Phenotypic discovery of GABAA receptor-mediated general anesthetics with analgesic activity

Phenotypic discovery of GABAA receptor-mediated general anesthetics with analgesic activity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Phenotypic discovery of GABA A receptor-mediated general anesthetics with analgesic activity Matthew McCarroll, Jarret Weinrich, Alyssa Marinas, Amanda Dombroski, and 26 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8341587/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract General anesthetics are essential for invasive medical procedures; however, there remains a need for safer agents. Using a bespoke 96-well-plate format platform enabling high-throughput imaging of larval zebrafish behavior, we screened 12,000 compounds and identified an isoxazole chemotype that phenocopies the intravenous anesthetics etomidate and propofol. Its optimization via medicinal chemistry yielded a novel anesthetic that we call nidradine. This anesthetic is efficacious in both zebrafish and mice and lacks the problematic adrenal suppression characteristic of etomidate. Mechanistic studies via electrophysiology and structural biology revealed that nidradine, like etomidate and propofol, is a positive allosteric modulator of the GABA A receptor. Remarkably, behavioral assays in mice demonstrate that nidradine differs from most general anesthetics in that it also produces analgesia. Biological sciences/Drug discovery/Drug screening/Phenotypic screening Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy Behavioral profiling anesthesia high throughput screening analgesia zebrafish GABAA Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Despite their widespread use, 1 general anesthetics have a suboptimal therapeutic profile. The most commonly used intravenous general anesthetic, propofol, causes unfavorable hemodynamics and respiratory depression, while etomidate causes adrenal suppression that is linked to increased mortality. 2 , 3 The GABA A receptor is the major target of many general anesthetics. 4 – 6 The GABA A receptor is a ligand-gated ion channel assembled as a pentamer of homologous subunits. The existence of 19 different receptor subunits in humans underlies an incredible diversity of GABA A receptors having distinct signaling properties and pharmacology. 7 The most abundant subtype in the human brain comprises α1, β2 and γ2 subunits. 8 , 9 GABA A receptor activation by GABA or exogenous molecules triggers opening of an intrinsic anion channel, which in most cases results in intracellular chloride conductance, dampening neuronal excitability. Notably, common intravenous anesthetics that act on the GABA A receptor, including etomidate and propofol, lack analgesic properties, 10,11 necessitating additional pain management measures, often via opioids. 12 The discovery and development of new anesthetics remain challenging given that anesthesia must be assessed in mammals via expensive and low-throughput experiments. In the past decade, aquatic vertebrates have emerged as an alternative to mammals in the discovery of neuroactive compounds. Indeed, early zebrafish screens established reproducible behavioral barcodes ( i.e ., phenotypes) that predict mechanism and revealed novel psychoactive scaffolds. 13 , 14 The fact that immersion dosing in these animals rapidly attains near-steady-state exposure in vivo facilitates chemical screens. 15 In neuroactive discovery, a focused zebrafish larval photomotor-response screen (~ 374 compounds) identified reversible anesthetic like sedative–hypnotics with activity spanning GABA A ​, neuronal nicotinic, and NMDA receptors, 15 illustrating that target-agnostic phenotypic assays can identify hits, including those that act through multiple targets.. Studies in zebrafish have even yielded propofol antagonists, fluorinated analogs that reverse propofol anesthesia, underscoring the capacity of these models to uncover both agonist and antagonist chemotypes. 16 We recently pioneered a novel screening approach that uses larval zebrafish, in which anesthetics produce rich behavioral fingerprints beyond simple immobility, namely measurable transient hyperexcitability or altered responses to specific stimuli. 17 By capturing these patterns, behavioral screening in zebrafish is amenable to high-throughput approaches for discovering new anesthetic drugs. As other vertebrates, zebrafish exhibit a complex repertoire of behaviors, yet their central nervous system is far simpler in architecture. 18 Because zebrafish larvae are small enough to fit in 96-well plates and are readily dosed with compounds dissolved directly in the water, it is possible to do high-through put screens of thousands of compounds. Indeed, zebrafish have been used to screen and identify novel pharmacological compounds, some of which have shown promising translational potential in mammalian models and have progressed to clinical trials ( e.g ., clemizole and lorcaserin for Dravet syndrome; and the mechanosensory hair-cell otoprotectant ORC-13661). 19 – 21 Importantly, the GABA A receptor gene family is highly conserved in zebrafish. Its genome contains at least 23 GABA A receptor subunits including orthologs of all mammalian subunits, except θ and ε, and an additional subunit β4. 22 Like mammals, the zebrafish α subfamily is the largest and most diverse of the subfamilies. Here, we report the execution of a novel high-throughput behavioral chemical screen in larval zebrafish that is predictive of general anesthetic activity. The larval hits that were also active in a secondary anesthesia screen in adult zebrafish proved to be efficacious in mice. Among them was an isoxazole whose analogs also had potent and reversible anesthetic effects. Medicinal chemistry efforts on the isoxazole scaffold yielded a lead compound that we named nidradine after Nidra, the Hindu deity of sleep. Biophysical experiments revealed that nidradine potentiates GABA A receptor function and shares a binding site with known anesthetics, including etomidate and propofol. However, compared to GABA A modulating anesthetics, nidradine not only exerted enhanced surgical anesthesia but also exhibited analgesic activity. RESULTS Discovery of novel anesthetics in zebrafish by phenocopying known anesthetics To investigate the behavioral modifications induced by intravenous anesthetics in fish, we used a suite of automated behavioral assays tailored to distinguish between a diverse array of neuroactive substances. 17 The methodology incorporated a variety of acoustic and visual stimuli to detect substances that either decrease motor activity or increase startle responses to low-intensity acoustic signals. We previously reported that etomidate and propofol, which act via GABA A receptors and provoke enhanced acoustic startle responses (eASRs) to a low-intensity solenoid tap, but not to other stimuli (Fig. 1a). 17 Here, we screened >10,000 structurally diverse compounds in larval zebrafish and examined their phenotypic similarity to the behavioral profile of etomidate (Fig. 1b). Sixty-nine compounds phenocopied etomidate and were designated as primary hits (refer to Supplementary Table 1). We next categorized these hits by structural similarity and identified 22 distinct clusters (Supplementary Figure 1, Supplementary Table 1). Certain clusters contained compounds having well-known pharmacophores. For example, Cluster 4 is enriched for piperidine–carbonyl scaffolds, a well-recognized CNS-active motif that includes local anesthetics (bupivacaine and ropivacaine). Cluster 17 was enriched for 8 related isoxazole scaffolds (Supplementary Table 1), which is a bioactive and CNS-privileged heterocycle found in both synthetic and natural neuroactive molecules. 23 From the sixty-nine primary hits,we selected 29 compounds representing the chemoinformatic clusters for further testing (Supplementary Table 2). We conducted dose response trials and confirmed that twenty-eight of the twenty-nine recapitulated the eASR phenotype (Fig. 1d, Supplementary Figure 2 and Supplementary Table 2), validating the reliability of the primary behavioral screen. As age-dependent neuropharmacology and pharmacokinetics can influence drug responses, 24–26 we next asked whether the hits anesthetized adult zebrafish. Our analysis was based on observations that etomidate reliably produces behavioral endpoints of the hypnotic component of general anesthesia ( i.e ., ability to produce loss of responsiveness), namely loss of righting reflex (LoRR) and immobility in adult fish (Fig. 1e, Supplementary Movie 1). Accordingly, we used LoRR and immobility as endpoints for a secondary screen, in which adult zebrafish were treated with each of the 29 primary screening hits at 50 µM (Fig. 1e). We identified 5 compounds that induced robust anesthesia (Fig. 1e,f); among them, we prioritized compound 9232649 (MM01) in hit-to-lead optimization efforts. Optimization of candidate chemotypes and anesthetic validation in mice Given the favorable phenotypic and chemical properties of the oxazole carboxamide hit, MM01, we conducted a structure-based analog search and identified 70 commercially available compounds having greater than 70% similarity to the parent molecule (Supplementary Table 3). Compounds were prioritized for purchase and evaluation via calculating the Tanimoto similarity scores and performing hierarchical clustering (Fig. 2a). This analysis revealed 8 distinct structural clusters, and we selected 1 or more representative analogs from each cluster for a total of 13 analogs (SupplementaryTable 3 and Supplementary Figure 3). From this analog-by-catalog screen, we identified 2 isoxazolecarboxamides [9267615 (MM02) and 9203415 (MM03)] that produced robust LoRR in adult zebrafish. Further characterization confirmed that these isoxazolecarboxamides were effective at sedating both larval and adult zebrafish (Fig. 2b, c). Notably, these compounds also showed improved aqueous solubility, enhancing their suitability for in vivo applications. Based on these results, we initiated synthesis of additional analogs to probe structure-activity relationships. In total, we synthesized 28 analogs within the oxazole and isoxazole chemical space (Figure 2d, Supplementary Figure 4 and Supplementary Table 4) and evaluated their behavioral effects in both larval and adult zebrafish (Figure 2e, f). From this set of compounds, we identified 3 analogs that produced clear LoRR phenotypes in adults and were soluble in aqueous media. Based on potency, solubility, and behavioral consistency, two of these were selected for further testing (Figure 2f, chemical structures shown). Next, we assessed compounds for general anesthetic activity in mice. To assess for both the presence of a general anesthetic effect and to estimate the dose that produces LORR in 50% of trials ( i.e ., the ED 50 ), we used Dixon’s up-down method. 27 IP injections of the parent molecule MM01, two of the analog by catalog hits, and the two optimized analogs produced LORR in mice indicating anesthetic properties (Figure 2c). The synthetic analog, AD-7-19 (nidradine) was the most easily formulated and thus subjected to further analyses. Pharmacokinetic analysis after IP injection of nidradine in mice demonstrated that the compound distributed to the plasma and brain, consistent with a CNS-mediated general anesthetic action (Supplementary Figure 5). Importantly, nidradine did not cause the problematic inhibition of cortisol biosynthesis underlying adrenal suppression like etomidate (see supporting information, Supplementary Figure 11). Nidradine potentiates the GABA A receptor through a membrane site As nidradine was efficacious in inducing anesthesia in both fish and mice in a manner comparable to etomidate, we selected it for detailed mechanistic analysis. Many general anesthetics, including etomidate, propofol, and barbiturates, act by potentiating the activity of the GABA A receptor, which led us to ask whether nidradine acted via the same target and mechanism. This prediction was further strengthened by our recent findings that most phenocopiers of etomidate from smaller behavioral screens in larval zebrafish screens are positive allosteric modulators of the GABA A receptors. 28 On these grounds, we tested the activity of nidradine on the GABA A receptor in whole-cell patch clamp electrophysiology experiments on HEK cells expressing the α1β2γ2 subtype of the receptor; this subunit assembly is the most abundant in the human brain and is targeted by the general anesthetics mentioned above. We used S -nidradine, as this enantiomer was most active in both fish and mice (Supplementary Figure 6). We began by testing the activity of S -nidradine on the GABA A receptor in the absence and presence of GABA. Figure 3a shows that S -nidradine has both positive allosteric modulator (PAM) and allosteric agonist activities. At lower concentrations, S -nidradine acts purely as a PAM; whereas, at higher concentrations it acts as both a PAM and as an agonist in a manner reminiscent of other general anesthetics acting through this receptor. 