Acute intoxication with picrotoxin or the rodenticide tetramethylenedisulfotetramine (TETS) can cause seizures that rapidly progress to
status epilepticus
and death. Both compounds inhibit ...γ-aminobutyric acid type-A (GABA
A
) receptors with similar potency. However, TETS is approximately 100 × more lethal than picrotoxin. Here, we directly compared the toxicokinetics of the two compounds following intraperitoneal administration in mice. Using LC/MS analysis we found that picrotoxinin, the active component of picrotoxin, hydrolyses quickly into picrotoxic acid, has a short in vivo half-life, and is moderately brain penetrant (brain/plasma ratio 0.3). TETS, in contrast, is not metabolized by liver microsomes and persists in the body following intoxication. Using both GC/MS and a TETS-selective immunoassay we found that mice administered TETS at the LD
50
of 0.2 mg/kg in the presence of rescue medications exhibited serum levels that remained constant around 1.6 μM for 48 h before falling slowly over the next 10 days. TETS showed a similar persistence in tissues. Whole-cell patch-clamp demonstrated that brain and serum extracts prepared from mice at 2 and 14 days after TETS administration significantly blocked heterologously expressed α
2
β
3
γ
2
GABA
A
-receptors confirming that TETS remains pharmacodynamically active in vivo
.
This observed persistence may contribute to the long-lasting and recurrent seizures observed following human exposures. We suggest that countermeasures to neutralize TETS or accelerate its elimination should be explored for this highly dangerous threat agent.
We have synthesized and established the structure of a long-suspected, but hitherto unknown, benzofuran side product (EBI) formed during the synthesis of NH-3. Understanding the mechanism of its ...formation has enabled isotope (D) labeling. We further developed a highly efficient method for separating EBI from NH-3. Interestingly, EBI was found to be a very potent thyroid hormone receptor (THR) agonist, while NH-3 is an antagonist. In this process, we have also achieved a significantly improved synthesis of NH-3.
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.
The chemical threat agent tetramethylenedisulfotetramine (TETS) is a γ-aminobutyric acid type A receptor (GABA AR) antagonist that causes life threatening seizures. Currently, there is no specific ...antidote for TETS intoxication. TETS-induced seizures are typically treated with benzodiazepines, which function as nonselective positive allosteric modulators (PAMs) of synaptic GABAARs. The major target of TETS was recently identified as the GABAAR α2β3γ2 subtype in electrophysiological studies using recombinantly expressed receptor combinations. Here, we tested whether these in vitro findings translate in vivo by comparing the efficacy of GABAAR subunit-selective PAMs in reducing TETS-induced seizure behavior in larval zebrafish. We tested PAMs targeting α1, α2, α2/3/5, α6, ß2/3, ß1/2/3, and δ subunits and compared their efficacy to the benzodiazepine midazolam (MDZ). The data demonstrate that α2- and α6-selective PAMs (SL-651,498 and SB-205384, respectively) were effective at mitigating TETS-induced seizure-like behavior. Combinations of SB-205384 and MDZ or SL-651,498 and 2–261 (ß2/3-selective) mitigated TETS-induced seizure-like behavior at concentrations that did not elicit sedating effects in a photomotor behavioral assay, whereas MDZ alone caused sedation at the concentration required to stop seizure behavior. Isobologram analyses suggested that SB-205384 and MDZ interacted in an antagonistic fashion, while the effects of SL-651,498 and 2–261 were additive. These results further elucidate the molecular mechanism by which TETS induces seizures and provide mechanistic insight regarding specific countermeasures against this chemical convulsant.
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•A zebrafish assay confirms the GABAAR subtype selectivity of the rodenticide TETS.•α2, α6 or ß2/3 selective PAMs mitigated TETS-induced seizure behavior in zebrafish.•The benzodiazepine midazolam (MDZ) caused sedation in photomotor assay.•Low dose MDZ combined with α6-selective PAM mitigates seizures with less sedation.•Combinations of α2 and ß2/3 selective PAMs mitigate seizures with less sedation.
