Novel cyclophilin D inhibitors derived from quinoxaline exhibit highly inhibitory activity against rat mitochondrial swelling and Ca2+ uptake/ release.

Aim: To investigate methods for identifying specific cyclophilin D (CypD) inhibitors derived from quinoxaline, thus developing possible lead compounds to inhibit mitochondrial permeability transition (MPT) pore opening.

Methods: Kinetic analysis of the CypD/inhibitor interaction was quantitatively performed by using surface plasmon resonance (SPR) and fluorescence titration (FT) techniques. IC(50) values of these inhibitors were determined by PPIase inhibition activity assays.

Results: All the equilibrium dissociation constants (KD) of the seven compounds binding to CypD were below 10 mumol/L. The IC(50) values were all consistent with the SPR and FT results. Compounds GW2, 5, 6, and 7 had high inhibition activities against Ca(2+)-dependent rat liver mitochondrial swelling and Ca(2+) uptake/release. Compound GW5 had binding selectivity for CypD over CypA.

Conclusion: The agreement between the measured IC(50) values and the results of SPR and FT suggests that these methods are appropriate and powerful methods for identifying CypD inhibitors. The compounds we screened using these methods (GW1-7) are reasonable CypD inhibitors. Its potent ability to inhibit mitochondrial swelling and the binding selectivity of GW5 indicates that GW5 could potentially be used for inhibiting MPT pore opening.