29 Comparing nidradine with etomidate (Fig. 3b-d, Supplementary Figure 7a-g), we found that S -nidradine has an approximately 3- to 4-fold lower PAM potency than etomidate (7 µM vs. 2 µM; Fig. 3b) yet it is similarly effective in enhancing the receptor’s sensitivity to GABA (Fig. 3c). Indeed, we also observed 3- to 5-fold potentiation in currents by nidradine in the presence of low GABA concentrations; however, at saturating GABA concentrations, S -nidradine does not further potentiate GABA A activity (Fig. 3d). The agonist potency of S -nidradine is ~10-fold weaker than that of etomidate (571 µM vs. 52.1 µM; Fig. 3e, Supplementary Figure 7h-i). As concentrations that induce agonist activity are unlikely to be reached in vivo , we suggest that the anesthetic effect in zebrafish and mice occurs via S -nidradine’s PAM activity. To gain a detailed understanding of how S -nidradine binds to these receptors and to inform future drug optimization, we resolved a 2.9 Å structure of the α1β2γ2 receptor bound to S -nidradine via cryo-EM (Supplementary Figure 9, Supplementary Table 5). We found that S -nidradine, like etomidate and propofol, binds to the β-α interfaces in the receptor’s transmembrane domain (TMD, Fig. 4a). The binding of molecules having quite different structures to the same site and inducing the same pharmacological effect is truly remarkable. Although nidradine binds to both β-α TMD interfaces, one binding site has higher ligand occupancy. From our cryo-EM density map and model, S -nidradine is positioned to make electrostatic interactions with N265 and T262 on the M2 helix of the β2 subunit via an amide oxygen and isoxazole nitrogen, respectively (Fig. 4b). Importantly, the key amino acids comprising the S -nidradine binding site are conserved across humans, mice, and zebrafish, which is consistent with nidradine’s cross-species activity and suggests strong potential for clinical utility (Fig. 4c). In any case, S -nidradine’s binding site partially overlaps with those of propofol and etomidate. However, this new compound binds deeper, towards the cytosol and slightly more inwards towards the pore than either propofol or etomidate (Fig. 4d). This binding mode allows S -nidradine to make hydrophobic interactions with residues located further down the receptor’s subunit interface, beyond where etomidate and propofol reach (Supplementary Figure 8a). Since the strong electrostatic interactions between S -nidradine and the N265 and T262 residues were suggested by the cryo-EM data to be determinants of the compound’s binding and activity, we substituted these amino acids via site-directed mutagenesis to test their importance. Specifically, we made N265I and T262V mutants and tested their function in electrophysiology experiments. To assess an influence on S -nidradine PAM activity, we co-applied GABA at EC5 (1 µM for WT, 0.5 µM for N265I, and 3 µM for T262V; Supplementary Figure 8b-d) with S -nidradine at 7 µM, then at 100 µM, on cells expressing WT and mutant receptors. Both the N265I and T262V mutants showed a significant decrease in sensitivity to 7 µM S -nidradine versus the WT receptors. However, when GABA was co-applied with S -nidradine at the higher 100 µM concentration, only the N265I mutant showed a significant decrease in PAM activity (Fig. 4e). To determine if these mutants also affected S -nidradine agonist activity, we applied S -nidradine alone at 300 µM and 1 mM on cells expressing WT and mutant receptors. Both mutants showed significant decreases in activity for both concentrations when compared to WT (Fig. 4e, Supplementary Figure 8e-g). We conclude that both N265 and T262 are key determinants of S -nidradine PAM and agonist activity, and that both activities likely arise through occupancy of the same sites. To gain a deeper understanding of how S -nidradine potentiates the receptor and how its mechanism compares to currently used general anesthetics, we next analyzed pore dimensions of the α1β2γ2 receptor bound to GABA alone, GABA + S -nidradine, GABA + etomidate, and GABA + propofol (Fig. 4f-g). While all of these structures are either in a desensitized or desensitized-like state, the GABA + S -nidradine structure has the narrowest -2ʹ desensitization gate, with a diameter of 2.4 Å, compared to GABA + etomidate, GABA + propofol and GABA-only pores having diameters of 2.9 Å, 3.3 Å, and 3.6 Å, 30 respectively, at this same location. Interestingly, at the 9ʹ activation gate, the GABA + S -nidradine structure has a pore diameter of 5.7 Å, which is similar to the etomidate-bound structure’s pore diameter of 6.2 Å. The GABA-only pore is smaller with a diameter of 4.6 Å, while the GABA + propofol structure’s pore is much wider with a diameter of 10.4 Å. These findings indicate that S -nidradine’s mechanism of potentiation may be similar to those of the currently used general anesthetics, where the compounds cause widening of the pore at the 9ʹ activation gate, likely destabilizing the resting state and sensitizing the channel to GABA potentiation. 31 Nidradine produces enhanced surgical anesthesia and analgesia An important feature of many general anesthetics is their ability to generate surgical anesthesia, namely loss of reflexive responses to noxious stimuli ( i.e ., nociceptive areflexia). For clinically relevant anesthetics (for example, volatile anesthetics, 32 propofol, 33,34 ketamine 35 ), the doses that induce surgical anesthesia are generally higher than those that induce hypnotic effects. To systematically determine whether nidradine produces surgical anesthesia in mice, we assessed for loss of reflexive withdrawal responses (LoWR) to 30 seconds of continuous strong pinch of the hindpaw. Interestingly, during our initial pilot testing of nidradine, we observed that mice lost withdrawal reflexes to noxious stimuli (hindpaw pinch) at the same dose that produces LoRR, which was unexpected. Therefore, we performed LoWR testing at approximately the ED 50 for LoRR, for both nidradine and etomidate (Fig. 5a). As expected, at the ED 50 dose for etomidate, mice that lost righting reflex continued to respond to strong hindpaw pinch (Fig. 5c). However, when the same test was conducted with nidradine, the withdrawal reflex to strong hindpaw pinch was absent in all mice that lost righting reflex (Fig. 5c). Surprisingly, this phenomenon was also observed in adult zebrafish (Fig. 5b, Supplementary Movie 2). To the best of our knowledge, the equipotent production of hypnosis and surgical anesthesia (areflexia) is unique to nidradine. As nidradine produces surgical anesthesia ( i.e ., blocks withdrawal of the paw to a strong pinch) at lower than expected doses, we next asked whether nidradine also blocks nocifensive responses, a reflex correlate of pain processing, at subanesthetic doses. Importantly, by assessing at nonsedating doses (ability to stay on rotarod) in awake mice (Supplementary Figure 10), any potential loss of responses to noxious stimuli will not erroneously be misclassified due to an inability of the mouse to respond. Here, we monitored nociceptive responses to a strong noxious heat stimulus produced by a short duration, high intensity infrared laser pulse in awake, freely moving mice. 36 As expected, subanesthetic doses of etomidate did not reduce nocifensive responses to the laser stimulus. In contrast, at subanesthetic doses, nidradine significantly reducedresponses to the laser stimulus, compared to vehicle controls (Fig. 4e). Follow up studies using light-induced acute noxious stimuli ( i.e ., optovin) confirmed that nidradine, but not etomidate, also reduces nocifensive responses in larval zebrafish (Fig. 5d). To better understand the mechanism underlying the anesthetic and analgesic actions of nidradine, we performed radioligand displacement profiling across a panel of neuroreceptors through the Psychoactive Drug Screening Program (PDSP). 37 Nidradine did not bind α2 adrenergic receptors, which are targets of established anesthetics and analgesics (Supplementary Figure 12), nor did it bind opioid receptors (mu, delta, kappa). Interestingly, nidradine did exhibit non-selective inhibition of voltage gated sodium (NaV) channels (NaV1.4, NaV1.5, NaV1.6, NaV1.8); however, the low potencies (~140 µM) measured indicate that NaV channels are unlikely to contribute to the hypnotic or analgesic action of nidradine (Supplementary Figure 13). DISCUSSION In this drug development effort, we discovered translatable chemotype that not only produces general anesthesia, but at lower doses, also analgesia. This discovery was enabled by a highly informative behavioral phenotype in larval zebrafish induced by known general anesthetics. Our high-throughput compatible platform for behavioral profiling enabled target agnostic screening of over 10,000 molecules in larval zebrafish. Our lead molecule developed from extensive hit-to-lead optimization efforts, nidradine, is a positive allosteric modulator of the GABA A receptor, and shares binding sites with other clinically relevant general anesthetics. Nidradine is distinct from clinically used anesthetics in that it produces hypnosis and areflexia at equipotent doses. Importantly, nidradine is analgesic at subanesthetic concentrations, greatly expanding its possible clinical translation. In the present study, our secondary screen of anesthetic-like hits in adult fish after the primary larval zebrafish screen was an essential step toward translating anesthetic candidates to mammals. During development, zebrafish undergo maturation of GABAergic circuitry and synaptic connectivity, which can alter drug sensitivity and network-level responses. 38–42 In addition, pharmacokinetic factors differ markedly between larvae and adults; larval fish are small, with limited diffusion barriers and have a rudimentary blood–brain barrier that may allow easier compound penetration. 24,43,44 For this reason, confirming that candidate compounds produce anesthesia in adult fish, particularly compounds that induce rapid LoRR within minutes, is key to identifying compounds with appropriate kinetics, bioavailability and pharmacodynamics for translation into mammals. The chemotypes discovered in the screen are structurally distinct from all known general anesthetics- the ethers (sevoflurane), alkyl phenols (propofol), cyclohexanones (ketamine) imidazoles (etomidate and dexmedetomidine), and the benzodiazepine remimazolam. Our primary hit MM01 contained an oxazole, whereas iterative SAR optimization converged on a related isoxazole scaffold. The isoxazole ring is a well-recognized CNS-privileged heteroaromatic motif and an adaptable medicinal chemistry scaffold, appearing in multiple synthetic and naturally occurring neuroactive compounds, including the GABAergic agonist muscimol from Amanita mushrooms. 45 Recent systematic analyses of isoxazole derivatives further highlight this chemotype’s broad bioactivity across CNS, anti-inflammatory, antimicrobial, and anticancer indications, underscoring its suitability as a privileged framework for many therapeutics including novel anesthetic leads. 23,45–49 All anesthetics that produce surgical anesthesia (loss of responses to noxious stimuli) do so at doses greater than those that induce hypnosis ( i.e ., loss of consciousness). 50 For example, for volatile anesthetics, the concentration of anesthetic that produces loss of volitional responses is generally 30-50% of the dose required for loss of response to surgical incision. 51,52 Similarly, intravenous general anesthetics, such a propofol 53–55 and etomidate, 56 also block reflexes to noxious stimuli at doses above those that induce hypnosis. In distinct contrast, nidradine blocks noxious stimulus-evoked reflexes at the lowest doses that block the righting reflex. Currently, we do not know the mechanism through which nidradine blocks nociceptive reflexes in the setting of surgical anesthesia. Interestingly, targeted mutations to the β2 and β3 subunits of the GABA A receptor (N265), which is a part of a shared binding site in the GABA A receptor targeted by etomidate, propofol, and nidradine, reduces, 57 or even abolishes, 58 the generation of surgical anesthesia by etomidate and propofol. This possible receptor interaction suggests that nidradine’s equipotent production of surgical anesthesia and hypnosis may be due to novel interactions with the GABA A receptor. It is also possible that nidradine’s analgesic effects occur through an undetermined receptor; however, we have ruled out voltage gated sodium channels, α2 adrenergic receptors, and opioid receptors. The ability of nidradine to produce analgesia at subsedative doses places it in a small class of anesthetics that can provide pain relief in awake animals. As others have shown, and we have confirmed with etomidate, most general anesthetics that act at the GABA A receptor are not analgesic at subsedative doses ( e.g ., propofol, 59 isoflurane, 60 and sevoflurane). 61 It is unlikely, therefore, that the analgesic effects of nidradine in awake animals is derived from GABA A receptor PAM activity. In future studies, we will investigate the molecular mechanism under nidradine’s production of analgesia. Importantly, by recapitulating the analgesic effects of nidradine in larval zebrafish, we will incorporate the ability to identify analgesic general anesthetics into our high throughput discovery pipeline. METHODS Fish maintenance, breeding and chemical treatments Maintenance and breeding of wild type zebrafish (Singapore) was performed as described 62 and staged in days post-fertilization (dpf). All embryos were raised on a 14/10-hour light/dark cycle at 28˚C until 7 dpf. Larvae were anesthetized with ice cold egg water and distributed 8 animals per well into square 96-well plates (GE Healthcare Life Sciences) with 300 µL of egg water. 62 Adult AbTL or TuAB fish and placed individually in the well of a round 6-well plate in 8 mL of egg water. Chemical treatments were applied directly to the egg water and larvae were incubated at room temp for 1 hour before behavioral analysis. Adult animals were assayed immediately following drug treatment. For secondary screening of hit compounds in adult zebrafish loss of righting reflex assays, single-animal experiments were performed at 50 µM. For Dixon ED 50 estimates, each compound was tested with n = 5 animals. All zebrafish procedures were approved by the UCSF Institutional Animal Care and Use Committee (protocol AN201827) and conducted in accordance with institutional and federal guidelines. Chemical libraries All chemical libraries were dissolved in DMSO. The Chembridge library (Chembridge Corporation) contains >10,000 compounds at 10 mM. All compounds were diluted in egg water and screened at 30 µM final concentration in < 1% DMSO. Controls were treated with an equal volume of DMSO. All previously annotated and novel hit compounds were validated in 3-12 replicate well plates in a dose response behavioral assay, 8 zebrafish larvae per well at concentrations range from 100 µM - 1.56 µM. Automated behavioral phenotyping assays Plates were illuminated with a 760 nm infrared light from a custom-built led array. Digital video was captured at 25 frames per second using an AVT Pike digital camera (Allied Vision). Assays’ duration was 30-120 seconds consisting of a combination of acoustic and light stimulus. Low (60db) and high (70 db) amplitude acoustic stimuli were delivered using push-style solenoids (12V) to tap a custom-built stage where the 96-well plate was placed. Light stimulus was delivered using high intensity LEDs (LEDENGIN) that delivered violet (405 nm, 11 μW/mm 2 ) blue (560 nm, 18 µW/mm 2 ), green (525 nm,11 μW/mm 2 ), and red (650 nm, 11 μW/mm 2 ) wavelengths. Stimuli and digital recordings were applied to the entire 96-well plate simultaneously. Instrument control and data acquisition were performed using custom python scripts. The zebrafish motion index (MI) was calculated as follows: MI = sum(abs(frame n – frame n−1 )). Normalized MI (nMI) was calculated as follows: nMI = (MI-min(MI))/max(MI)). For motion estimation expressed as a CD(10), value was estimated as the count of pixels that changed from the previous frame by intensity ≥ 10 / 