Quinoxalinedione deprotonation is important for glutamate receptor binding.

Quinoxalinediones are an important class of competitive antagonists at ionotropic glutamate receptors (iGluRs), where they are widely used to block α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptor responses. In this study we utilize two prototypic quinoxalinedione antagonists, namely DNQX and CNQX, which quench the intrinsic fluorescence of the ligand binding domain (LBD), to perform in vitro binding assays. We find that binding of DNQX and CNQX at the AMPA receptor GluA2 LBD is strongly pH dependent, whereas glutamate binding is not affected by pH. We also show that the deprotonation of DNQX, CNQX and other quinoxalinediones (NBQX and YM90K) occurs close to physiological pH, which can be explained by the lactam-lactim tautomerization of the quinoxalinedione scaffold. Analysis of our binding data indicates that quinoxalinedione deprotonation is a key requirement for binding, as we find a >100-fold higher affinity for binding of the monoanionic form compared to the neutral form. This suggests a large electrostatic contribution to the interaction with a conserved arginine residue located in the binding pocket of iGluRs. The strong pH dependence of quinoxalinedione binding, which has not previously been reported, is relevant for structure-function studies, but also for the use of quinoxalinediones in physiological experiments and envisioned therapeutic applications.

3-Cyano-3-aza-ß-amino Acid Derivatives as Inhibitors of Human Cysteine Cathepsins.

Nitrile-type inhibitors are known to interact with cysteine proteases in a covalent-reversible manner. The chemotype of 3-cyano-3-aza-ß-amino acid derivatives was designed in which the N-cyano group is centrally arranged in the molecule to allow for interactions with the nonprimed and primed binding regions of the target enzymes. These compounds were evaluated as inhibitors of the human cysteine cathepsins K, S, B, and L. They exhibited slow-binding behavior and were found to be exceptionally potent, in particular toward cathepsin K, with second-order rate constants up to 52 900 × 10(3) M(-1) s(-1).