| α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are one of the three subtypes of ionotropic glutamate receptors. AMPA receptors mediate fast synaptic neurotransmission in the central nervous system (CNS). Over-activation of calcium permeable AMPA receptors causes intracellular calcium overload, which leads to neurodegeneration and cell death. As such, AMPA receptors have been implicated in a number of neurological disorders and diseases, such as epilepsy, amyotrophic lateral sclerosis (ALS), and Parkinson's disease. 2,3-Benzodiazepine derivatives (or GYKI compounds) are a group of structurally similar compounds synthesized as inhibitors of AMPA receptors, and they have been used as potential drug candidates for the treatment of various neurological disorders involving excessive activity of AMPA receptors. However, the detailed mechanism of action of these inhibitors has not been well understood, and a structure-activity relationship has not been defined at the molecular level.;To investigate the mechanism of action and establish a detailed structure-activity relationship at the molecular level, I used rapid kinetic techniques, primarily a laser-pulse photolysis technique that provided a microsecond time resolution, combined with whole-cell patch clamp recording. Specifically, I investigated the effect of an inhibitor on the channel-opening rate process that occurs within the microsecond-to-millisecond time span. I particularly focused on a group of compounds that all contain a C-4 methyl and additional functional groups attached to the N-3 position on the diazepine ring.;My results show several key findings. (i) These compounds are noncompetitive inhibitors of AMPA receptors with a preference of closed-channel over the open-channel conformation. (ii) All of these compounds show a strong specificity for GluA2 and GluA1 AMPA receptor subunits but not GluA3 and GluA4. (iii) The R configuration, but not the S, of the 4-methyl group on the 2,3-benzodiazepine ring is crucial for the activity of resulting compounds as AMPA receptor inhibitors. (iv) All of these 4-methyl group-containing compounds bind to the same noncompetitive site on the GluA2 receptors. This is the same site where GYKI 52446 binds as well. (v) The N-3 on the 2,3-benzodiazepine ring is a desirable position in that attaching functional groups to this position strengthens potency, as compared with GYKI 52466, which is the prototypic compound of this family and is underivatized at N-3 position. Among those, compounds with heterocyclic functional groups attached to the N-3 position exhibit the strongest inhibitory potency.;These results are useful for design of new 2,3-benzodiazepine compounds with predictable molecular properties, including selectivity, potency and conformational specificity. My results also suggest new directions for developing more effective 2,3-benzodiazepine compounds as potential drug candidates for the treatment of neurological disorders and diseases involving over-activation of AMPA receptors. |