255. Detailed descriptions of the behavioral assays are described here. 17,63–65 Mouse husbandry All mouse husbandry and surgical procedures adhered to the regulatory standards of the Institutional Animal Care and Use Committee of the University of California San Francisco (UCSF; protocol AN203168). Mice were kept on a 12-hour light/dark cycle, with access to food and water ad libitum . Mouse behavioral assays Compound solubilization for mouse behavior testing: Prior to testing, compounds were solubilized in 1.5% ethanol, 11% kolliphor EL (Sigma-Aldrich, C5135-500G) and 87.5% sterile saline. Dry compounds were first mixed with solution of ethanol and kolliphor, then diluted with saline immediately prior to testing. Maximum intraperitoneal injection (IP) volume was set to 0.5cc. LoRR Testing: To assess the hypnotic effects of known and novel general anesthetics, we assayed mice for the presence of loss of righting reflex (LoRR) after compound administration. Mice are tested in a behavioral arena that consists of a 4-walled, 12” by 12” acrylic chamber. The chamber is placed onto a heating pad whose temperature is maintained by circulating warm water (40˚C). To initiate the testing session, compounds were administered via IP injection, after which mice were carefully placed into the behavioral arena and closely monitored for 10 minutes post-injection. Mice were assayed for LoRR approximately every 30 seconds. During assessment for LoRR the tester attempts to gently place the mice into the supine position. A positive LoRR response is scored as the continuous, unaided maintenance of the supine position for 30 seconds. After the 10-minute testing session, mice are monitored for regain of righting reflex (RoRR). Positive RoRR is defined as 3 consecutive failed LoRR tests after the mice first reestablish the prone position (all 4 paws on the floor), tested every 30 seconds. LoWR testing: To assess for nociceptive areflexia, we assayed mice for the presence of loss of withdrawal reflexes (LoWR) after compound administration. First mice are injected (IP) with compound then assessed for LoRR within 10 minutes after injection within the same arena described above. After LoRR is achieved, LoWR testing begins. The LoWR test consists of pinching the mouse hindpaw with an extra-long hemostat (23cm), without engaging the locking mechanism. Positive LoWR is defined as lack of observable reflexive hindlimb withdrawal response to 30 seconds of continuous pinch. Pain testing: To assess for analgesic effects of subsedative doses of general anesthetics, we assessed for changes to pain responses following to acute noxious thermal stimuli delivered by high-intensity, short-duration infrared laser pulse, as previously described. 66 The laser stimulus is generated by a LASMED (Lass-7M) 7W 975 nm laser set to a laser power of 1750 mA and pulse duration of 300 ms. Briefly, mice are placed within a chamber with a glass floor that allows for the presentation of the noxious laser stimulus to the plantar surface of the hindpaw. During a testing session, the laser stimulus is presented to one of the two hindpaws 5 times, with a cooldown period of at least 3 minutes between individual trials. The left and right hindpaws are stimulated in an alternating fashion. Positive nociceptive responses to laser stimuli are considered as the withdrawal of the hindpaw from the glass floor, shaking of the hindpaw or hindlimb, and licking of the hindpaw. Laser testing sessions began 30 minutes after the injection (IP) of either vehicle, etomidate, or nidradine. Prior to the testing day, mice are habituated to the testing chamber for at least 30 minutes. Rotarod testing: To establish the sedative doses of known and novel general anesthetics, we performed rotarod testing. 67 Here, mice are placed onto an accelerating spinning rod, and the time to fall from the rod is recorded. Mice are considered sedated if they fall off the rotarod within 120 seconds of testing. Mice are tested 30 minutes after compound injection (IP). Prior to testing mice are trained to perform the rotarod test, and mice are considered trained only after they can remain on the rotarod without falling for at least 5 minutes. Estimation of ED 50 values using Dixon’s Up/Down method: To estimate the effective dose that produces a desired effect ( i.e. , LoRR) during 50% of testing sessions (ED 50 ), we used Dixon’s Up/Down method. 27 To perform the Dixon method, first, the test initial test is performed at the desired starting dose. If the test is positive for the effect ( i.e ., LoRR occurs), the dose is halved for the subsequent test. If the test is negative ( i.e ., no LoRR), then the dose is doubled. This testing paradigm continues until the first change in response direction ( i.e ., in a series of positive tests, the first negative test, or vice versa), and then 4 more tests are performed after this change in direction. After this, the response observations (X for a positive response, O for a negative), the dosing scale (d, equal to 2), and the concentration of drug on the final test (x final ), are recorded. The response observations are compared to a look up table generated in the original Dixon paper to generate the k-value. Then, to generate an estimate of the ED 50 , the x final , d, and k values are applied to the Dixon equation: log 2 (ED 50 ) = x final + (d * k) Voltage gated sodium channel profiling Cell Culture Human Embryonic Kidney (HEK) 293 cells stably expressing human NaV1.4/β1, NaV1.5/β1 and NaV1.6/ β1 (SB Drug Discovery, Glasgow, United Kingdom) and Chinese Hamster Ovary (CHO) cells stably expressing human NaV1.8/β3 in a tetracycline-inducible system (ChanTest, Cleveland, OH, United States) were cultured in Minimum Essential Media Eagle (MEM) with 10% fetal bovine serum (FBS), 2 mM ʟ-glutamine and selection antibiotics as per manufacturer's recommendations. Cells were grown in a 5% CO2 incubator at 37 °C and passaged every 3–4 days at 70–80 % confluency using TrypLE Express (Invitrogen). hNav1.8 expression was induced by addition of tetracycline (1 μg/mL) and incubation at 28 °C for 48 h prior to assays. Electrophysiology Automated whole-cell patch-clamp recordings were performed with a QPatch II automated electrophysiology platform (Sophion Bioscience, Ballerup, Denmark) using single-hole (QPlate 16 with a standard resistance of 2 ± 0.4 MΩ) or multi-hole (QPlate 16X with a standard resistance 0.2 ± 0.04 MΩ, NaV1.8 only). Whole-cell currents were filtered at 8 kHz and acquired at 25 kHz and the linear leak was corrected by P/4 subtraction (leak potential −90 mV, leak sweep amplitude 10 %). Series resistance across recorded cells ranged between 5 and 10 MΩ. The extracellular solution (ECS) consisted of (in mM) 145 NaCl, 4 KCl, 2 CaCl 2 , 1 MgCl 2 , 10 HEPES, and 10 glucose, pH to 7.4 with NaOH (adjusted to 305 mOsm/L with sucrose). The intracellular solution (ICS) consisted of (in mM) 140 CsF, 1 EGTA, 5 CsOH, 10 HEPES, and 10 NaCl, pH to 7.3 with CsOH (adjusted to 320 mOsm/L with sucrose). TTX (1 μM) was added to the ECS for NaV1.8 recordings to inhibit background endogenous TTX sensitive current in CHO cells. Cumulative concentration–response curves were obtained at a holding potential of −90 mV using 50 ms depolarizing pulses to −20 mV (or +10 mV for NaV1.8) delivered every 20 s (0.05 Hz). A 1:1 mixture of Nidradine (R)- and (S)-enantiomers, prepared from 100 mM DMSO stocks, was diluted in ECS containing 0.1% Pluronic F-127 and applied to cells for 2 minutes at the indicated concentrations. Peak currents were normalized to the buffer control and fitted with a four-parameter Hill equation with a variable Hill coefficient. In vitro mammalian receptor profiling In vitro binding assays and Ki data was generated by the National Institute of Mental Health's Psychoactive Drug Screening Program (PDSP), contract no. HHSN-271-2008-00025-C (NIMH PDSP), for assay details:http://pdsp.med.unc.edu/PDSP%20Protocols%20II%202013-03-28.pdf. Identification of phenotypically related compounds To compare multi-dimensional behavioral profiles from the large-scale screen, we first defined a reference profile against which all other wells were evaluated. The prototypical profile for etomidate was generated by averaging six replicate wells treated with 6.25 µM etomidate. Phenoscore distances were then computed between each test well and this reference profile using the Pearson correlation distance (implemented in the SciPy package, Python). The phenoscore ranges from −1 to +1, with positive and negative values indicating positive or negative correlation, respectively; negative scores reflect anti-correlation. In practice, phenoscores saturated at ~0.7, which indicates strong positive correlation given that the MI time series comprises vectors with >10,000 values.Ranking the screening hits.Phenoscores were computed to assign each compound in the screening library a rank order. Hit compounds were defined as the top 69 scoring compounds from this ranked list. Cortisol detection assay Isolation and detection of whole larval cortisol levels were performed as described. 68 30 animals were used per condition and treated with their respective compounds for 1 hour. Animals were then anesthetized in ice-cold egg water after excess water was removed from samples and were frozen in an ethanol/dry ice bath. Animals were then homogenized in 100 uL of H2O, 1 mL of ethyl acetate was added to dissolve cortisol from the sample the supernatant was collected and vaporized. Cortisol was dissolved in 0.2% Bovine serum albumin (A7030, Sigma) and frozen. For cortisol ELISA experiments the commercially available colorimetric competitive enzyme immunoassay kit (Enzo-ADI-900-071) was used as instructed.Reaction was stopped using 1 M H2SO4. Absorbance was read at 450nm in an ELISA plate reader (Biotek H4). Structural clustering Structural clustering was performed using custom written Python scripts. The RDKit Morgan fingerprint function was used to convert molecular structures into a digital hash. The SciPy hierarchical clustering (fcluster, linkage) and distance (pdist, parameters: metric = ‘jaccard’) functions were used to cluster the resulting molecular fingerprints. α1β2γ2 GABA A receptor expression and purification A tri-cistronic construct was used to express the α1β2γ2 GABA A R as described previously. 69 In brief, the three genes coding for each subunit were cloned into the pSBtet expression vector in the following order: β2-γ2-α1. For cryo-EM experiments, the intracellular loop between M3 and M4 for all subunits was replaced with a 7 amino acid linker, SQPARAA. 70,71 This EM construct also contains a strep tag on the γ2 subunit. Stable cell lines were created via a Sleeping Beauty transposon system. 72,73 Adherent HEK293S GnTI- cells were co-transfected with 1.9 µg of the pSBtet vector (pSBtet-GP, item #60495) carrying the EM construct as well as 0.1 µg of the transposase carrying vector, SB100X (pCMV(CAT)T7-SB100, item #34879). Lipofectamine2000 (Invitrogen) was used for transfection following the manufacturer’s protocol. 24 hours after transfection, selection of cells was initiated by incubation with 1 µg/mL puromycin. Selection was monitored by cell fluorescence and was terminated when all cells showed fluorescence. The adherent HEK293 GNTI- cells were then moved into suspension and expanded to a total of 6.4 L. When they reached a density of 3.5-4x10 6 cells/mL, protein expression was induced with 2 µg/mL doxycycline and the cells were incubated with shaking at 30°C with 8% CO 2 . Cells expressing GABA A Rs were harvested through centrifugation and resuspended in 20 mM Tris pH 7.4, 150 mM NaCl (TBS buffer) containing 1 mM phenylmethanesulfonyl fluoride (PMSF; Sigma-Aldrich) and 2 mM GABA (Sigma-Aldrich) before lysing with an Avestin Emulsiflex. The lysed cells were then centrifuged at 8,000 rpm for 15 min at 4°C. The resulting membrane-containing supernatant was then centrifuged at 40,000 rpm for 2 hours at 4°C. The membrane pellets were then homogenized using a Dounce homogenizer and solubilized in TBS buffer enriched with 40 mM n-dodecyl-β-D-maltoside (DDM, Anatrace), 1 mM PMSF and 2 mM GABA for 1 h at 4°C with nutation. Solubilized membranes were then centrifuged at 40,000 rpm for 40 min at 4°C. The supernatant was passed though Strep-Tactin XT Superflow affinity resin (IBA-GmbH). The resin was washed previously with TBS buffer containing 2 mM DDM, 0.01% (w/v) porcine brain polar lipids (Avanti) and 2 mM GABA. Protein was eluted with TBS buffer supplemented with 2 mM DDM, 0.01% (w/v) porcine brain polar lipids, 2 mM GABA and 50 mM biotin (Sigma-Aldrich). Nanodisc reconstitution The plasmid used for saposin A expression was kindly provided by Salipro Biotech AB. Nanodisc reconstitution followed the same protocol as described previously. 74,75 Concentrated receptors were first mixed with porcine brain polar lipids before incubation at room temperature for 10 min. Saposin was then added and the mixture was incubated for another 2 min. The reconstitution reaction was prepared in a 1:230:30 molar ratio of protein, lipids, and saposin A. To help initiate reconstitution, the mixture was diluted ~10-fold with TBS buffer supplemented with 2 mM GABA and 100 µM S -nidradine. Bio-Beads SM-2 (Bio-Rad) were added at ~200 mg/mL to remove detergent. The reconstitution reaction was rotated overnight at 4°C. The following morning, the Bio-beads were removed and the sample was collected for size-exclusion chromatography. Monoclonal antibody digestion and Fab purification 1F4 monoclonal antibody (mAB) against the α 1 subunit of the GABA A receptor (IgG2b, κ) was raised using standard methods (Monoclonal Core, Vaccine and Gene Therapy Institute, Oregon Health & Science University). Fab fragments were purified through papain cleavage. For 2 h at 37°C, 0.5 mg/mL of mAb was incubated with papain in a 1:30 ratio (w/w) in 50 mM NaPO 4 , pH 7.0, 1 mM EDTA and 10 mM cysteine. The reaction was quenched by incubation with 30 mM iodoacetamide at 25°C for 10 min. Fab was purified through anion exchange chromatography using a HiTrap Q HP (GE Healthcare) column in 10 mM Tris, pH 8.0 with a NaCl gradient elution. Cryo-EM sample preparation The reconstituted receptors were mixed with 1F4 Fab in a 3:1 (w/w) ratio and incubated on ice for 15 min. The sample was concentrated to 0.5-1 mL and injected into a Superose 6 Increase 10/300 GL column (GE Healthcare) which had previously been equilibrated with TBS buffer supplemented with 2 mM GABA and 100 µM S -nidradine. Peak fractions were analyzed by fluorescence-detection size-exclusion chromatography, using tryptophan fluorescence. Target fractions were pooled and concentrated to ~2 mg/mL then another 100 µM of S -nidradine was spiked in to a final concentration of 200 µM. The sample was supplemented with 0.5 mM fluorinated Fos-Choline-8 (Anatrace) to induce random orientations immediately prior to freezing grids. Copper R2/1 200 mesh 2 nm continuous carbon grids (Quantifoil) were glow-discharged (PELCO easiGlow) for 30 seconds at 10 mA before addition of 3 μL of sample followed by plunge-freezing in liquid ethane using a Vitrobot Mark IV (FEI). The grids were blotted for 3 seconds with a blot force of -15 at 100% humidity and 4°C. Cryo-EM data collection and processing Cryo-EM data was collected at the University of California, San Diego Cryo-EM facility for 24 h on a 300 kV Titan Krios Microscope (FEI) equipped with a Falcon 4 direct electron detector and Selectris X energy filter. The total exposure was 50 e − Å −2 and the defocus range was set to -1.2 µm to -2.0 µm. The dataset was processed using CryoSPARC v4.6.2 and v.4.7. The images were motion and gain corrected using patch motion correction. Patch CTF was used for contrast transfer function estimation. Particles were selected and 2x Fourier binned before three rounds of 2D classification. The selected particles were then re-extracted at full size which were then aligned with non-uniform refinement. Two focused 3D classification jobs were run with focus masks on the two β-α TMD interfaces. Classes with well-ordered TMDs were combined and duplicates removed. These particles were then aligned through another non-uniform refinement. This resulted in the final unsharpened map at a resolution of 2.9 Å, which was further sharped through a DeepEMhancer job. Model building, refinement, and validation An initial model was built for the S -nidradine structure using ModelAngelo. 