Mechanism of inhibition of the GluA2 AMPA receptor channel opening: consequences of adding an N-3 methylcarbamoyl group to the diazepine ring of 2,3-benzodiazepine derivatives.

The fundamental postulate of the structure-activity relationship is that molecular activity is a function of its structure; consequently, structurally similar molecules have similar functions. The ultimate goal of studying the structure-activity relationship is to establish predictability for designing better regulatory agents, such as inhibitors, that have higher potency and tighter selectivity toward a common protein target. To achieve such predictability, a set of similar chemical structures is constructed, and rigorous studies, such as the study of the mode of action for these structures, are required to develop atom-based “descriptors” for the structure-activity relationship. For this reason, here we describe a rapid kinetic investigation of the functional consequences of adding an N-3 methylcarbamoyl group to the 2,3-benzodiazepine ring, as part of our comprehensive study of the structure-activity relationship of 2,3-benzodiazepine compounds.1–2 2,3-Benzodiazepine derivatives, also known as GYKI compounds, are synthesized as a group of drug candidates that target α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. AMPA receptors, together with N-methyl-d-aspartate (NMDA) and kainate receptors, belong to the ionotropic glutamate receptor family.3–5 AMPA receptors mediate most excitatory neurotransmission in the brain and are indispensible for brain development and function, such as memory and learning.4 However, excessive activity of AMPA receptors leads to extra calcium entry, and the calcium build-up inside the cell results in cell death.6–7 This phenomenon, known as excitotoxicity, is a general pathogenic mechanism that underlies several neurological disorders and diseases, including epilepsy, cerebral ischemia, and amyotrophic lateral sclerosis.4 Thus, compounds that inhibit AMPA receptors, especially those of the GluA2 subunit that control the Ca2+ permeability,4, 8 are highly sought out as potential drug candidates. Among different types of AMPA receptor inhibitors, 2,3-benzodiazepine compounds are one of the best because of their tight selectivity for AMPA receptors. Their superior selectivity is due in part to their binding to site(s) distinct from where agonist binds on AMPA receptors.9–10 Tests of these compounds in animal models also show promising neuroprotection.10–11 Consequently, hundreds of 2,3-benzodiazepine derivatives have been synthesized based on the prototypic structure of 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine, also known as GYKI 5246611 (Figure 1). To make new compounds that are more potent inhibitors, it is important to characterize the structure-activity relationship for this group of inhibitors. Figure 1 Chemical structures of GYKI 52466 and BDZ-f. The chemical names of these compounds are given in the text. To date, the structure-activity relationship for these compounds has not been systematically characterized on a time scale comparable to that of the opening of an AMPA receptor channel. AMPA receptors open their channels, in response to the binding of glutamate, the endogenous neurotransmitter, in the microsecond (µs) time scale but desensitize in the millisecond (ms) time scale.12 Therefore a kinetic investigation of the mode of action of a 2,3-benzodiazepine compound must be carried out using a technique that provides sufficient time resolution to measure the channel-opening rate of an AMPA receptor.1 However, commonly used techniques, such as solution flow and single-channel recording, do not have sufficient time resolution for characterizing the effect of a 2,3-benzodiazepine compound on the channel-opening rate process. To overcome that limitation, we used a laser-pulse photolysis technique in this study, together with a photolabile precursor of glutamate or caged glutamate.13–14 This technique provides a time resolution of ~60 µs,12, 14 which is sufficient for measuring the rate of channel opening and for investigating the mechanism of inhibition without the complication of channel desensitization in the millisecond time scale.1, 14 In this study, we investigated the mechanism by which the GluA2Qflip AMPA receptor channel-opening rate process is inhibited by (−)1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-4,5-dihydro-3-methylcarbamoyl-2,3-benzodiazepine or BDZ-f (this compound is also named GYKI 53784 or LY 303070; see Figure 1). The questions we asked are: What is the mechanism of action of BDZ-f? Does the addition of an N-3 methylcarbamoyl group affect potency and specificity for the open-channel and the closed-channel conformations? Do GYKI 52466 and BDZ-f bind to the same site, or does the addition of an N-3 methylcarbamoyl group affect the binding site? Answers to these questions will allow us to determine whether addition of this group at the N-3 position in the diazepine ring will make BDZ-f a better inhibitor than the parent compound, i.e., GYKI 52466. This report is the third in a comprehensive mechanistic study to establish a more quantitative structure-activity relationship for a series of 2,3-benzodiazepine compounds.1–2