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Hexahydro-1,3,5-trinitro-1,3,5-triazine, or Royal Demolition Explosive (RDX), is a major component of plastic explosives such as C-4. Acute exposures from intentional or accidental ingestion are a documented clinical concern, especially among young male U.S. service members in the armed forces. When ingested in large enough quantity, RDX causes tonic-clonic seizures. Previous in silico and in vitro experiments predict that RDX causes seizures by inhibiting α1β2γ2 γ-aminobutyric acid type A (GABAA) receptor-mediated chloride currents. To determine whether this mechanism translates in vivo, we established a larval zebrafish model of RDX-induced seizures. After a 3 h of exposure to 300 µM RDX, larval zebrafish exhibited a significant increase in motility in comparison to vehicle controls. Researchers blinded to experimental group manually scored a 20-min segment of video starting at 3.5 h post-exposure and found significant seizure behavior that correlated with automated seizure scores. Midazolam (MDZ), an nonselective GABAAR positive allosteric modulator (PAM), and a combination of Zolpidem (α1 selective PAM) and compound 2-261 (β2/3-selective PAM) were effective in mitigating RDX-triggered behavioral and electrographic seizures. These findings confirm that RDX induces seizure activity via inhibition of the α1β2γ2 GABAAR and support the use of GABAAR-targeted anti-seizure drugs for the treatment of RDX-induced seizures.

Transient receptor potential vanilloid 1 (TRPV1) ion channel is a nociceptor critically involved in pain sensation. Direct blockade of TRPV1 exhibits significant analgesic effects but also incurs severe side effects such as hyperthermia, causing failures of TRPV1 inhibitors in clinical trials. In order to selectively target TRPV1 channels that are actively involved in pain-sensing, peptidic positive allosteric modulators (PAMs) based on the high-resolution structure of the TRPV1 intracellular ankyrin-repeat like domain are de novo designed. The hotspot centric approach is optimized for protein design; its usage in Rosetta increases the success rate in protein binder design. It is demonstrated experimentally, with a combination of fluorescence resonance energy transfer (FRET) imaging, surface plasmon resonance, and patch-clamp recording, that the designed PAMs bind to TRPV1 with nanomolar affinity and allosterically enhance its response to ligand activation as it is designed. It is further demonstrated that the designed PAM exhibits long-lasting in vivo analgesic effects in rats without changing their body temperature, suggesting that they have potentials for developing into novel analgesics.

GABAB (γ-aminobutyric acid B) receptors may be a therapeutic target for anxiety disorders. Here we characterized the effects of the GABAB receptor positive allosteric modulator (PAM) BHF177 on conditioned and unconditioned physiological responses to threat in the light-enhanced startle (LES), stress-induced hyperthermia, and fear-potentiated startle (FPS) procedures in rats. The effects of BHF177 on LES were compared with those of the GABAB receptor agonists baclofen and CGP44532, and the positive control buspirone, a 5-HT1A receptor partial agonist with anxiolytic activity in humans. Baclofen (0.4, 0.9 and 1.25 mg/kg) and CGP44532 (0.065, 0.125 and 0.25 mg/kg) administration had significant sedative, but not anxiolytic, activity reflected in overall decrease in the startle response in the LES tests. BHF177 (10, 20 and 40 mg/kg) had no effect on LES, nor did it produce an overall sedative effect. Interesting, however, when rats were grouped by high and low LES responses, BHF177 had anxiolytic-like effects only on LES in high, but not low, LES responding rats. BHF177 also blocked stress-induced hyperthermia, but had no effect on conditioned fear responses in the FPS test. Buspirone (1 and 3 mg/kg) had an anxiolytic-like profile in both LES and FPS tests. These results indicate that BHF177 may specifically attenuate unconditioned anxiety in individuals that exhibit a high anxiety state, and has fewer sedative effects than direct agonists. Thus, BHF177 or other GABAB receptor PAMs may be promising compounds for alleviating increased anxiety seen in various psychiatric disorders with a superior side-effect profile compared to GABAB receptor agonists.