76 This model was then docked into the S -nidradine experimental map and was manually adjusted in Coot, 77 with the GABA A receptor in complex with 1F4 fab, GABA and etomidate (PDB ID: 6X3V) as reference. The model was subjected to global real space and B-factor refinement with stereochemistry restraints in Phenix. 78 Geometry restraints for S -nidradine were generated using the Grade Web Server (Smart, O.S., Sharff A., Holstein, J., Womack, T.O., Flensburg, C., Keller, P., Paciorek, W., Vonrhein, C. and Bricogne G. (2021) Grade2 version 1.7.0. Cambridge, United Kingdom: Global Phasing Ltd). Model quality was checked with Phenix and Molprobity. 79 Sequences for sequence alignments were downloaded from the UniProt database and alignments were made with Clustal Omega. 80 Hole2 was used to generate pore radius profiles. 81 UCSF ChimeraX 82 was used to generate structural figures. Densities consistent with some phospholipids were present in the unsharpened cryo-EM density map. While these densities were not particularly strong, we built them into the model based on consistency with earlier models. 83 Electrophysiology of the α1β2γ2 GABA A receptor Whole-cell voltage-clamp electrophysiology recordings were collected from adherent HEK293S GnTI - cells. Recordings for WT receptors were collected from stable cell lines created with a Sleeping Beauty transposon system. For the mutant receptors, adherent HEK293S GnTI- cells were transiently transfected with a tri-cistronic pEZT construct encoding the mutant receptors and a GFP protein in pEZT for selection. After protein expression was induced, the cells were incubated at 30°C for 48-72 h. On the day of recording, cells were plated onto 35 mm dishes and washed with bath solution (140 mM NaCl, 4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM glucose and 10 mM HEPES, pH 7.4). Borosilicate pipettes were pulled and polished to an initial resistance of 2.5-3.5 MΩ. Pipettes were filled with pipette solution (110 mM CsF, 10 mM CsCl, 10 mM NaCl, 10 mM HEPES pH 7.2 and 10 mM EGTA). Cells were clamped at -75mV. Whole cell currents were recorded with an Axopatch 200B amplifier, sampled at 10 kHz, and low pass filtered at 2 kHz using a Digidata 1440 A (Molecular Devices), and analyzed with Clampfit 11 software (Molecular Devices). Solutions were exchanged using a gravity-driven RSC-200 rapid solution changer (Bio-Logic). Ligand solutions were prepared in bath solution. Statistical analysis of the GABA A receptor electrophysiology data Statistical analysis was performed using GraphPad Prism 10.6.0 software (GraphPad Software, Inc., La Jolla, CA). Data are expressed as mean valuesstandard error of the mean of at least four recordings from independent cells. Two-tailed Welch’s t-tests were used. A p -value of < 0.05 was considered statistically significant. General procedure for synthesis of oxazolecarboxamides and isoxazolecarboxamides and spectral data of synthesized intermediates see Supplementary Materials. Declarations AUTHORS AND AFFILIATIONS Department of Pharmaceutical Chemistry, University of California, San Francisco, CA, 94158, USA Matthew N. McCarroll, Xilin Gu, Elizabeth Sisko, Lain X. Pierce, Ralph Zhang, Ricardo Da Luz, Jason K. Sello, Cole Helsell, Douglas Myers-Turnbull, Brian K. Shoichet Department of Chemistry, Brown University, Providence, RI, 02912, USA Amanda Dombroski Department of Anesthesia and Perioperative Care University of California, San Francisco, California 94143, USA Jarret A.P. Weinrich, Michael P. Bokoch Department of Anatomy, University of California, San Francisco, California 94143, USA Jarret A.P. Weinrich, Allan I. Basbaum, Madison Jewell, Karnika Bhardwaj, Sian Rodriguez-Rosado, Department of Neurobiology, University of California, San Diego, CA, 92093, USA Ryan E. Hibbs, Alyssa Marinas, Jinfeng Teng, Megan J. Larmore Institute for Neurodegenerative Diseases, University of California, San Francisco, California 94143, USA Dave Kokel, Jack C. Taylor, Amanda Carbajal, Reid Kinser Institute for Molecule Bioscience, University of Queensland, St Lucia QLD 4072, Australia Jennifer Deuis, Asa Andersson, Irina Vetter Blavatnik Institute of Neurobiology, Harvard Medical School, Boston, MA, 02115, USA Bruce Palmer Bean AUTHOR CONTRIBUTIONS Conceptualization, M.N.N., D.K., J.K.S., J.A.P.W.; Methodology M.N.M., J.A.P.W., R.E.H., A.H.M., M.L., Jf.T.; Software, M.N.M., D.M.T., C.H., J.A.P.W., R.K.; Formal Analysis, M.N.M., A.H.M., Jf.T., J.A.P.W., R.K.; Investigation, M.N.M, J.A.P.W., X.G., A.D., A.H.M., E.S., Jf.T., J.C.T., L.X.P., C.A., A.C., R.D.L., R.Z., M.J., K.B., S.R.R., J.D., A.A.; Resources, M.N.M, D.K., J.K.S., R.E.H, I.V., B.P.B., A.I.B.; Data Curation, M.N.M, D.M.T., C.H., A.H.M., Jf.T.; Writing - Original Draft, M.N.M., J.A.P.W., A.H.M.; Writing - Reviewing and Editing, M.N.M, R.E.H., J.A.P.W., M.B., A.I.B., A.H.M., J.K.S.; Visualization, M.N.M., J.A.P.W., A.H.M., Jf.T.; Supervision, M.N.M., R.E.H., D.K., A.I.B., B.P.B., I.V., B.K.S., J.K.S.; Project Administration, M.N.M., A.I.B., R.E.H., J.K.S.; Funding Acquisition, M.N.M, D.K., A.I.B., B.K.S., B.P.B., I.V., M.B., R.E.H., J.K.S. CORRESPONDING AUTHORS Correspondence to Matthew N. McCarroll ( [email protected] ) Ryan E. Hibbs ( [email protected] ), Allan I. Basbaum ( [email protected] ), and Jason K. Sello ( [email protected] ). ACKNOWLEDGMENTS We thank Louie Ramos for exceptional animal care and husbandry. ELISA plate reader analysis was performed at the UCSF center for advanced technology, supported by UCSF PBBR, RRP IMIA, and NIH 1S10OD028511-01 grants. This work was supported by the Defense Advanced Research Projects Agency (DARPA) grant: W911NF-24-2-0109 (MNM, JKS, AIB, REH, BKS MPB) and the US National Institutes of Health (NIH) grants: DP1DA058350 (MNM), AI123400-03 (JKS), R01AA022583 (DK), F31NS145564 and T32GM007752 (AHM), and R01DA047325 (REH). This work was also supported by the Chan Zuckerberg Initiative (JKS), an advised fund of the Silicon Valley Community Foundation and Ono pharmaceuticals (JKS). COMPETING INTERESTS The University of California San Francisco and the Chan Zuckerberg Biohub have filed a patent application for compounds related to the new anesthetics with analgesic potential. Drs. McCarroll, Sello, Kokel, Basbaum, and Weinrich are named as co-inventors. Other authors declare no competing interests. DATA AVAILABILITY All motion index time series, behavioral screening data, and cryo-EM maps and models are available for peer review upon request. Cryo-EM map and model will be deposited in the electron microscopy data bank and protein data bank, respectively, upon acceptance of the manuscript for publication. CODE AVAILABILITY All code is available upon request. SUPPORTING INFORMATION The supporting information includes Supplementary Figures showing selected chemical structures, mouse pharmacokinetics, stereoisomer behavioral analysis, electrophysiology representative traces, electrophysiology at mutant GABA A receptors, Cryo-EM data processing workflow, cortisol level analysis, PDSP data and voltage gated sodium channel electrophysiology. Also included are Supplementary Tables with chemical names, SMILES, SAR information, and extended methods and spectral Analysis of oxazoles and isoxazoles synthesis. References McQueen, K. A. K. Anesthesia and the global burden of surgical disease. Int. Anesthesiol. Clin. 48 , 91–107 (2010). Gu, W.-J., Wang, F., Tang, L. & Liu, J.-C. Single-dose etomidate does not increase mortality in patients with sepsis: a systematic review and meta-analysis of randomized controlled trials and observational studies. Chest 147 , 335–346 (2015). Bruder, E. A., Ball, I. M., Ridi, S., Pickett, W. & Hohl, C. 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Protein Soc. 30 , 70–82 (2021). Legesse, D. H. et al. Structural insights into opposing actions of neurosteroids on GABAA receptors. Nat. Commun. 14 , 5091 (2023). Additional Declarations Yes there is potential Competing Interest. The University of California San Francisco and the Chan Zuckerberg Biohub have filed a patent application for compounds related to the new anesthetics with analgesic potential. Drs. McCarroll, Sello, Kokel, Basbaum, and Weinrich are named as co-inventors. Other authors declare no competing interests. Supplementary Files MovieS2.mp4 Movie S2. Loss of Withdrawl Reflex Test of Adult Zebrafish. MovieS1.mp4 Movie S1. Loss of Righting Reflex Test of Adult Zebrafish. 20251201NidradineSupplementaryMaterial.docx SUPPORTING INFORMATION D1000300933valreportfullP1.pdf PDB Validation report 20260105NidradineSupplementaryMaterial.docx Supplementary Materials Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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1","display":"","copyAsset":false,"role":"figure","size":474931,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHigh throughput behavioral screening identifies anesthesia phenocopy molecules in zebrafish. a\u003c/strong\u003e, Time-series behavioral profiles (motion index, MI; y-axis) of larval zebrafish during a multi-stimulus battery delivered at defined time points (x-axis). The stimulus track below marks timing: gray bars = solenoid taps; orange/blue/purple = visible-light stimuli. Traces show vehicle (gray) and anesthetic dosing: etomidate (red, top) and propofol (red, bottom). \u003cem\u003en\u003c/em\u003e = 4–12 wells per condition; 8 larvae per well. \u003cstrong\u003eb\u003c/strong\u003e, Rank-ordered scatter of phenoscores (y-axis) across all screened wells (x-axis, sorted low-high). The black curve shows a smoothed trend. Dotted lines mark the vehicle (solvent) mean, vehicle mean + 3 SD, and the pre-specified hit threshold (phenoscore = 0.55). \u003cstrong\u003ec\u003c/strong\u003e, Dose response heatmap for 29 retest compounds (rows), concentration is in µM. Each cell shows the mean phenoscore (color bar) across n = 3 wells at the indicated concentrations (columns); 0.0 denotes on-plate vehicle controls. \u003cstrong\u003ed\u003c/strong\u003e, Adult zebrafish validation. Mean motion index (MI) per compound for the retest set; lower MI indicates reduced locomotor activity. Etomidate is the positive control; vehicle (solvent) is the negative control. Labeled structures of the five strongest adult hits are shown.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8341587/v1/c1a7d9ba7fbc421caabc4342.png"},{"id":100366024,"identity":"1b2a487a-15e5-43d1-9cab-b1c114c27352","added_by":"auto","created_at":"2026-01-16 07:55:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":539323,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalog development campaign of the primary oxazole lead (MM01). a\u003c/strong\u003e, Structural clustering of 70 commercially available analog by catalog compounds \u0026gt;70% similar to MM01 (y-axis) forms 8 clusters using a Tanimoto similarity metric (x-axis). \u003cstrong\u003eb\u003c/strong\u003e, Dose response heatmap for 18 analog by catalog compounds (rows). Each cell shows the mean etomidate similarity phenoscore (color bar) across n = 3 wells at the indicated concentrations (columns); 0.0 denotes on-plate vehicle controls. \u003cstrong\u003ec\u003c/strong\u003e, Adult zebrafish validation of catalog analogs. Average motion index (MI) per compound; lower MI indicates reduced locomotor activity. Etomidate served as the positive control, vehicle as the negative control. Structures of the two strongest analogs are shown above the plot, with labels highlighted in red. \u003cstrong\u003ed\u003c/strong\u003e, Structural clustering of 29 in-house–synthesized analogs, forming five clusters by Tanimoto similarity. \u003cstrong\u003ee\u003c/strong\u003e, Dose response heatmap for 29 in house synthesized analogs (rows). Each cell shows the mean etomidate similarity phenoscore (color bar) across n = 3 wells, 8 fish/well at the indicated concentrations (columns); 0.0 denotes vehicle controls. \u003cstrong\u003ef\u003c/strong\u003e, Adult zebrafish validation of in-house analogs. Average MI per compound with labeled structures of the top two analogs (highlighted in red). \u003cstrong\u003eg, h,\u003c/strong\u003e Bar plots showing ED\u003csub\u003e50\u003c/sub\u003e of values (y-axis) in µM for zebrafish (\u003cstrong\u003eg) \u003c/strong\u003eor mg/kg in mice (\u003cstrong\u003eh\u003c/strong\u003e) for the parent molecule MM01, and the top analogs.