Rationale: Cyclophilin D (CypD), a peptidyl-prolyl isomerase (PPIase), is an integral modulatory component of the mitochondrial permeability transition pore (MPTP) which regulates cell death. Indeed, CypD knockout mice have been shown to more resistant to traumatic brain injury compared to controls (PMID: 25528329; PMID: 27489538). Further, CypD inhibition appears to ameliorate pathological conditions associated with mitochondrial dysfunction (PMID: 30558250), including Parkinson’s disease (PMID: 21529244), Alzheimer’s disease (PMID: 18806802), amyotrophic lateral sclerosis (PMID: 22961554) and Huntington disease (PMID: 20558522). We have previously shown that pharmacological inhibition of CypD with cyclosporin A (CsA) or N-methyl-4-isoleucine-cyclosporin (NIM811) renders anti-seizure effects in epileptic Kcna1-null mice (PMID: 25899847). However, as current inhibitors of CypD have several disadvantages such as off-target effects, a narrow therapeutic index, and/or immunosuppressive effects, there is a need to develop novel CypD inhibitors with a more favorable safety and pharmacokinetics profile. Here, we evaluated the effect of two novel CypD inhibitors CC-2055 and CC-4126 (Cypralis Ltd.) on the Ca2+-induced permeability transition using acutely isolated brain mitochondria from control and CypD KO mice. Methods: Mitochondria were acutely isolated from whole brain from control (C3H) and CypD KO mice aged P35 (males, N=6; females, N=3) as previously reported (PMID: 12783416). Ca2+-induced MPTP thresholds were measured with a Shimadzu RF-5301PC spectrofluorophotometer (PMID: 16517608). Statistical analysis was performed using GraphPad Prism. The significance threshold was set at .05. Results: While thecalcium retention capacity (CRC) in brain mitochondria isolated from WT mice was significantly increased by treatment with 1 µM CC-2205 (~11%, p< 0.05) and 1 µM CC-4126 (~15%, p< 0.01), the Ca2+ buffering capacity of brain mitochondria from CypD KO mice increased by ~ 20% (p< 0.01). Neither 1 µM CC-2205 nor 1 µM CC-4126 affected CRC in mitochondria isolated from CypD KO mice, confirming that both CC-2205 and CC-4126 selectively targets CypD to decrease Ca2+ sensitivity to MPTP opening. We found no sex-related differences (males vs. females) in MPTP thresholds when CC-2205 and CC-4126 were applied to mitochondria from either WT or CypD KO mice. Conclusions: Both CC-2205 and CC-4126 (at 1 µM) inhibit Ca2+-mediated MPTP opening via CypD binding with similar effectiveness. These novel compounds may represent potential therapies for a broad array of conditions associated with mitochondrial dysfunction and neurodegeneration. Funding: Please list any funding that was received in support of this abstract.: University of Calgary, Alberta Children’s Hospital Research Institute
