Novel Quinoxaline Derivatives As Potent Cyclophilin D Inhibitors Exhibit Highly Inhibitory Activity Against Ca～(2+)-dependent Rat Mitochondral Swelling And Ca～(2+) Uptake/release

The mitochondrial permeability transition pore (MPTP), a non-specific pore will open in the inner mitochondrial membrane with high concentration of matrix Ca²⁺, especially companied by cellular stress or damage. Opening of MPTP may cause dissipation of the mitochondrial membrane potential, depleted adenine nucleotides, uncoupling of oxidative phosphorylation, large amplitude mitochondrial swelling, rupture of the outer membrane and release of apoptogenic proteins from the mitochondrial intermembrane space. In most models, it is suggested that MPTP is comprised by three major proteins: the adenine nucleotide translocator (ANT) in the inner membrane, the voltage-dependent anion channel (VDAC) in the outer membrane and cyclophilin D (CypD) in the matrix of mitochondria. Like other cyclophilins, CypD has also peptidyl-prolyl cis-trans isomerase activity (PPIase), it catalyses the cis-trans isomerization of peptidyl-prolyl (Xaa-Pro) bonds. The natural ligand of CypD, CSA can inhibit the PPIase activity of CypD and attenuate the binding ability between CypD and ANT, thereby inhibiting the MPTP opening . Components of MPTP can be regarded as potential targets for pharmacological intervention in human conditions in which apoptotic or necrotic cell death is implicated, such as ischemial/reperfusion injury, trauma and neurodegenerative diseases. The CypD's activity pocket is big and shallow. It seems that only the sum of multiple interactions between a ligand and CypD is capable of leading to a high binding. As a consequence, it is difficult to find a small drug-like, low-molecular weight compound with decent affinity to CypD. Recently, CypD-binding compounds are described in two patents. The KDs for CypD-binding are in the range of 100 μM. We found seven novel quinoxaline derivatives that can highly inhibit the PPIase activity of CypD. By using surface plasmon resonance (SPR) and fluorescence titration techniques, the kinetic analysis of the CypD/inhibitor interaction was investigated. The inhibition ability against the rat liver Ca2+-depedent mitochondrial swelling and Ca2+ uptake/release for the compounds was also determined. The binding selectivity of CypD over CypA for the tested compounds was analyzed, and well explained based on the molecular docking technique. It is hoped that this present research might provide a useful approach in the discovery of cyclophilin D...