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8341587/v1/24914e00e51099a232339874.png"},{"id":100366619,"identity":"9a7cb1a5-63d1-43cc-a945-3cc7539acb66","added_by":"auto","created_at":"2026-01-16 07:56:24","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":185964,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-nidradine activity on the α1β2γ2 GABA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e receptor. a\u003c/strong\u003e, 2D chemical structure of \u003cem\u003eS\u003c/em\u003e-nidradine (upper) and whole cell patch clamp electrophysiology showing activity of \u003cem\u003eS\u003c/em\u003e-nidradine on the GABAA receptor at different concentrations and in the absence and presence of GABA. \u003cstrong\u003eb\u003c/strong\u003e, Electrophysiology showing dose-dependent effects of \u003cem\u003eS\u003c/em\u003e-nidradine and \u003cem\u003eR\u003c/em\u003e-etomidate (etomidate) on the receptor in the presence of GABA at EC5 (1 µM). Etomidate is more efficacious and more potent. \u003cstrong\u003ec\u003c/strong\u003e, Electrophysiology showing how fixed concentrations of \u003cem\u003eS\u003c/em\u003e-nidradine and etomidate affect the dose-dependent activity of GABA. Both compounds enhance GABA activity with the greatest effects occurring at low [GABA]. \u003cstrong\u003ed\u003c/strong\u003e, Effects of \u003cem\u003eS\u003c/em\u003e-nidradine (\u003cem\u003eS\u003c/em\u003e-nid) and etomidate on the receptor in the presence of high [GABA]; here, neither compound further increases GABAA receptor activity; p = 0.7609 for GABA alone vs. GABA + \u003cem\u003eS\u003c/em\u003e-nid, p = 0.8691 for GABA alone vs. GABA + etomidate. \u003cstrong\u003ee\u003c/strong\u003e, Both compounds are agonists on the receptor at concentrations well above the range where they are active as PAMs. n = 4-5, error bars represent mean ± SEM for all conditions.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8341587/v1/328672b55b6e8079a80e01f4.jpeg"},{"id":100133546,"identity":"26178394-af4b-46ae-981e-3c4592c8407a","added_by":"auto","created_at":"2026-01-13 10:27:55","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":706885,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStructural details of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-nidradine binding and mechanisms of potentiation. a, \u003c/strong\u003eSide view of the cryo-EM density map of the α1β2γ2 receptor with \u003cem\u003eS\u003c/em\u003e-nidradine bound. α1 shown in green, β2 shown in blue, γ2 shown in purple and \u003cem\u003eS\u003c/em\u003e-nidradine in yellow. \u003cstrong\u003eb\u003c/strong\u003e, Details of the \u003cem\u003eS\u003c/em\u003e-nidradine binding site with some interacting residues shown. \u003cstrong\u003ec\u003c/strong\u003e, Sequence alignment of the \u003cem\u003eS\u003c/em\u003e-nidradine binding site, showing conservation across humans, mice, and zebrafish; residues important for \u003cem\u003eS\u003c/em\u003e-nidradine binding are highlighted and residues likely making electrostatic interactions with \u003cem\u003eS\u003c/em\u003e-nidradine are labelled with a dotted line. \u003cstrong\u003ed\u003c/strong\u003e, Superposition of the \u003cem\u003eS\u003c/em\u003e-nidradine, etomidate, and propofol binding sites. \u003cstrong\u003ee\u003c/strong\u003e, Electrophysiology comparing \u003cem\u003eS\u003c/em\u003e-nidradine PAM and agonist activities on the WT, N265I and T262V mutated receptors. GABA was co-applied at EC5 (1 µM for WT, 0.5 µM for N265I, and 3 µM for T262V) with \u003cem\u003eS\u003c/em\u003e-nidradine at 7 µM then at 100 µM. \u003cem\u003eS\u003c/em\u003e-nidradine was then applied alone at 300 µM and 1 mM. n = 4-6 cells, error bars represent mean ± SEM for all conditions; *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001. \u003cstrong\u003ef\u003c/strong\u003e, HOLE representations of the α1β2γ2 GABAA receptor in the presence of GABA alone, GABA + \u003cem\u003eS\u003c/em\u003e-nidradine, GABA + etomidate, and GABA + propofol (PDB IDs: 6X3Z, 9YNN, 6X3V, 6X3T). The diameters of the 9ʹ activation and -2ʹ desensitization gates are shown. \u003cstrong\u003eg\u003c/strong\u003e, HOLE plot showing the radius along the pore for the GABA only, GABA + \u003cem\u003eS\u003c/em\u003e-nidradine, GABA + etomidate, and GABA + propofol structures.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8341587/v1/0b789a88f28d2dda86568e66.jpeg"},{"id":100366108,"identity":"876e8bb6-d467-4d78-801c-deebda02628c","added_by":"auto","created_at":"2026-01-16 07:55:58","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":296326,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNidradine produces surgical anesthesia and analgesia in fish and mice. a, \u003c/strong\u003eDixon estimates of the loss of righting reflex (LORR) ED\u003csub\u003e50\u003c/sub\u003e in both adult zebrafish (µM) and mice (mg/kg). \u003cstrong\u003eb,\u003c/strong\u003e In fish, withdrawal reflexes to noxious pinch are not present during administration of nidradine (25µM) but are present during etomidate (3.125µM) (nidradine: 0/5 fish responding; etomidate: 5/5 fish responding, p ≤ 0.0079, Fisher’s exact test). \u003cstrong\u003ec,\u003c/strong\u003e In mice, withdrawal reflexes to noxious pinch are not present during administration of nidradine (115mg/kg) but are present during etomidate (12mg/kg) (nidradine: 0/5 mice responding; etomidate: 6/6 mice responding, p ≤ 0.0022, Fisher’s exact test). \u003cstrong\u003ed, \u003c/strong\u003eSub-sedative doses of nidradine reduce positive responses to noxious stimuli in mice (vehicle: median = 90%, 25 Percentile = 80%, 75 Percentile = 100%, N = 4 mice; nidradine: median =\u0026nbsp; 40%, 25 percentile = 25%, 75 percentile\u0026nbsp; = 55%, N = 4 mice; p ≤ 0.0286, Mann Whitney test) but etomidate does not (vehicle: median = 90%, 25 Percentile = 80%, 75 Percentile = 100%, N = 4 mice; etomidate: median =\u0026nbsp; 100%, 25 percentile = 100%, 75 percentile\u0026nbsp; = 100%, N = 4 mice; p ≤ 4286, Mann Whitney test). \u003cstrong\u003ee, \u003c/strong\u003eSub-sedative doses of nidradine reduce positive responses to noxious stimuli in fish (normalized to vehicle control; vehicle: mean ± standard error = 100 ± 5.9%, N = 4 fish; nidradine: mean ± standard error = 65.11 ± 4.696%, N = 4 fish; p ≤ 0.0041, Welch’s test) but etomidate does not (normalized to vehicle control; vehicle: mean ± standard error = 100 ± 5.320%, N = 4 fish; etomidate: mean ± standard error = 107.3 ± 4.033%, N = 4 fish; p ≤ 0.3216, Welch’s test).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8341587/v1/e78fb4340bde2ad4636d8713.png"},{"id":100382426,"identity":"c046e3bf-3085-4f36-9ff7-b905a288eac0","added_by":"auto","created_at":"2026-01-16 10:42:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3953975,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8341587/v1/8316c40e-64f5-434f-bd9d-b3ce50b1df11.pdf"},{"id":100133543,"identity":"aafc5613-1e58-4113-b674-226089a57bf7","added_by":"auto","created_at":"2026-01-13 10:27:55","extension":"mp4","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1350171,"visible":true,"origin":"","legend":"Movie S2. Loss of Withdrawl Reflex Test of Adult Zebrafish.","description":"","filename":"MovieS2.mp4","url":"https://assets-eu.researchsquare.com/files/rs-8341587/v1/cef624687bf643257ca24fa7.mp4"},{"id":100133549,"identity":"87352b34-c07e-43fc-a15a-6f69f99db826","added_by":"auto","created_at":"2026-01-13 10:27:55","extension":"mp4","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2966767,"visible":true,"origin":"","legend":"Movie S1. Loss of Righting Reflex Test of Adult Zebrafish.","description":"","filename":"MovieS1.mp4","url":"https://assets-eu.researchsquare.com/files/rs-8341587/v1/09d666dc8d4a02c9f315bdec.mp4"},{"id":100367417,"identity":"ac28c2c7-a6cb-45da-ba97-2723b79d792f","added_by":"auto","created_at":"2026-01-16 07:57:02","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":6145351,"visible":true,"origin":"","legend":"SUPPORTING INFORMATION","description":"","filename":"20251201NidradineSupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-8341587/v1/183ad3083ac123094ce38f63.docx"},{"id":100133548,"identity":"ca551cc0-c5d1-4237-bf04-2fc5c7898889","added_by":"auto","created_at":"2026-01-13 10:27:55","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":2213233,"visible":true,"origin":"","legend":"PDB Validation report","description":"","filename":"D1000300933valreportfullP1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8341587/v1/9bda62f810104c133c936372.pdf"},{"id":100366467,"identity":"08bc2502-e2cb-45b3-a7bd-55f95f81b6dd","added_by":"auto","created_at":"2026-01-16 07:56:19","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":6145910,"visible":true,"origin":"","legend":"Supplementary Materials","description":"","filename":"20260105NidradineSupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-8341587/v1/6bfb5655b935f0197d86388f.docx"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nThe University of California San Francisco and the Chan Zuckerberg Biohub have filed a patent application for compounds related to the new anesthetics with analgesic potential. Drs. McCarroll, Sello, Kokel, Basbaum, and Weinrich are named as co-inventors. Other authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003ePhenotypic discovery of GABA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e receptor-mediated general anesthetics with analgesic activity\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eDespite their widespread use,\u003csup\u003e1\u003c/sup\u003e general anesthetics have a suboptimal therapeutic profile. The most commonly used intravenous general anesthetic, propofol, causes unfavorable hemodynamics and respiratory depression, while etomidate causes adrenal suppression that is linked to increased mortality.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e The GABA\u003csub\u003eA\u003c/sub\u003e receptor is the major target of many general anesthetics.\u003csup\u003e\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e The GABA\u003csub\u003eA\u003c/sub\u003e receptor is a ligand-gated ion channel assembled as a pentamer of homologous subunits. The existence of 19 different receptor subunits in humans underlies an incredible diversity of GABA\u003csub\u003eA\u003c/sub\u003e receptors having distinct signaling properties and pharmacology.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e The most abundant subtype in the human brain comprises α1, β2 and γ2 subunits.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e GABA\u003csub\u003eA\u003c/sub\u003e receptor activation by GABA or exogenous molecules triggers opening of an intrinsic anion channel, which in most cases results in intracellular chloride conductance, dampening neuronal excitability. Notably, common intravenous anesthetics that act on the GABA\u003csub\u003eA\u003c/sub\u003e receptor, including etomidate and propofol, lack analgesic properties,\u003csup\u003e10,11\u003c/sup\u003e necessitating additional pain management measures, often via opioids.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe discovery and development of new anesthetics remain challenging given that anesthesia must be assessed in mammals via expensive and low-throughput experiments. In the past decade, aquatic vertebrates have emerged as an alternative to mammals in the discovery of neuroactive compounds. Indeed, early zebrafish screens established reproducible behavioral barcodes (\u003cem\u003ei.e\u003c/em\u003e., phenotypes) that predict mechanism and revealed novel psychoactive scaffolds.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e The fact that immersion dosing in these animals rapidly attains near-steady-state exposure \u003cem\u003ein vivo\u003c/em\u003e facilitates chemical screens.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e In neuroactive discovery, a focused zebrafish larval photomotor-response screen (~\u0026thinsp;374 compounds) identified reversible anesthetic like sedative\u0026ndash;hypnotics with activity spanning GABA\u003csub\u003eA\u003c/sub\u003e​, neuronal nicotinic, and NMDA receptors,\u003csup\u003e15\u003c/sup\u003e illustrating that target-agnostic phenotypic assays can identify hits, including those that act through multiple targets.. Studies in zebrafish have even yielded propofol antagonists, fluorinated analogs that reverse propofol anesthesia, underscoring the capacity of these models to uncover both agonist and antagonist chemotypes.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eWe recently pioneered a novel screening approach that uses \u003cem\u003elarval\u003c/em\u003e zebrafish, in which anesthetics produce rich behavioral fingerprints beyond simple immobility, namely measurable transient hyperexcitability or altered responses to specific stimuli.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e By capturing these patterns, behavioral screening in zebrafish is amenable to high-throughput approaches for discovering new anesthetic drugs. As other vertebrates, zebrafish exhibit a complex repertoire of behaviors, yet their central nervous system is far simpler in architecture.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e Because zebrafish larvae are small enough to fit in 96-well plates and are readily dosed with compounds dissolved directly in the water, it is possible to do high-through put screens of thousands of compounds. Indeed, zebrafish have been used to screen and identify novel pharmacological compounds, some of which have shown promising translational potential in mammalian models and have progressed to clinical trials (\u003cem\u003ee.g\u003c/em\u003e., clemizole and lorcaserin for Dravet syndrome; and the mechanosensory hair-cell otoprotectant ORC-13661).\u003csup\u003e\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e Importantly, the GABA\u003csub\u003eA\u003c/sub\u003e receptor gene family is highly conserved in zebrafish. Its genome contains at least 23 GABA\u003csub\u003eA\u003c/sub\u003e receptor subunits including orthologs of all mammalian subunits, except θ and ε, and an additional subunit β4.\u003csup\u003e22\u003c/sup\u003e Like mammals, the zebrafish α subfamily is the largest and most diverse of the subfamilies.\u003c/p\u003e \u003cp\u003eHere, we report the execution of a novel high-throughput behavioral chemical screen in larval zebrafish that is predictive of general anesthetic activity. The larval hits that were also active in a secondary anesthesia screen in adult zebrafish proved to be efficacious in mice. Among them was an isoxazole whose analogs also had potent and reversible anesthetic effects. Medicinal chemistry efforts on the isoxazole scaffold yielded a lead compound that we named nidradine after Nidra, the Hindu deity of sleep. Biophysical experiments revealed that nidradine potentiates GABA\u003csub\u003eA\u003c/sub\u003e receptor function and shares a binding site with known anesthetics, including etomidate and propofol. However, compared to GABA\u003csub\u003eA\u003c/sub\u003e modulating anesthetics, nidradine not only exerted enhanced surgical anesthesia but also exhibited analgesic activity.