Molecular Pharmacology Fast Forward. Published on October 27, 2020 as DOI: 10.1124/molpharm.120.000090 This article has not been copyedited and formatted. The final version may differ from this version. Identification of the Functional Binding Site for the Convulsant Tetramethylenedisulfotetramine in the Pore of the α2β3γ2 GABA-A Receptor Brandon Pressly, Ruth D. Lee, Bogdan Barnych, Bruce D. Hammock, Heike Wulff Downloaded from Department of Pharmacology (B.P., R.D.L, H.W.), University of California, Davis, California, USA Department of Entomology and Nematology, and Comprehensive Cancer Center, (B.B., B.D.H.) molpharm.aspetjournals.org University of California, Davis, USA at ASPET Journals on September 24, 2021 1 Molecular Pharmacology Fast Forward. Published on October 27, 2020 as DOI: 10.1124/molpharm.120.000090 This article has not been copyedited and formatted. The final version may differ from this version. Running Title: The Binding Site of TETS Address correspondence to: Heike Wulff, Department of Pharmacology, Genome and Biomedical Sciences Facility, Room 3502, 451 Health Sciences Drive, University of California, Davis, Davis, CA 95616; phone: 530- 754-6136; email: [email protected] Downloaded from Number of Text Pages: 41 Number of Tables: 0 Number of Figures: 8 molpharm.aspetjournals.org Number of References: 43 Number of Words in the Abstract: 217 Number of Words in the Introduction: 725 at ASPET Journals on September 24, 2021 Number of Words in the Discussion: 1,645 Nonstandard abbreviations used: AUC, area under the curve; DMSO, dimethyl sulfoxide; EBOB, 1-(4-ethynylphenyl)-4-n-propyl-2,6,7-trioxabicyclo[2.2.2]octane; ECD, extracellular domain; GABA, gamma-aminobutyric acid; GABAA, GABA receptor type A; HEK, human embryonic kidney; NCA, non-competitive antagonist; pdb, Protein data bank; PAM, positive allosteric modulator; PTX, picrotoxin; REU, Rosetta energy unit; TBPS, tert- butylbicyclophosphorothionate; TETS; tetramethylenedisulfotetramine; TMD, transmembrane domain. 2 Molecular Pharmacology Fast Forward. Published on October 27, 2020 as DOI: 10.1124/molpharm.120.000090 This article has not been copyedited and formatted. The final version may differ from this version. Abstract Tetramethylenedisulfotetramine (TETS) is a so-called “caged” convulsant, that is responsible for thousands of accidental and malicious poisonings. Similar to the widely used GABAA antagonist picrotoxinin, TETS has been proposed to bind to the non-competitive antagonist (NCA) site in the pore of the gamma-aminobutyric acid (GABA) receptor channel. However, the TETS binding site has never been experimentally mapped, and we here set out to gain atomistic level insights Downloaded from into how TETS inhibits the human α2β3γ2 GABAA receptor. Using the Rosetta molecular modeling suite, we generated three homology models of the α2β3γ2 receptor in the open, molpharm.aspetjournals.org 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, where it at ASPET Journals on September 24, 2021 shows perfect space-complementarity and is forming hydrogen bonds or making hydrophobic interactions with all five pore-lining threonine residues of the pentameric receptor. Mutating T6' in either the α2 or β3 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. 3 Molecular Pharmacology Fast Forward. Published on October 27, 2020 as DOI: 10.1124/molpharm.120.000090 This article has not been copyedited and formatted. The final version may differ from this version. Significance Statement Our study identifies the binding site of the highly toxic convulsant TETS (Tetramethylenedisulfotetramine), 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 α2β3γ2 Downloaded from GABAA receptor at the so-called T6' ring, where five threonine residues line the permeation pathway of the pentameric receptor channel. This knowledge could help inform the design of a TETS binding therapeutic protein. molpharm.aspetjournals.org at ASPET Journals on September 24, 2021 4 Molecular Pharmacology Fast Forward. Published on October 27, 2020 as DOI: 10.1124/molpharm.120.000090 This article has not been copyedited and formatted. The final version may differ from this version. Introduction GABAA receptors are pentameric ligand-gated chloride channels (Olsen and Sieghart, 2008) that are activated by gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in Downloaded from the mammalian and insect nervous system. GABAA receptors