Mechanism of Inhibition of the GluR2 AMPA Receptor Channel Opening by 2,3-Benzodiazepine Derivatives

Abstract

2,3-Benzodiazepine derivatives are drug candidates synthesized for 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 GluR2Qflip AMPA receptor channel opening by two 2,3-benzodiazepine derivatives that are structurally similar (BDZ-2 and BDZ-3). Using a laser-pulse photolysis technique with a time resolution of approximately 60 mus, we measured the effects of these inhibitors on both the channel opening rate and the whole-cell current amplitude. We found that both compounds preferably inhibit the open-channel state, although BDZ-2 is a more potent inhibitor in that it inhibits the open-channel state approximately 5-fold stronger than BDZ-3 does. Both compounds bind to the same noncompetitive site. Binding of an inhibitor to the receptor involves the formation of a loose, partially conducting channel intermediate, which rapidly isomerizes to a tighter complex. The isomerization reaction is identified as the main step at which the receptor distinguishes the structural difference between the two compounds. These results suggest that addition of a bulky group at the N-3 position on the diazepine ring, as in BDZ-3, does not alter the mechanism of action, or the site of binding, but does lower the inhibitory potency, possibly due to an unfavorable interaction of a bulky group at the N-3 position with the receptor site. The new mechanistic revelation about the structure-reactivity relationship is useful in designing conformation-specific, more potent noncompetitive inhibitors for the GluR2 AMPA receptor.

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

“…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. 40 Together, these data show that the single stereoisomeric difference at the C-4 position gives rise to the difference in biological activity of BDZ-d and BDZ-e in that the S configuration significantly diminishes the activity of the enantiomer on the same receptor.…”

“…For each compound, we further measured the inhibition constant for both the closed-channel (Figure 2b) and openchannel state (Figure 2c) of GluA2Q flip by setting the glutamate concentration at 100 μM and 3 mM, respectively. 15,16,33,34 As such, the inhibition constant of BDZ-d was found to be 15 ± 1 μM for the closed-channel state (K I ) and 30 ± 4 μM for the open-channel state (K̅ I ), by the use of eq 1 (all equations, including eq 1, are listed and described in the Supporting Information). Likewise, a K I of 201 ± 27 μM and a K̅ I of 304 ± 32 μM were determined for BDZ-e (see also the summary of these data in Table 1).…”

“…41 The time resolution, represented by the time constant of the photolysis reaction, was sufficient to enable us to measure the channel-opening reaction of the GluA2 receptor 42 and the effect of an inhibitor on the channel-opening rate process. 15,16,33,34 Shown in Figure 4a are representative whole-cell current traces obtained from the laser-pulse photolysis measurement. As a control (upper trace in Figure 4a), the rise of the glutamate-evoked whole-cell current reflected the opening of the GluA2 channels from a single HEK-293 cell.…”

“…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.…”

Potent and Selective Inhibition of a Single α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptor Subunit by an RNA Aptamer

“…(For the purpose of this work, the closed-channel conformation is defined as the unliganded, resting form of the receptor; this is "A" as in a general mechanism of channel opening in the "Experimental Procedures.") The choice of this conformation was based on our earlier hypothesis that the closed-channel conformation is more flexible or more modifiable in the context of inhibitor binding/inhibition (28). Therefore, the closed-channel conformation would be a better structural scaffold for geometrical complementarity selection (29).…”