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003eDiscovery of novel anesthetics in zebrafish by phenocopying known anesthetics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e To investigate the behavioral modifications induced by intravenous anesthetics in fish, we used a suite of automated behavioral assays tailored to distinguish between a diverse array of neuroactive substances.\u003csup\u003e17\u003c/sup\u003e The methodology incorporated a variety of acoustic and visual stimuli to detect substances that either decrease motor activity or increase startle responses to low-intensity acoustic signals. We previously reported that etomidate and propofol, which act via GABA\u003csub\u003eA\u003c/sub\u003e receptors and provoke enhanced acoustic startle responses (eASRs) to a low-intensity solenoid tap, but not to other stimuli (Fig. 1a).\u003csup\u003e17\u003c/sup\u003e Here, we screened \u0026gt;10,000 structurally diverse compounds in larval zebrafish and examined their phenotypic similarity to the behavioral profile of etomidate (Fig. 1b). Sixty-nine compounds phenocopied etomidate and were designated as primary hits (refer to Supplementary Table 1).\u003c/p\u003e\n\u003cp\u003eWe next categorized these hits by structural similarity and identified 22 distinct clusters (Supplementary Figure 1, Supplementary Table 1). Certain clusters contained compounds having well-known pharmacophores. For example, Cluster 4 is enriched for piperidine\u0026ndash;carbonyl scaffolds, a well-recognized CNS-active motif that includes local anesthetics (bupivacaine and ropivacaine). Cluster 17 was enriched for 8 related isoxazole scaffolds (Supplementary Table 1), which is a bioactive and CNS-privileged heterocycle found in both synthetic and natural neuroactive molecules.\u003csup\u003e23\u003c/sup\u003e From the sixty-nine primary hits,we selected 29 compounds representing the chemoinformatic clusters for further testing (Supplementary Table 2). We conducted dose response trials and confirmed that twenty-eight of the twenty-nine recapitulated the eASR phenotype (Fig. 1d, Supplementary Figure 2 and Supplementary Table 2), validating the reliability of the primary behavioral screen.\u003c/p\u003e\n\u003cp\u003e As age-dependent neuropharmacology and pharmacokinetics can influence drug responses,\u003csup\u003e24\u0026ndash;26\u003c/sup\u003e we next asked whether the hits anesthetized adult zebrafish. Our analysis was based on observations that etomidate reliably produces behavioral endpoints of the hypnotic component of general anesthesia (\u003cem\u003ei.e\u003c/em\u003e., ability to produce loss of responsiveness), namely loss of righting reflex (LoRR) and immobility in adult fish (Fig. 1e, Supplementary Movie 1). Accordingly, we used LoRR and immobility as endpoints for a secondary screen, in which adult zebrafish were treated with each of the 29 primary screening hits at 50 \u0026micro;M (Fig. 1e). We identified 5 compounds that induced robust anesthesia (Fig. 1e,f); among them, we prioritized compound 9232649 (MM01) in hit-to-lead optimization efforts.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOptimization of candidate chemotypes and anesthetic validation in mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e Given the favorable phenotypic and chemical properties of the oxazole carboxamide hit, MM01, we conducted a structure-based analog search and identified 70 commercially available compounds having greater than 70% similarity to the parent molecule (Supplementary Table 3). Compounds were prioritized for purchase and evaluation via calculating the Tanimoto similarity scores and performing hierarchical clustering (Fig. 2a). This analysis revealed 8 distinct structural clusters, and we selected 1 or more representative analogs from each cluster for a total of 13 analogs (SupplementaryTable 3 and Supplementary Figure 3).\u003c/p\u003e\n\u003cp\u003eFrom this analog-by-catalog screen, we identified 2 isoxazolecarboxamides [9267615 (MM02) and 9203415 (MM03)] that produced robust LoRR in adult zebrafish. Further characterization confirmed that these isoxazolecarboxamides were effective at sedating both larval and adult zebrafish (Fig. 2b, c). Notably, these compounds also showed improved aqueous solubility, enhancing their suitability for \u003cem\u003ein vivo\u003c/em\u003e applications. Based on these results, we initiated synthesis of additional analogs to probe structure-activity relationships. In total, we synthesized 28 analogs within the oxazole and isoxazole chemical space (Figure 2d, Supplementary Figure 4 and Supplementary Table 4) and evaluated their behavioral effects in both larval and adult zebrafish (Figure 2e, f). From this set of compounds, we identified 3 analogs that produced clear LoRR phenotypes in adults and were soluble in aqueous media. Based on potency, solubility, and behavioral consistency, two of these were selected for further testing (Figure 2f, chemical structures shown).\u003c/p\u003e\n\u003cp\u003e Next, we assessed compounds for general anesthetic activity in mice. To assess for both the presence of a general anesthetic effect and to estimate the dose that produces LORR in 50% of trials (\u003cem\u003ei.e\u003c/em\u003e., the ED\u003csub\u003e50\u003c/sub\u003e), we used Dixon\u0026rsquo;s up-down method.\u003csup\u003e27\u003c/sup\u003e IP injections of the parent molecule MM01, two of the analog by catalog hits, and the two optimized analogs produced LORR in mice indicating anesthetic properties (Figure 2c). The synthetic analog, AD-7-19 (nidradine) was the most easily formulated and thus subjected to further analyses. Pharmacokinetic analysis after IP injection of nidradine in mice demonstrated that the compound distributed to the plasma and brain, consistent with a CNS-mediated general anesthetic action (Supplementary Figure 5). Importantly, nidradine did not cause the problematic inhibition of cortisol biosynthesis underlying adrenal suppression like etomidate (see supporting information, Supplementary Figure 11).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eNidradine potentiates the GABA\u003csub\u003eA\u003c/sub\u003e receptor through a membrane site\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e As nidradine was efficacious in inducing anesthesia in both fish and mice in a manner comparable to etomidate, we selected it for detailed mechanistic analysis. Many general anesthetics, including etomidate, propofol, and barbiturates, act by potentiating the activity of the GABA\u003csub\u003eA\u003c/sub\u003e receptor, which led us to ask whether nidradine acted via the same target and mechanism. This prediction was further strengthened by our recent findings that most phenocopiers of etomidate from smaller behavioral screens in larval zebrafish screens are positive allosteric modulators of the GABA\u003csub\u003eA\u003c/sub\u003e receptors.\u003csup\u003e28\u003c/sup\u003e On these grounds, we tested the activity of nidradine on the GABA\u003csub\u003eA\u003c/sub\u003e receptor in whole-cell patch clamp electrophysiology experiments on HEK cells expressing the \u0026alpha;1\u0026beta;2\u0026gamma;2 subtype of the receptor; this subunit assembly is the most abundant in the human brain and is targeted by the general anesthetics mentioned above. We used \u003cem\u003eS\u003c/em\u003e-nidradine, as this enantiomer was most active in both fish and mice (Supplementary Figure 6).\u003c/p\u003e\n\u003cp\u003e We began by testing the activity of \u003cem\u003eS\u003c/em\u003e-nidradine on the GABA\u003csub\u003eA\u003c/sub\u003e receptor in the absence and presence of GABA. Figure 3a shows that \u003cem\u003eS\u003c/em\u003e-nidradine has both positive allosteric modulator (PAM) and allosteric agonist activities. At lower concentrations, \u003cem\u003eS\u003c/em\u003e-nidradine acts purely as a PAM; whereas, at higher concentrations it acts as both a PAM and as an agonist in a manner reminiscent of other general anesthetics acting through this receptor.\u003csup\u003e29\u003c/sup\u003e Comparing nidradine with etomidate (Fig. 3b-d, Supplementary Figure 7a-g), we found that \u003cem\u003eS\u003c/em\u003e-nidradine has an approximately 3- to 4-fold lower PAM potency than etomidate (7 \u0026micro;M vs. 2 \u0026micro;M; Fig. 3b) yet it is similarly effective in enhancing the receptor\u0026rsquo;s sensitivity to GABA (Fig. 3c). Indeed, we also observed 3- to 5-fold potentiation in currents by nidradine in the presence of low GABA concentrations; however, at saturating GABA concentrations, \u003cem\u003eS\u003c/em\u003e-nidradine does not further potentiate GABA\u003csub\u003eA\u003c/sub\u003e activity (Fig. 3d). The agonist potency of \u003cem\u003eS\u003c/em\u003e-nidradine is ~10-fold weaker than that of etomidate (571 \u0026micro;M vs. 52.1 \u0026micro;M; Fig. 3e, Supplementary Figure 7h-i). As concentrations that induce agonist activity are unlikely to be reached \u003cem\u003ein vivo\u003c/em\u003e, we suggest that the anesthetic effect in zebrafish and mice occurs via \u003cem\u003eS\u003c/em\u003e-nidradine\u0026rsquo;s PAM activity.\u003c/p\u003e\n\u003cp\u003e To gain a detailed understanding of how \u003cem\u003eS\u003c/em\u003e-nidradine binds to these receptors and to inform future drug optimization, we resolved a 2.9 \u0026Aring; structure of the \u0026alpha;1\u0026beta;2\u0026gamma;2 receptor bound to \u003cem\u003eS\u003c/em\u003e-nidradine via cryo-EM (Supplementary Figure 9, Supplementary Table 5). We found that \u003cem\u003eS\u003c/em\u003e-nidradine, like etomidate and propofol, binds to the \u0026beta;-\u0026alpha; interfaces in the receptor\u0026rsquo;s transmembrane domain (TMD, Fig. 4a). The binding of molecules having quite different structures to the same site and inducing the same pharmacological effect is truly remarkable. Although nidradine binds to both \u0026beta;-\u0026alpha; TMD interfaces, one binding site has higher ligand occupancy. From our cryo-EM density map and model, \u003cem\u003eS\u003c/em\u003e-nidradine is positioned to make electrostatic interactions with N265 and T262 on the M2 helix of the \u0026beta;2 subunit via an amide oxygen and isoxazole nitrogen, respectively (Fig. 4b). Importantly, the key amino acids comprising the \u003cem\u003eS\u003c/em\u003e-nidradine binding site are conserved across humans, mice, and zebrafish, which is consistent with nidradine\u0026rsquo;s cross-species activity and suggests strong potential for clinical utility (Fig. 4c). In any case, \u003cem\u003eS\u003c/em\u003e-nidradine\u0026rsquo;s binding site partially overlaps with those of propofol and etomidate. However, this new compound binds deeper, towards the cytosol and slightly more inwards towards the pore than either propofol or etomidate (Fig. 4d). This binding mode allows \u003cem\u003eS\u003c/em\u003e-nidradine to make hydrophobic interactions with residues located further down the receptor\u0026rsquo;s subunit interface, beyond where etomidate and propofol reach (Supplementary Figure 8a).\u003c/p\u003e\n\u003cp\u003e Since the strong electrostatic interactions between \u003cem\u003eS\u003c/em\u003e-nidradine and the N265 and T262 residues were suggested by the cryo-EM data to be determinants of the compound\u0026rsquo;s binding and activity, we substituted these amino acids via site-directed mutagenesis to test their importance. Specifically, we made N265I and T262V mutants and tested their function in electrophysiology experiments. To assess an influence on \u003cem\u003eS\u003c/em\u003e-nidradine PAM activity, we co-applied GABA at EC5 (1 \u0026micro;M for WT, 0.5 \u0026micro;M for N265I, and 3 \u0026micro;M for T262V; Supplementary Figure 8b-d) with \u003cem\u003eS\u003c/em\u003e-nidradine at 7 \u0026micro;M, then at 100 \u0026micro;M, on cells expressing WT and mutant receptors. Both the N265I and T262V mutants showed a significant decrease in sensitivity to 7 \u0026micro;M \u003cem\u003eS\u003c/em\u003e-nidradine versus the WT receptors. However, when GABA was co-applied with \u003cem\u003eS\u003c/em\u003e-nidradine at the higher 100 \u0026micro;M concentration, only the N265I mutant showed a significant decrease in PAM activity (Fig. 4e). To determine if these mutants also affected \u003cem\u003eS\u003c/em\u003e-nidradine agonist activity, we applied \u003cem\u003eS\u003c/em\u003e-nidradine alone at 300 \u0026micro;M and 1 mM on cells expressing WT and mutant receptors. Both mutants showed significant decreases in activity for both concentrations when compared to WT (Fig. 4e, Supplementary Figure 8e-g). We conclude that both N265 and T262 are key determinants of \u003cem\u003eS\u003c/em\u003e-nidradine PAM and agonist activity, and that both activities likely arise through occupancy of the same sites.\u003c/p\u003e\n\u003cp\u003e To gain a deeper understanding of how \u003cem\u003eS\u003c/em\u003e-nidradine potentiates the receptor and how its mechanism compares to currently used general anesthetics, we next analyzed pore dimensions of the \u0026alpha;1\u0026beta;2\u0026gamma;2 receptor bound to GABA alone, GABA + \u003cem\u003eS\u003c/em\u003e-nidradine, GABA + etomidate, and GABA + propofol (Fig. 4f-g). While all of these structures are either in a desensitized or desensitized-like state, the GABA + \u003cem\u003eS\u003c/em\u003e-nidradine structure has the narrowest -2ʹ desensitization gate, with a diameter of 2.4 \u0026Aring;, compared to GABA + etomidate, GABA + propofol and GABA-only pores having diameters of 2.9 \u0026Aring;, 3.3 \u0026Aring;, and 3.6 \u0026Aring;,\u003csup\u003e30\u003c/sup\u003e respectively, at this same location. Interestingly, at the 9ʹ activation gate, the GABA + \u003cem\u003eS\u003c/em\u003e-nidradine structure has a pore diameter of 5.7 \u0026Aring;, which is similar to the etomidate-bound structure\u0026rsquo;s pore diameter of 6.2 \u0026Aring;. The GABA-only pore is smaller with a diameter of 4.6 \u0026Aring;, while the GABA + propofol structure\u0026rsquo;s pore is much wider with a diameter of 10.4 \u0026Aring;. These findings indicate that \u003cem\u003eS\u003c/em\u003e-nidradine\u0026rsquo;s mechanism of potentiation may be similar to those of the currently used general anesthetics, where the compounds cause widening of the pore at the 9ʹ activation gate, likely destabilizing the resting state and sensitizing the channel to GABA potentiation.