Mechanism of Inhibition of the GluA2 AMPA Receptor Channel Opening: The Role of 4-Methyl versus 4-Carbonyl Group on the Diazepine Ring of 2,3-Benzodiazepine Derivatives

Abstract

2,3-Benzodiazepine derivatives are synthesized as drug candidates for a potential treatment of various neurodegenerative diseases involving the excessive activity of AMPA receptors. Here, we describe a rapid kinetic investigation of the mechanism of inhibition of the GluA2Qflip AMPA receptor channel opening by two 2,3-benzodiazepine derivatives, i.e. the prototypic 2,3-benzodiazepine compound GYKI 52466 [(1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine)] and 1-(4-aminophenyl)-3,5-dihydro-7,8-methylenedioxy-4H-2,3-benzodiazepin-4-one (BDZ-2). GYKI 52466 and BDZ-2 are structurally similar in that the 4-methyl group in the diazepine ring of GYKI 52466 is replaced by a carbonyl group, yielding BDZ-2. Using a laser-pulse photolysis technique with ∼60 μs time resolution, we characterize the effect of the two compounds individually on the channel-opening process of the GluA2Qflip receptor expressed in HEK-293 cells. We find that BDZ-2 preferentially inhibits the open-channel state, whereas GYKI 52466 is more selective for the closed-channel state of the GluA2Qflip receptors. Each inhibitor binds independently to its own noncompetitive site, yet the two sites do not interact allosterically. The significance of these results in the context of both the structure-activity relationship and the properties of the GluA2Qflip receptor channels is presented.

Mechanism of Inhibition of the GluA2 AMPA Receptor Channel Opening by Talampanel and Its Enantiomer: The Stereochemistry of the 4-Methyl Group on the Diazepine Ring of 2,3-Benzodiazepine Derivatives

“…Based on the fact that BDZ-d showed similar inhibition constants for the closed-channel and the open-channel state of the flip and flop isoforms of GluA2, we concluded that BDZ-d did not discriminate between the flip and flop isoforms of GluA2. This result is consistent with our observation from the study of other 2,3-benzodiazepine compounds for the flip and flop of GluA2, 15,16,33,34 and suggests that the flip/flop sequence cassette most likely is not involved in the site of binding. 15,16,33,34 Apparently, these compounds cannot be used to control the difference in various functional properties between the flip and flop isoforms of GluA2, such as desensitization 35−39 and channel opening reaction.…”

“…The fact that BDZ-d inhibited both k cl and k op was consistent with a noncompetitive mechanism of inhibition for BDZ-d acting on the GluA2 AMPA receptor. 15,16,33,34 Together, both the amplitude and rate data from our experiments further clarified that BDZ-d directly inhibits GluA2 AMPA receptor by binding to a noncompetitive site, contrary to what has been perceived. 11,20 BDZ-d Inhibited the Channel Opening of GluA2Q flip by a Two-Step Process.…”

“…As such, the structural change from GYKI 52466 to the resulting compound or 1-(4-aminophenyl)-3,5-dihydro-7,8-methylenedioxy-4H-2,3-benzodiazepin-4-one is in fact a replacement of the azomethine moiety with a ε-lactam moiety. 15 By this structure−activity relationship, it is therefore not surprising that both GYKI 52466 and BDZ-d compete for binding to the same noncompetitive site or the "M" site on the GluA2Q flip receptor. All of these sites are schematically shown in Figure 7.…”

“…15,16,33,34 By this mechanism, the inhibition of GluA2Q flip channels by BDZ-d involves two steps, and each step contributes to the overall inhibition of the receptor ( Figure 5). The first step involves the formation of an inhibitor-receptor intermediate, producing partial inhibition; and the second step involves a rapid isomerization from the loose inhibitor-receptor complex into a tighter, nonconducting complex that yields additional inhibition.…”

“…13,14 In other words, 2,3-benzodiazepine compounds with a C-4 carbonyl moiety bind to one site on the receptor, while those with a C-4 methyl group bind to a different site, as shown in our previous studies. 15,16 The importance of the C-4 methyl group is further reflected by its stereochemistry, as the C-4 methyl group can adopt two stereoisomeric configurations, provided that the 3,4-double bond is saturated (see the chemical structure of GYKI 52466 in Figure 1). However, the 3,4-dihydro analogue of GYKI 52466, known as GYKI 52895, turns out to be a selective dopamine uptake inhibitor.…”