Identification of the Functional Binding Site for the Convulsant Tetramethylenedisulfotetramine in the Pore of the α 2 ß 3 γ 2 GABAA Receptor.

Tetramethylenedisulfotetramine (TETS) is a so-called "caged" convulsant that is responsible for thousands of accidental and malicious poisonings. Similar to the widely used GABA receptor type A (GABAA) antagonist picrotoxinin, TETS has been proposed to bind to the noncompetitive antagonist (NCA) site in the pore of the receptor channel. However, the TETS binding site has never been experimentally mapped, and we here set out to gain atomistic level insights into how TETS inhibits the human α 2 ß 3 γ 2 GABAA receptor. Using the Rosetta molecular modeling suite, we generated three homology models of the α 2 ß 3 γ 2 receptor in the open, desensitized, and closed/resting state. Three different ligand-docking algorithms (RosettaLigand, Glide, and Swissdock) identified two possible TETS binding sites in the channel pore. Using a combination of site-directed mutagenesis, electrophysiology, and modeling to probe both sites, we demonstrate that TETS binds at the T6' ring in the closed/resting-state model, in which it shows perfect space complementarity and forms hydrogen bonds or makes hydrophobic interactions with all five pore-lining threonine residues of the pentameric receptor. Mutating T6' in either the α 2 or ß 3 subunit reduces the IC50 of TETS by ∼700-fold in whole-cell patch-clamp experiments. TETS is thus interacting at the NCA site in the pore of the GABAA receptor at a location that is overlapping but not identical to the picrotoxinin binding site. SIGNIFICANCE STATEMENT: Our study identifies the binding site of the highly toxic convulsant tetramethylenedisulfotetramine (TETS), which is classified as a threat agent by the World Health Organization. Using a combination of homology protein modeling, ligand docking, site-directed mutagenesis, and electrophysiology, we show that TETS is binding in the pore of the α2ß3γ2 GABA receptor type A receptor at the so-called T6' ring, wherein five threonine residues line the permeation pathway of the pentameric receptor channel.

The efficacy of γ-aminobutyric acid type A receptor (GABA AR) subtype-selective positive allosteric modulators in blocking tetramethylenedisulfotetramine (TETS)-induced seizure-like behavior in larval zebrafish with minimal sedation.

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 α2ß3γ2 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.

Comparison of the toxicokinetics of the convulsants picrotoxinin and tetramethylenedisulfotetramine (TETS) in mice.

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 (GABAA) 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 LD50 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 α2ß3γ2 GABAA-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.

GABAA receptor subtype selectivity of the proconvulsant rodenticide 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 GABAA receptors. To determine whether TETS exhibits subtype selectivity for a particular GABAA receptor combination, we used whole-cell patch-clamp to determine the potency of TETS on the major synaptic and extrasynaptic GABAA receptors associated with convulsant activity. The active component of picrotoxin, picrotoxinin, was used as a control. While picrotoxinin did not differentiate well between 13 GABAA receptors, TETS exhibited the highest activity on α2ß3γ2 (IC50 480 nM, 95% CI 320-640 nM) and α6ß3γ2 (IC50 400 nM, 95% CI 290-510 nM). Introducing ß1 or ß2 subunits into these receptor combinations reduced or abolished TETS sensitivity, suggesting that TETS preferentially affects receptors with α2/ß3 or α6/ß3 composition. Since α2ß3γ2 receptors make up 15-20% of the GABAA receptors in the mammalian CNS, we suggest that α2ß3γ2 is probably the most important GABAA receptor for the seizure-inducing activity of TETS.

Rapid Throughput Analysis of GABAA Receptor Subtype Modulators and Blockers Using DiSBAC1(3) Membrane Potential Red Dye.

Fluorometric imaging plate reader membrane potential dye (FMP-Red-Dye) is a proprietary tool for basic discovery and high-throughput drug screening for G-protein-coupled receptors and ion channels. We optimized and validated this potentiometric probe to assay functional modulators of heterologous expressed GABAA receptor (GABAAR) isoforms (synaptic α1ß3γ2, extrasynaptic α4ß3δ, and ß3 homopentomers). High-resolution mass spectrometry identified FMP-Red-Dye as 5,5'-(1-propen-1-yl-3-ylidene)bis[1,3-dimethyl-2-thio-barbituric acid]. GABAAR-expressing cells equilibrated with FMP-Red-Dye exhibited depolarized equilibrium membrane potentials compared with GABAAR-null cells. The channel blockers picrotoxin, fipronil, and tetramethylenedisulfotetramine, and the competitive antagonist bicuculline reduced fluorescence near the levels in GABAAR-null cells indicating that FMR-Red-Dye, a barbiturate derivative, activates GABAAR-mediated outward Cl- current in the absence of GABA. GABA caused concentration-dependent increases in fluorescence with rank order of potencies among GABAAR isoforms consistent with results from voltage-clamp experiments (EC50 values for α4ß3δ, α1ß3γ2, and ß3 homopentamers were 6 ± 1, 40 ± 11, and >18 mM, respectively), whereas GABAAR-null cells were unresponsive. Neuroactive steroids (NAS) increased fluorescence of GABAAR expressing cells in the absence of GABA and demonstrated positive allosteric modulation in the presence of GABA, whereas benzodiazepines only exhibited positive allosteric modulator (PAM) activity. Of 20 NAS tested, allopregnanolone, (3α,5α,20E)-3-hydroxy-13,24-cyclo-18-norcholan-20-ene-21-carbonitrile, eltanolone, 5ß-pregnan-3α,21-diol-20-one, and ganaxolone showed the highest potency. The FMP-Red-Dye-based assay described here provides a sensitive and quantitative method of assessing the activity of GABAAR agonists, antagonists, and PAMs on diverse GABAAR isoforms. The assay has a wide range of applications, including screening for antiseizure agents and identifying channel blockers of interest to insecticide discovery or biosecurity.