\u003csup\u003e31\u003c/sup\u003e\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eNidradine produces enhanced surgical anesthesia and analgesia\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e An important feature of many general anesthetics is their ability to generate surgical anesthesia, namely loss of reflexive responses to noxious stimuli (\u003cem\u003ei.e\u003c/em\u003e., nociceptive areflexia). For clinically relevant anesthetics (for example, volatile anesthetics,\u003csup\u003e32\u003c/sup\u003e propofol,\u003csup\u003e33,34\u003c/sup\u003e ketamine\u003csup\u003e35\u003c/sup\u003e), the doses that induce surgical anesthesia are generally higher than those that induce hypnotic effects. To systematically determine whether nidradine produces surgical anesthesia in mice, we assessed for loss of reflexive withdrawal responses (LoWR) to 30 seconds of continuous strong pinch of the hindpaw. Interestingly, during our initial pilot testing of nidradine, we observed that mice lost withdrawal reflexes to noxious stimuli (hindpaw pinch) at the same dose that produces LoRR, which was unexpected. Therefore, we performed LoWR testing at approximately the ED\u003csub\u003e50\u003c/sub\u003e for LoRR, for both nidradine and etomidate (Fig. 5a). As expected, at the ED\u003csub\u003e50\u003c/sub\u003e dose for etomidate, mice that lost righting reflex continued to respond to strong hindpaw pinch (Fig. 5c). However, when the same test was conducted with nidradine, the withdrawal reflex to strong hindpaw pinch was absent in all mice that lost righting reflex (Fig. 5c). Surprisingly, this phenomenon was also observed in adult zebrafish (Fig. 5b, Supplementary Movie 2). To the best of our knowledge, the equipotent production of hypnosis and surgical anesthesia (areflexia) is unique to nidradine.\u003c/p\u003e\n\u003cp\u003e As nidradine produces surgical anesthesia (\u003cem\u003ei.e\u003c/em\u003e., blocks withdrawal of the paw to a strong pinch) at lower than expected doses, we next asked whether nidradine also blocks nocifensive responses, a reflex correlate of pain processing, at subanesthetic doses. Importantly, by assessing at nonsedating doses (ability to stay on rotarod) in awake mice (Supplementary Figure 10), any potential loss of responses to noxious stimuli will not erroneously be misclassified due to an inability of the mouse to respond. Here, we monitored nociceptive responses to a strong noxious heat stimulus produced by a short duration, high intensity infrared laser pulse in awake, freely moving mice.\u003csup\u003e36\u003c/sup\u003e As expected, subanesthetic doses of etomidate did not reduce nocifensive responses to the laser stimulus. In contrast, at subanesthetic doses, nidradine significantly reducedresponses to the laser stimulus, compared to vehicle controls (Fig. 4e). Follow up studies using light-induced acute noxious stimuli (\u003cem\u003ei.e\u003c/em\u003e., optovin) confirmed that nidradine, but not etomidate, also reduces nocifensive responses in larval zebrafish (Fig. 5d).\u003c/p\u003e\n\u003cp\u003e To better understand the mechanism underlying the anesthetic and analgesic actions of nidradine, we performed radioligand displacement profiling across a panel of neuroreceptors through the Psychoactive Drug Screening Program (PDSP).\u003csup\u003e37\u003c/sup\u003e Nidradine did not bind \u0026alpha;2 adrenergic receptors, which are targets of established anesthetics and analgesics (Supplementary Figure 12), nor did it bind opioid receptors (mu, delta, kappa). Interestingly, nidradine did exhibit non-selective inhibition of voltage gated sodium (NaV) channels (NaV1.4, NaV1.5, NaV1.6, NaV1.8); however, the low potencies (~140 \u0026micro;M) measured indicate that NaV channels are unlikely to contribute to the hypnotic or analgesic action of nidradine (Supplementary Figure 13). \u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn this drug development effort, we discovered translatable chemotype that not only produces general anesthesia, but at lower doses, also analgesia. This discovery was enabled by a highly informative behavioral phenotype in larval zebrafish induced by known general anesthetics. Our high-throughput compatible platform for behavioral profiling enabled target agnostic screening of over 10,000 molecules in larval zebrafish. Our lead molecule developed from extensive hit-to-lead optimization efforts, nidradine, is a positive allosteric modulator of the GABA\u003csub\u003eA\u003c/sub\u003e receptor, and shares binding sites with other clinically relevant general anesthetics. Nidradine is distinct from clinically used anesthetics in that it produces hypnosis and areflexia at equipotent doses. Importantly, nidradine is analgesic at subanesthetic concentrations, greatly expanding its possible clinical translation.\u003c/p\u003e\n\u003cp\u003eIn the present study, our secondary screen of anesthetic-like hits in adult fish after the primary larval zebrafish screen was an essential step toward translating anesthetic candidates to mammals. During development, zebrafish undergo maturation of GABAergic circuitry and synaptic connectivity, which can alter drug sensitivity and network-level responses.\u003csup\u003e38\u0026ndash;42\u003c/sup\u003e In addition, pharmacokinetic factors differ markedly between larvae and adults; larval fish are small, with limited diffusion barriers and have a rudimentary blood\u0026ndash;brain barrier that may allow easier compound penetration.\u003csup\u003e24,43,44\u003c/sup\u003e For this reason, confirming that candidate compounds produce anesthesia in adult fish, particularly compounds that induce rapid LoRR within minutes, is key to identifying compounds with appropriate kinetics, bioavailability and pharmacodynamics for translation into mammals.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe chemotypes discovered in the screen are structurally distinct from all known general anesthetics- the ethers (sevoflurane), alkyl phenols (propofol), cyclohexanones (ketamine) imidazoles (etomidate and dexmedetomidine), and the benzodiazepine remimazolam. Our primary hit MM01 contained an oxazole, whereas iterative SAR optimization converged on a related isoxazole scaffold. The isoxazole ring is a well-recognized CNS-privileged heteroaromatic motif and an adaptable medicinal chemistry scaffold, appearing in multiple synthetic and naturally occurring neuroactive compounds, including the GABAergic agonist muscimol from Amanita mushrooms.\u003csup\u003e45\u003c/sup\u003e Recent systematic analyses of isoxazole derivatives further highlight this chemotype\u0026rsquo;s broad bioactivity across CNS, anti-inflammatory, antimicrobial, and anticancer indications, underscoring its suitability as a privileged framework for many therapeutics including novel anesthetic leads.\u003csup\u003e23,45\u0026ndash;49\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eAll anesthetics that produce surgical anesthesia (loss of responses to noxious stimuli) do so at doses greater than those that induce hypnosis (\u003cem\u003ei.e\u003c/em\u003e., loss of consciousness).\u003csup\u003e50\u003c/sup\u003e For example, for volatile anesthetics, the concentration of anesthetic that produces loss of volitional responses is generally 30-50% of the dose required for loss of response to surgical incision.\u003csup\u003e51,52\u003c/sup\u003e Similarly, intravenous general anesthetics, such a propofol\u003csup\u003e53\u0026ndash;55\u003c/sup\u003e and etomidate,\u003csup\u003e56\u003c/sup\u003e also block reflexes to noxious stimuli at doses above those that induce hypnosis. In distinct contrast, nidradine blocks noxious stimulus-evoked reflexes at the lowest doses that block the righting reflex. Currently, we do not know the mechanism through which nidradine blocks nociceptive reflexes in the setting of surgical anesthesia. Interestingly, targeted mutations to the \u0026beta;2 and \u0026beta;3 subunits of the GABA\u003csub\u003eA\u003c/sub\u003e receptor (N265), which is a part of a shared binding site in the GABA\u003csub\u003eA\u003c/sub\u003e receptor targeted by etomidate, propofol, and nidradine, reduces,\u003csup\u003e57\u003c/sup\u003e or even abolishes,\u003csup\u003e58\u003c/sup\u003e the generation of surgical anesthesia by etomidate and propofol. This possible receptor interaction suggests that nidradine\u0026rsquo;s equipotent production of surgical anesthesia and hypnosis may be due to novel interactions with the GABA\u003csub\u003eA\u003c/sub\u003e receptor. It is also possible that nidradine\u0026rsquo;s analgesic effects occur through an undetermined receptor; however, we have ruled out voltage gated sodium channels, \u0026alpha;2 adrenergic receptors, and opioid receptors.\u003c/p\u003e\n\u003cp\u003eThe ability of nidradine to produce analgesia at subsedative doses places it in a small class of anesthetics that can provide pain relief in awake animals. As others have shown, and we have confirmed with etomidate, most general anesthetics that act at the GABA\u003csub\u003eA\u003c/sub\u003e receptor are not analgesic at subsedative doses (\u003cem\u003ee.g\u003c/em\u003e., propofol,\u003csup\u003e59\u003c/sup\u003e isoflurane,\u003csup\u003e60\u003c/sup\u003e and sevoflurane).\u003csup\u003e61\u003c/sup\u003e It is unlikely, therefore, that the analgesic effects of nidradine in awake animals is derived from GABA\u003csub\u003eA\u003c/sub\u003e receptor PAM activity. In future studies, we will investigate the molecular mechanism under nidradine\u0026rsquo;s production of analgesia. Importantly, by recapitulating the analgesic effects of nidradine in larval zebrafish, we will incorporate the ability to identify analgesic general anesthetics into our high throughput discovery pipeline.\u0026nbsp;\u003c/p\u003e"},{"header":"METHODS","content":"\u003cp\u003e\u003cstrong\u003eFish maintenance, breeding and chemical treatments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMaintenance and breeding of wild type zebrafish (Singapore) was performed as described\u003csup\u003e62\u003c/sup\u003e and staged in days post-fertilization (dpf). All embryos were raised on a 14/10-hour light/dark cycle at 28˚C until 7 dpf. Larvae were anesthetized with ice cold egg water and distributed 8 animals per well into square 96-well plates (GE Healthcare Life Sciences) with 300 \u0026micro;L of egg water.\u003csup\u003e62\u003c/sup\u003e Adult AbTL or TuAB fish and placed individually in the well of a round 6-well plate in 8 mL of egg water. Chemical treatments were applied directly to the egg water and larvae were incubated at room temp for 1 hour before behavioral analysis. Adult animals were assayed immediately following drug treatment. For secondary screening of hit compounds in adult zebrafish loss of righting reflex assays, single-animal experiments were performed at 50 \u0026micro;M. For Dixon ED\u003csub\u003e50\u003c/sub\u003e estimates, each compound was tested with n = 5 animals. All zebrafish procedures were approved by the UCSF Institutional Animal Care and Use Committee (protocol AN201827) and conducted in accordance with institutional and federal guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChemical libraries\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll chemical libraries were dissolved in DMSO. The Chembridge library (Chembridge Corporation) contains \u0026gt;10,000 compounds at 10 mM. All compounds were diluted in egg water and screened at 30 \u0026micro;M final concentration in \u0026lt; 1% DMSO. Controls were treated with an equal volume of DMSO. All previously annotated and novel hit compounds were validated in 3-12 replicate well plates in a dose response behavioral assay, 8 zebrafish larvae per well at concentrations range from 100 \u0026micro;M - 1.56 \u0026micro;M.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAutomated behavioral phenotyping assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlates were illuminated with a 760 nm infrared light from a custom-built led array. Digital video was captured at 25 frames per second using an AVT Pike digital camera (Allied Vision). Assays\u0026rsquo; duration was 30-120 seconds consisting of a combination of acoustic and light stimulus. Low (60db) and high (70 db) amplitude acoustic stimuli were delivered using push-style solenoids (12V) to tap a custom-built stage where the 96-well plate was placed. Light stimulus was delivered using high intensity LEDs (LEDENGIN) that delivered violet (405 nm, 11 \u0026mu;W/mm\u003csup\u003e2\u003c/sup\u003e) blue (560 nm, 18 \u0026micro;W/mm\u003csup\u003e2\u003c/sup\u003e), green (525 nm,11 \u0026mu;W/mm\u003csup\u003e2\u003c/sup\u003e), and red (650 nm, 11 \u0026mu;W/mm\u003csup\u003e2\u003c/sup\u003e) wavelengths. Stimuli and digital recordings were applied to the entire 96-well plate simultaneously. Instrument control and data acquisition were performed using custom python scripts. The zebrafish motion index (MI) was calculated as follows: MI = sum(abs(frame\u003csub\u003en\u003c/sub\u003e \u0026ndash; frame\u003csub\u003en\u0026minus;1\u003c/sub\u003e)). Normalized MI (nMI) was calculated as follows: nMI = (MI-min(MI))/max(MI)). For motion estimation expressed as a CD(10), value was estimated as the count of pixels that changed from the previous frame by intensity \u0026ge; 10 / 255. Detailed descriptions of the behavioral assays are described here.\u003csup\u003e17,63\u0026ndash;65\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMouse husbandry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll mouse husbandry and surgical procedures adhered to the regulatory standards of the Institutional Animal Care and Use Committee of the University of California San Francisco (UCSF; protocol AN203168). Mice were kept on a 12-hour light/dark cycle, with access to food and water \u003cem\u003ead libitum\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMouse behavioral assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompound solubilization for mouse behavior testing:\u003c/strong\u003ePrior to testing, compounds were solubilized in 1.5% ethanol, 11% kolliphor EL (Sigma-Aldrich, C5135-500G) and 87.5% sterile saline. Dry compounds were first mixed with solution of ethanol and kolliphor, then diluted with saline immediately prior to testing. Maximum intraperitoneal injection (IP) volume was set to 0.5cc.