity was assessed by the standard chemotrypsin-coupled assay. The effects of C31 on mPTP opening were investigated in isolated mouse cardiac mitochondria by measuring mitochondrial swelling and calcium retention capacity (CRC) in rat H9C2 cardiomyoblasts and in adult mouse cardiomyocytes by fluorescence microscopy in isolated perfused mouse hearts and ex vivo after drug infusion in mice. C31 potently inhibited CypD PPIase activity and mitochondrial swelling. C31 was more effective at increasing mitochondrial CRC than CsA and was still able to increase CRC in (CypD-inactivated) cardiac mitochondria. C31 delayed both mPTP opening and cell death in cardiomyocytes subjected to hypoxia reoxygenation. However, high concentrations of both drugs were necessary to reduce mPTP opening in isolated perfused hearts, and neither CsA nor C31 inhibited mPTP opening in heart after in vivo infusion, underlying the importance of myocardial drug distribution for cardioprotection. C31 is an original inhibitor of mPTP opening involving both CypD-dependent and -independent mechanisms. It constitutes a promising new cytoprotective agent. Optimization of its pharmacokinetic properties is now required prior to its use against cardiac ischemia-reperfusion injury. SIGNIFICANCE STATEMENT: This study demonstrates that the new cyclophilin inhibitor C31 potently inhibits cardiac mitochondrial permeability transition pore (mPTP) opening in vitro and ex vivo. The dual mechanism of action of C31 allows the prevention of mPTP opening beyond cyclophilin D inhibition. Further development of the compound might bring promising drug candidates for cardioprotection. However, the lack of effect of both C31 and cyclosporine A after systemic administration demonstrates the difficulties of targeting myocardial mitochondria in vivo and should be taken into account in cardioprotective strategies.
线粒体通透性转换孔（mPTP）的开放是心肌缺血再灌注过程中细胞死亡的关键事件。环孢素 A（CsA）抑制其调节剂亲环素 D（CypD）可减少缺血再灌注损伤。虽然 CsA 在这种情况下的使用存在争议，但靶向 mPTP 仍然是实现的主要目标。我们研究了新的原创小分子亲环素抑制剂 C31 的保护作用，C31 是专门设计用于靶向 CypD 的。通过标准胰凝乳蛋白酶偶联测定评估 CypD 肽基脯氨酰顺反异构酶（PPIase）活性。通过测量分离的小鼠心肌线粒体的线粒体肿胀和钙保留能力（CRC），在大鼠 H9C2 心肌细胞和在分离的灌注小鼠心脏中的荧光显微镜和在小鼠体内药物输注后的离体心脏中研究 C31 对 mPTP 开放的影响。C31 强烈抑制 CypD PPIase 活性和线粒体肿胀。C31 增加线粒体 CRC 的效果优于 CsA，并且仍然能够增加（CypD 失活）心肌线粒体的 CRC。C31 延迟缺氧复氧后心肌细胞的 mPTP 开放和细胞死亡。然而，在分离的灌注心脏中减少 mPTP 开放需要两种药物的高浓度，并且 CsA 或 C31 都不能抑制体内输注后心脏的 mPTP 开放，这突显了心肌药物分布对心脏保护的重要性。C31 是一种涉及 CypD 依赖性和非依赖性机制的 mPTP 开放的原创抑制剂。它构成了一种有前途的新细胞保护剂。在将其用于对抗心肌缺血再灌注损伤之前，现在需要优化其药代动力学特性。意义声明：本研究表明，新型亲环素抑制剂 C31 可有效抑制体外和离体心脏线粒体通透性转换孔（mPTP）的开放。C31 的双重作用机制允许在环孢素 D 抑制之外预防 mPTP 开放。该化合物的进一步开发可能为心脏保护带来有前途的药物候选物。然而，全身给予 C31 和环孢素 A 均无效表明了在体内靶向心肌线粒体的困难，应在心脏保护策略中加以考虑。