Structural Insights into the Atomistic Mechanisms of Action of Small Molecule Inhibitors Targeting the KCa3.1 Channel Pore.

The intermediate-conductance Ca2+-activated K+ channel (KCa3.1) constitutes an attractive pharmacological target for immunosuppression, fibroproliferative disorders, atherosclerosis, and stroke. However, there currently is no available crystal structure of this medically relevant channel that could be used for structure-assisted drug design. Using the Rosetta molecular modeling suite we generated a molecular model of the KCa3.1 pore and tested the model by first confirming previously mapped binding sites and visualizing the mechanism of TRAM-34 (1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole), senicapoc (2,2-bis-(4-fluorophenyl)-2-phenylacetamide), and NS6180 (4-[[3-(trifluoromethyl)phenyl]methyl]-2H-1,4-benzothiazin-3(4H)-one) inhibition at the atomistic level. All three compounds block ion conduction directly by fully or partially occupying the site that would normally be occupied by K+ before it enters the selectivity filter. We then challenged the model to predict the receptor sites and mechanisms of action of the dihydropyridine nifedipine and an isosteric 4-phenyl-pyran. Rosetta predicted receptor sites for nifedipine in the fenestration region and for the 4-phenyl-pyran in the pore lumen, which could both be confirmed by site-directed mutagenesis and electrophysiology. While nifedipine is thus not a pore blocker and might be stabilizing the channel in a nonconducting conformation or interfere with gating, the 4-phenyl-pyran was found to be a classical pore blocker that directly inhibits ion conduction similar to the triarylmethanes TRAM-34 and senicapoc. The Rosetta KCa3.1 pore model explains the mechanism of action of several KCa3.1 blockers at the molecular level and could be used for structure-assisted drug design.

Chasing the Elusive Benzofuran Impurity of the THR Antagonist NH-3: Synthesis, Isotope Labeling, and Biological Activity.

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.

The seizure-inducing plastic explosive RDX inhibits the α1ß2γ2 GABAA receptor.

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 α1ß2γ2 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 α1ß2γ2 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 ß2 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.

Structure-Activity Relationship of Neuroactive Steroids, Midazolam, and Perampanel Toward Mitigating Tetramine-Triggered Activity in Murine Hippocampal Neuronal Networks.

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.

Susceptibility of larval zebrafish to the seizurogenic activity of GABA type A receptor antagonists.

Previous studies demonstrated that pentylenetetrazole (PTZ), a GABA type A receptor (GABAAR) antagonist, elicits seizure-like phenotypes in larval zebrafish (Danio rerio). Here, we determined whether the GABAAR antagonists, tetramethylenedisulfotetramine (TETS) and picrotoxin (PTX), both listed as credible chemical threat agents, similarly trigger seizures in zebrafish larvae. Larvae of three, routinely used laboratory zebrafish lines, Tropical 5D, NHGRI and Tupfel long fin, were exposed to varying concentrations of PTZ (used as a positive control), PTX or TETS for 20 min at 5 days post fertilization (dpf). Acute exposure to PTZ, PTX or TETS triggered seizure behavior in the absence of morbidity or mortality. While the concentration-effect relationship for seizure behavior was similar across zebrafish lines for each GABAAR antagonist, significantly less TETS was required to trigger seizures relative to PTX or PTZ. Recordings of extracellular field potentials in the optic tectum of 5 dpf Tropical 5D zebrafish confirmed that all three GABAAR antagonists elicited extracellular spiking patterns consistent with seizure activity, although the pattern varied between chemicals. Post-exposure treatment with the GABAAR positive allosteric modulators (PAMs), diazepam, midazolam or allopregnanolone, attenuated seizure behavior and activity but did not completely normalize electrical field recordings in the optic tectum. These data are consistent with observations of seizure responses in mammalian models exposed to these same GABAAR antagonists and PAMs, further validating larval zebrafish as a higher throughput-screening platform for antiseizure therapeutics, and demonstrating its appropriateness for identifying improved countermeasures for TETS and other convulsant chemical threat agents that trigger seizures via GABAAR antagonism.