constitute the targets of many widely used drugs as well as for major household and agricultural pesticides. GABAA receptor agonists like barbiturates or positive allosteric modulators (PAMs) as exemplified by the various molpharm.aspetjournals.org typical and atypical benzodiazepines reduce neuronal excitability and are widely used as anesthetics, anxiolytics and anticonvulsants (Krall et al., 2015; Olsen, 2015). In contrast, compounds that inhibit GABAA receptor functions increase neuronal firing. Depending on their species specificity, GABAA antagonist accordingly either constitute useful insecticides like at ASPET Journals on September 24, 2021 fipronil or afoxalaner (Casida and Durkin, 2015), or, if they inhibit mammalian GABAA receptors like picrotoxinin or pentylenetetrazol, induce seizures and, at higher concentrations, death. The extremely toxic tetramethylenedisulfotetramine (TETS) has been known since the early 1940’s and was initially used as an anti-mold agent for furniture. TETS was subsequently investigated both as a rodenticide and as a pesticide (for an extensive review see (Laukova et al., 2019)). However, since human intoxications resulting in death, seizures, and, if victims survived exposure, in neurological deficits and spontaneous recurrent seizures have repeatedly occurred, the World Health Organization therefore banned the use of TETS (Laukova et al., 2019). But, due to its ease of manufacture and its impressive effectiveness in killing rats and mice, TETS continues to be used as a rodenticide in China and presents a real exposure risk to humans and a viable threat agent for inciting a mass casualty incident. Pharmacological experiments performed in the late 1950s demonstrating that sub-lethal doses of TETS could 5 Molecular Pharmacology Fast Forward. Published on October 27, 2020 as DOI: 10.1124/molpharm.120.000090 This article has not been copyedited and formatted. The final version may differ from this version. reverse pentobarbital-induced decreases in blood pressure and respiration in dogs (Haskell and Voss, 1957) suggested that TETS might be targeting GABAA receptors. This was later confirmed in binding assays. TETS, which is far more toxic to mice than to insects (Esser et al., 1991), has affinity for both mammalian GABAA receptors and the insect RDL receptor. While the insect RDL receptor is a homopentamer consisting of 5 identical subunits, mammalian GABAA receptors are typically composed of two α, two β and one γ or subunit assembled as a pentamer around a central chloride conducting pore (Olsen and Sieghart, 2008). In binding Downloaded from 3 assays with rat brain synaptosomes TETS displaces GABAA receptor blockers like [ H]EBOB and [35S]TBPS (Esser et al., 1991; Zhao et al., 2014), most likely by binding to the non- molpharm.aspetjournals.org competitive antagonist site (NCA) in the GABAA receptor pore. However, it was not known whether TETS displayed any subtype selectivity and where exactly it was binding. To address the first question our group recently tested the potency of TETS on the major synaptic and extrasynaptic GABAA receptors associated with convulsant activity using whole-cell patch-clamp at ASPET Journals on September 24, 2021 and reported that TETS is most active on α2β3γ2L and α6β3γ2L GABAA receptors (Pressly et al., 2018). Based on the observation that α2β3γ2 receptors make up 15-20% of the GABAA receptors in the mammalian CNS, we suggested that this receptor combination probably constitutes the most important GABAA receptor target for the seizure inducing activity of TETS (Pressly et al., 2018). The results still left the question of the binding site unanswered. The GABAA field has recently advanced substantially through the publication of numerous new structures (Garcia-Nafria and Tate, 2019). As of now, 19 GABAA receptor structures have been resolved in the closed, open or desensitized state. While the first structure was a homopentamer (Miller and Aricescu, 2014), more physiologically relevant heteropentamers containing α, β and γ subunits have been elucidated using cryo-EM and have started to provide the field molecular insights into both ligand gating and the actions of clinically widely used drugs such as the benzodiazepines (Garcia-Nafria and Tate, 2019; Masiulis et al., 2019). Using three recently published GABAA receptor structures as templates we here 6 Molecular Pharmacology Fast Forward. Published on October