Identifying Molecular Targets for the Proconvulsant Activity of TETS

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Inhibition of γ-aminobutyric acid type A (GABAA) receptors (GABAAR) is the presumed mechanism of the seizure-inducing activity of the natural product picrotoxin (PTX), the rodenticide tetramethylenedisulfotetramine (TETS) and the high-energy explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). TETS, which has been banned from production worldwide, is still readily available on the black market, and is associated with thousands of human poisonings per year. It is incredibly stable in the environment. RDX is an environmental contaminant found in both groundwater and soil due to its worldwide military and civilian use, and it is an illicit abuse substance. PTX is of toxicological concern, because like TETS and RDX, it is listed as a credible threat agent by the United States Department of Homeland Security. All three compounds can cause seizures that rapidly progress to status epilepticus and death; however, TETS is by far the most potent with a lethal dose of 7 to 10 mg in humans and an LD50 of 0.1 mg/kg in rodents, which makes it roughly ~40x more potent than PTX and ~1000x more potent than RDX. There currently is no approved medical countermeasure for individuals acutely intoxicated with these convulsant chemicals. While some information is available regarding the molecular site of action and the GABAAR subtype selectivity of PTX, practically nothing is known about the molecular mechanism of action of TETS and RDX other than that they are most probably GABAAR inhibitors. We intend to use molecular modeling, whole-cell patch-clamp electrophysiology and gene knockdown techniques in zebrafish to identify the subunit specificity of TETS and RDX interactions with GABAA receptors and to determine whether it differs from that of picrotoxin. Treatment with subtype selective compounds/drugs should allow us to confirm which GABAA receptor subunit combinations are important for the seizure activity of TETS and RDX, and whether they differ from the receptor subunit profile that mediate PTX action. Detailed understanding of the molecular mechanism(s) of action of TETS and RDX will not only provide novel insight as to the biological reasons for the toxicologic differences between these agents, but will also be important for evaluating the validity of “read across” risk assessment approaches for GABAA receptor antagonists, and for developing effective medical countermeasures for terminating SE in intoxicated individuals.