Abstract
Tetramethylenedisulfotetramine (tetramine or TETS), a potent convulsant, triggers abnormal electrical spike activity (ESA) and synchronous Ca2+ oscillation (SCO) patterns in cultured ...neuronal networks by blocking gamma-aminobutyric acid (GABAA) receptors. Murine hippocampal neuronal/glial cocultures develop extensive dendritic connectivity between glutamatergic and GABAergic inputs and display two distinct SCO patterns when imaged with the Ca2+ indicator Fluo-4: Low amplitude SCO events (LASE) and High amplitude SCO events (HASE) that are dependent on TTX-sensitive network electrical spike activity (ESA). Acute TETS (3.0 µM) increased overall network SCO amplitude and decreased SCO frequency by stabilizing HASE and suppressing LASE while increasing ESA. In multielectrode arrays, TETS also increased burst frequency and synchronicity. In the presence of TETS (3.0 µM), the clinically used anticonvulsive perampanel (0.1–3.0 µM), a noncompetitive AMPAR antagonist, suppressed all SCO activity, whereas the GABAA receptor potentiator midazolam (1.0-30 µM), the current standard of care, reciprocally suppressed HASE and stabilized LASE. The neuroactive steroid (NAS) allopregnanolone (0.1–3.0 µM) normalized TETS-triggered patterns by selectively suppressing HASE and increasing LASE, a pharmacological pattern distinct from its epimeric form eltanolone, ganaxolone, alphaxolone, and XJ-42, which significantly potentiated TETS-triggered HASE in a biphasic manner. Cortisol failed to mitigate TETS-triggered patterns and at >1 µM augmented them. Combinations of allopregnanolone and midazolam were significantly more effective at normalizing TETS-triggered SCO patterns, ESA patterns, and more potently enhanced GABA-activated Cl− current, than either drug alone.
The rodenticide tetramethylenedisulfotetramine (TETS) is a potent convulsant (lethal dose in humans 7–10 mg) that is listed as a possible threat agent by the United States Department of Homeland ...Security. TETS has previously been studied in vivo for toxicity and in vitro in binding assays, with the latter demonstrating it to be a non-competitive antagonist on GABA
A
receptors. To determine whether TETS exhibits subtype selectivity for a particular GABA
A
receptor combination, we used whole-cell patch-clamp to determine the potency of TETS on the major synaptic and extrasynaptic GABA
A
receptors associated with convulsant activity. The active component of picrotoxin, picrotoxinin, was used as a control. While picrotoxinin did not differentiate well between 13 GABA
A
receptors, TETS exhibited the highest activity on α2β3γ2 (IC
50
480 nM, 95% CI 320–640 nM) and α6β3γ2 (IC
50
400 nM, 95% CI 290–510 nM). Introducing β1 or β2 subunits into these receptor combinations reduced or abolished TETS sensitivity, suggesting that TETS preferentially affects receptors with α2/β3 or α6/β3 composition. Since α2β3γ2 receptors make up 15–20% of the GABA
A
receptors in the mammalian CNS, we suggest that α2β3γ2 is probably the most important GABA
A
receptor for the seizure-inducing activity of TETS.
The rodenticide tetramethylenedisulfotetramine (TETS) is a potent convulsant (lethal dose in humans 7-10 mg) that is listed as a possible threat agent by the United States Department of Homeland ...Security. TETS has previously been studied in vivo for toxicity and in vitro in binding assays, with the latter demonstrating it to be a non-competitive antagonist on GABA
receptors. To determine whether TETS exhibits subtype selectivity for a particular GABA
receptor combination, we used whole-cell patch-clamp to determine the potency of TETS on the major synaptic and extrasynaptic GABA
receptors associated with convulsant activity. The active component of picrotoxin, picrotoxinin, was used as a control. While picrotoxinin did not differentiate well between 13 GABA
receptors, TETS exhibited the highest activity on α2β3γ2 (IC
480 nM, 95% CI 320-640 nM) and α6β3γ2 (IC
400 nM, 95% CI 290-510 nM). Introducing β1 or β2 subunits into these receptor combinations reduced or abolished TETS sensitivity, suggesting that TETS preferentially affects receptors with α2/β3 or α6/β3 composition. Since α2β3γ2 receptors make up 15-20% of the GABA
receptors in the mammalian CNS, we suggest that α2β3γ2 is probably the most important GABA
receptor for the seizure-inducing activity of TETS.
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