The seizure‐inducing plastic explosive RDX inhibits the α1β2γ2 GABA_A receptor

Abstract

Objective Royal demolition explosive (RDX) can induce seizures in wildlife and humans following release into the environment or after voluntary consumption. During the Vietnam War, RDX intoxication was the most common cause of generalized seizures in US service personnel, and in some sections of the armed forces, eating of RDX has continued as “a dare” to this day. After its mechanism of action was long unknown, RDX was recently shown to be a GABAA receptor antagonist. We here determined the GABAA receptor subtype‐selectivity of RDX and mapped its functional binding site. Methods We used whole‐cell patch‐clamp to determine the potency of RDX on 10 recombinantly expressed GABAA receptors and mapped the RDX binding site using a combination of Rosetta molecular modeling and site‐directed mutagenesis. Results RDX was found to reversibly inhibit the α1β2γ2 GABAA receptor with an IC50 of 23 μmol/L (95% CI 15.1–33.3 μmol/L), whereas α4 and α6 containing GABAA receptor combinations were 4–10‐fold less sensitive. RDX is binding to the noncompetitive antagonist (NCA) site in the pore. In a molecular model based on the cryo‐EM structure of the resting state of the α1β2γ2 receptor, RDX forms two hydrogen bonds with the threonines at the T6’ ring and makes hydrophobic interactions with the valine and alanine in 2′ position of the α1 or β2 subunits. Interpretation Our findings characterize the mechanism of action of RDX at the atomistic level and suggest that RDX‐induced seizures should be susceptible to treatment with GABAA modulating drugs such as benzodiazepines, barbiturates, propofol, or neurosteroids.

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) causes seizure activity in larval zebrafish via antagonism of γ-aminobutyric acid type A receptor α1β2γ2

“…However, we have previously demonstrated that Zolpidem is not effective at mitigating seizures induced by TETS, a GABA A R antagonist that selectively targets α2 and α6 subunits (Mundy et al 2021 ). Our observations that Zolpidem is effective at mitigating RDX-induced seizures supports our previous in vitro and in silico modeling work (Pressly et al 2022 ) that identified the α1 GABA A R subunit as a major target of RDX.…”

“…The significant difference between EC 50 values measured for Zolpidem and 1:1 Zolpidem:compound 2-261, with the EC 50 of the 1:1 combination shifted to the left, suggests that modulation of both subunits is necessary for a more efficient mitigation of seizures. Consistent with the in vitro findings that RDX does not target α6-containing GABA A R (Pressly et al 2022 ), SB-205384 (an α-6 select PAM) was unable to mitigate seizure-like activity. Combined, these data support the hypothesis that the molecular target of RDX is α1β2γ2.…”

“…Based on data obtained using both competitive binding assays and rat brain slice recordings from the amygdala, (Williams et al 2011 ), it was suggested that RDX binds to the non-competitive antagonist (NCA) site in the pore of γ-aminobutyric acid type A receptors (GABA A R). Recent in silico and in vitro studies from our laboratory corroborated these conclusions (Pressly et al 2022 ). Mutations at the so-called “threonine ring” in the pore of the GABA A R drastically reduced RDX potency.…”

“…Additionally, 10 µM of the α1-selective Zolpidem, 1 µM of the β2/3-selective compound 2–261, or 1 µM of a 1:1 combination of Zolpidem and compound 2–261 decreased seizure activity to approximately the same degree as treatment with 3 µM MDZ. Treatment with the α6-selective PAM SB-205384 was included as a negative control because in vitro studies suggested RDX does not target α6-containing GABA A R (Pressly et al 2022 ). SB-205384 was unable to decrease seizure activity to the same extent as 3 µM MDZ at any of the concentrations tested.…”

“…Mutations at the so-called “threonine ring” in the pore of the GABA A R drastically reduced RDX potency. Further, using whole-cell patch-clamp analyses of recombinantly expressed GABA A R, we found that RDX preferentially blocked the GABA A R subtype α1β2γ2 in a fully reversible manner (Pressly et al 2022 ). However, it remains unknown whether this interaction between RDX and α1β2γ2 GABA A R mediates the in vivo seizurogenic activity of RDX.…”