Small-molecule inhibitors of cyclophilins block opening of the mitochondrial permeability transition pore and protect mice from hepatic ischemia–reperfusion injury

Abstract Background & Aims Hepatic ischemia–reperfusion injury is a complication of liver surgery that involves mitochondrial dysfunction resulting from mitochondrial permeability transition pore (mPTP) opening. Cyclophilin D (PPIF or CypD) is a peptidyl-prolyl cis-trans isomerase that regulates mPTP opening in the inner mitochondrial membrane. We investigated whether and how recently created small-molecule inhibitors of CypD prevent opening of the mPTP in hepatocytes and the resulting effects in cell models and livers of mice undergoing ischemia–reperfusion injury. Methods We measured the activity of 9 small-molecule inhibitors of Cyps in an assay of CypD activity. The effects of the small-molecule CypD inhibitors or vehicle on mPTP opening were assessed by measuring mitochondrial swelling and calcium retention in isolated liver mitochondria from C57BL/6J (wild-type) and Ppif –/– (CypD knock-out) mice, and in primary mouse and human hepatocytes by fluorescence microscopy. We induced ischemia–reperfusion injury in livers of mice given a small-molecule CypD inhibitor or vehicle before and during reperfusion, and collected samples of blood and liver for histologic analysis. Results The compounds inhibited peptidyl-prolyl isomerase activity (IC 50 values, 0.2 to 16.2 μM) and, as a result, calcium-induced mitochondrial swelling, by preventing mPTP opening (IC 50 values, 1.4 to 132 μM) in a concentration-dependent manner. The most potent inhibitor (C31) bound CypD with high affinity and inhibited swelling in mitochondria from livers of wild-type and Ppif –/– mice (indicating an additional, CypD-independent effect on mPTP opening) and in primary human and mouse hepatocytes. Administration of C31 in mice with ischemia–reperfusion injury before and during reperfusion restored hepatic calcium retention capacity and oxidative phosphorylation parameters and reduced liver damage compared with vehicle. Conclusions Recently created small-molecule inhibitors of CypD reduced calcium-induced swelling in mitochondria from mouse and human liver tissues. Administration of these compounds to mice during ischemia–reperfusion restored hepatic calcium retention capacity and oxidative phosphorylation parameters and reduced liver damage. These compounds might be developed to protect patients from ischemia–reperfusion injury after liver surgery or for other hepatic or non-hepatic disorders related to abnormal mPTP opening.