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLoRR Testing:\u003c/strong\u003e To assess the hypnotic effects of known and novel general anesthetics, we assayed mice for the presence of loss of righting reflex (LoRR) after compound administration. Mice are tested in a behavioral arena that consists of a 4-walled, 12\u0026rdquo; by 12\u0026rdquo; acrylic chamber. The chamber is placed onto a heating pad whose temperature is maintained by circulating warm water (40˚C). To initiate the testing session, compounds were administered via IP injection, after which mice were carefully placed into the behavioral arena and closely monitored for 10 minutes post-injection. Mice were assayed for LoRR approximately every 30 seconds. During assessment for LoRR the tester attempts to gently place the mice into the supine position. A positive LoRR response is scored as the continuous, unaided maintenance of the supine position for 30 seconds. After the 10-minute testing session, mice are monitored for regain of righting reflex (RoRR). Positive RoRR is defined as 3 consecutive failed LoRR tests after the mice first reestablish the prone position (all 4 paws on the floor), tested every 30 seconds.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLoWR testing:\u003c/strong\u003e To assess for nociceptive areflexia, we assayed mice for the presence of loss of withdrawal reflexes (LoWR) after compound administration. First mice are injected (IP) with compound then assessed for LoRR within 10 minutes after injection within the same arena described above. After LoRR is achieved, LoWR testing begins. The LoWR test consists of pinching the mouse hindpaw with an extra-long hemostat (23cm), without engaging the locking mechanism. Positive LoWR is defined as lack of observable reflexive hindlimb withdrawal response to 30 seconds of continuous pinch.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePain testing:\u003c/strong\u003e To assess for analgesic effects of subsedative doses of general anesthetics, we assessed for changes to pain responses following to acute noxious thermal stimuli delivered by high-intensity, short-duration infrared laser pulse, as previously described.\u003csup\u003e66\u003c/sup\u003e The laser stimulus is generated by a LASMED (Lass-7M) 7W 975 nm laser set to a laser power of 1750 mA and pulse duration of 300 ms. Briefly, mice are placed within a chamber with a glass floor that allows for the presentation of the noxious laser stimulus to the plantar surface of the hindpaw. During a testing session, the laser stimulus is presented to one of the two hindpaws 5 times, with a cooldown period of at least 3 minutes between individual trials. The left and right hindpaws are stimulated in an alternating fashion. Positive nociceptive responses to laser stimuli are considered as the withdrawal of the hindpaw from the glass floor, shaking of the hindpaw or hindlimb, and licking of the hindpaw. Laser testing sessions began 30 minutes after the injection (IP) of either vehicle, etomidate, or nidradine. Prior to the testing day, mice are habituated to the testing chamber for at least 30 minutes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRotarod testing:\u003c/strong\u003e To establish the sedative doses of known and novel general anesthetics, we performed rotarod testing.\u003csup\u003e67\u003c/sup\u003e Here, mice are placed onto an accelerating spinning rod, and the time to fall from the rod is recorded. Mice are considered sedated if they fall off the rotarod within 120 seconds of testing. Mice are tested 30 minutes after compound injection (IP). Prior to testing mice are trained to perform the rotarod test, and mice are considered trained only after they can remain on the rotarod without falling for at least 5 minutes.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003cstrong\u003eEstimation of ED\u003csub\u003e50\u003c/sub\u003e values using Dixon\u0026rsquo;s Up/Down method:\u003c/strong\u003eTo estimate the effective dose that produces a desired effect (\u003cem\u003ei.e.\u003c/em\u003e, LoRR) during 50% of testing sessions (ED\u003csub\u003e50\u003c/sub\u003e), we used Dixon\u0026rsquo;s Up/Down method.\u003csup\u003e27\u003c/sup\u003e To perform the Dixon method, first, the test initial test is performed at the desired starting dose. If the test is positive for the effect (\u003cem\u003ei.e\u003c/em\u003e., LoRR occurs), the dose is halved for the subsequent test. If the test is negative (\u003cem\u003ei.e\u003c/em\u003e., no LoRR), then the dose is doubled. This testing paradigm continues until the first change in response direction (\u003cem\u003ei.e\u003c/em\u003e., in a series of positive tests, the first negative test, or vice versa), and then 4 more tests are performed after this change in direction. After this, the response observations (X for a positive response, O for a negative), the dosing scale (d, equal to 2), and the concentration of drug on the final test (x\u003csub\u003efinal\u003c/sub\u003e), are recorded. The response observations are compared to a look up table generated in the original Dixon paper to generate the k-value. Then, to generate an estimate of the ED\u003csub\u003e50\u003c/sub\u003e, the x\u003csub\u003efinal\u003c/sub\u003e, d, and k values are applied to the Dixon equation:\u003c/p\u003e\n\u003cp\u003elog\u003csub\u003e2\u003c/sub\u003e(ED\u003csub\u003e50\u003c/sub\u003e) = x\u003csub\u003efinal\u003c/sub\u003e + (d * k)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVoltage gated sodium channel profiling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCell Culture\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eHuman Embryonic Kidney (HEK) 293 cells stably expressing human NaV1.4/\u0026beta;1, NaV1.5/\u0026beta;1 and NaV1.6/ \u0026beta;1 (SB Drug Discovery, Glasgow, United Kingdom) and Chinese Hamster Ovary (CHO) cells stably expressing human NaV1.8/\u0026beta;3 in a tetracycline-inducible system (ChanTest, Cleveland, OH, United States) were cultured in Minimum Essential Media Eagle (MEM) with 10% fetal bovine serum (FBS), 2 mM ʟ-glutamine and selection antibiotics as per manufacturer\u0026apos;s recommendations. Cells were grown in a 5% CO2 incubator at 37 \u0026deg;C and passaged every 3\u0026ndash;4 days at 70\u0026ndash;80 % confluency using TrypLE Express (Invitrogen). hNav1.8 expression was induced by addition of tetracycline (1 \u0026mu;g/mL) and incubation at 28 \u0026deg;C for 48 h prior to assays.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eElectrophysiology\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAutomated whole-cell patch-clamp recordings were performed with a QPatch II automated electrophysiology platform (Sophion Bioscience, Ballerup, Denmark) using single-hole (QPlate 16 with a standard resistance of 2 \u0026plusmn; 0.4 M\u0026Omega;) or multi-hole (QPlate 16X with a standard resistance 0.2 \u0026plusmn; 0.04 M\u0026Omega;, NaV1.8 only). Whole-cell currents were filtered at 8 kHz and acquired at 25 kHz and the linear leak was corrected by P/4 subtraction (leak potential \u0026minus;90 mV, leak sweep amplitude 10 %). Series resistance across recorded cells ranged between 5 and 10 M\u0026Omega;.\u003c/p\u003e\n\u003cp\u003eThe extracellular solution (ECS) consisted of (in mM) 145 NaCl, 4 KCl, 2 CaCl\u003csub\u003e2\u003c/sub\u003e, 1 MgCl\u003csub\u003e2\u003c/sub\u003e, 10 HEPES, and 10 glucose, pH to 7.4 with NaOH (adjusted to 305 mOsm/L with sucrose). The intracellular solution (ICS) consisted of (in mM) 140 CsF, 1 EGTA, 5 CsOH, 10 HEPES, and 10 NaCl, pH to 7.3 with CsOH (adjusted to 320 mOsm/L with sucrose). TTX (1 \u0026mu;M) was added to the ECS for NaV1.8 recordings to inhibit background endogenous TTX sensitive current in CHO cells.\u003c/p\u003e\n\u003cp\u003eCumulative concentration\u0026ndash;response curves were obtained at a holding potential of \u0026minus;90 mV using 50 ms depolarizing pulses to \u0026minus;20 mV (or +10 mV for NaV1.8) delivered every 20 s (0.05 Hz). A 1:1 mixture of Nidradine (R)- and (S)-enantiomers, prepared from 100 mM DMSO stocks, was diluted in ECS containing 0.1% Pluronic F-127 and applied to cells for 2 minutes at the indicated concentrations. Peak currents were normalized to the buffer control and fitted with a four-parameter Hill equation with a variable Hill coefficient.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vitro mammalian receptor profiling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn vitro binding assays and Ki data was generated by the National Institute of Mental Health\u0026apos;s Psychoactive Drug Screening Program (PDSP), contract no. HHSN-271-2008-00025-C (NIMH PDSP), for assay details:http://pdsp.med.unc.edu/PDSP%20Protocols%20II%202013-03-28.pdf.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIdentification of phenotypically related compounds\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo compare multi-dimensional behavioral profiles from the large-scale screen, we first defined a reference profile against which all other wells were evaluated. The prototypical profile for etomidate was generated by averaging six replicate wells treated with 6.25 \u0026micro;M etomidate. Phenoscore distances were then computed between each test well and this reference profile using the Pearson correlation distance (implemented in the SciPy package, Python). The phenoscore ranges from \u0026minus;1 to +1, with positive and negative values indicating positive or negative correlation, respectively; negative scores reflect anti-correlation. In practice, phenoscores saturated at ~0.7, which indicates strong positive correlation given that the MI time series comprises vectors with \u0026gt;10,000 values.Ranking the screening hits.Phenoscores were computed to assign each compound in the screening library a rank order. Hit compounds were defined as the top 69 scoring compounds from this ranked list.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCortisol detection assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIsolation and detection of whole larval cortisol levels were performed as described.\u003csup\u003e68\u003c/sup\u003e 30 animals were used per condition and treated with their respective compounds for 1 hour. Animals were then anesthetized in ice-cold egg water after excess water was removed from samples and were frozen in an ethanol/dry ice bath. Animals were then homogenized in 100 uL of H2O, 1 mL of ethyl acetate was added to dissolve cortisol from the sample the supernatant was collected and vaporized. Cortisol was dissolved in 0.2% Bovine serum albumin (A7030, Sigma) and frozen. For cortisol ELISA experiments the commercially available colorimetric competitive enzyme immunoassay kit (Enzo-ADI-900-071) was used as instructed.Reaction was stopped using 1 M H2SO4. Absorbance was read at 450nm in an ELISA plate reader (Biotek H4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStructural clustering\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStructural clustering was performed using custom written Python scripts. The RDKit Morgan fingerprint function was used to convert molecular structures into a digital hash. The SciPy hierarchical clustering (fcluster, linkage) and distance (pdist, parameters: metric = \u0026lsquo;jaccard\u0026rsquo;) functions were used to cluster the resulting molecular fingerprints.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026alpha;1\u0026beta;2\u0026gamma;2 GABA\u003csub\u003eA\u003c/sub\u003e receptor expression and purification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA tri-cistronic construct was used to express the \u0026alpha;1\u0026beta;2\u0026gamma;2 GABA\u003csub\u003eA\u003c/sub\u003eR as described previously.\u003csup\u003e69\u003c/sup\u003e In brief, the three genes coding for each subunit were cloned into the pSBtet expression vector in the following order: \u0026beta;2-\u0026gamma;2-\u0026alpha;1. For cryo-EM experiments, the intracellular loop between M3 and M4 for all subunits was replaced with a 7 amino acid linker, SQPARAA.\u003csup\u003e70,71\u003c/sup\u003e This EM construct also contains a strep tag on the \u0026gamma;2 subunit. Stable cell lines were created via a Sleeping Beauty transposon system.\u003csup\u003e72,73\u003c/sup\u003e Adherent HEK293S GnTI- cells were co-transfected with 1.9 \u0026micro;g of the pSBtet vector (pSBtet-GP, item #60495) carrying the EM construct as well as 0.1 \u0026micro;g of the transposase carrying vector, SB100X (pCMV(CAT)T7-SB100, item #34879). Lipofectamine2000 (Invitrogen) was used for transfection following the manufacturer\u0026rsquo;s protocol. 24 hours after transfection, selection of cells was initiated by incubation with 1 \u0026micro;g/mL puromycin. Selection was monitored by cell fluorescence and was terminated when all cells showed fluorescence. The adherent HEK293 GNTI- cells were then moved into suspension and expanded to a total of 6.4 L. When they reached a density of 3.5-4x10\u003csup\u003e6\u003c/sup\u003e cells/mL, protein expression was induced with 2 \u0026micro;g/mL doxycycline and the cells were incubated with shaking at 30\u0026deg;C with 8% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003eCells expressing GABA\u003csub\u003eA\u003c/sub\u003eRs were harvested through centrifugation and resuspended in 20 mM Tris pH 7.4, 150 mM NaCl (TBS buffer) containing 1 mM phenylmethanesulfonyl fluoride (PMSF; Sigma-Aldrich) and 2 mM GABA (Sigma-Aldrich) before lysing with an Avestin Emulsiflex. The lysed cells were then centrifuged at 8,000 rpm for 15 min at 4\u0026deg;C. The resulting membrane-containing supernatant was then centrifuged at 40,000 rpm for 2 hours at 4\u0026deg;C. The membrane pellets were then homogenized using a Dounce homogenizer and solubilized in TBS buffer enriched with 40 mM n-dodecyl-\u0026beta;-D-maltoside (DDM, Anatrace), 1 mM PMSF and 2 mM GABA for 1 h at 4\u0026deg;C with nutation. Solubilized membranes were then centrifuged at 40,000 rpm for 40 min at 4\u0026deg;C. The supernatant was passed though Strep-Tactin XT Superflow affinity resin (IBA-GmbH). The resin was washed previously with TBS buffer containing 2 mM DDM, 0.01% (w/v) porcine brain polar lipids (Avanti) and 2 mM GABA. Protein was eluted with TBS buffer supplemented with 2 mM DDM, 0.01% (w/v) porcine brain polar lipids, 2 mM GABA and 50 mM biotin (Sigma-Aldrich).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNanodisc reconstitution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe plasmid used for saposin A expression was kindly provided by Salipro Biotech AB. Nanodisc reconstitution followed the same protocol as described previously.\u003csup\u003e74,75\u003c/sup\u003e Concentrated receptors were first mixed with porcine brain polar lipids before incubation at room temperature for 10 min. Saposin was then added and the mixture was incubated for another 2 min. The reconstitution reaction was prepared in a 1:230:30 molar ratio of protein, lipids, and saposin A. To help initiate reconstitution, the mixture was diluted ~10-fold with TBS buffer supplemented with 2 mM GABA and 100 \u0026micro;M \u003cem\u003eS\u003c/em\u003e-nidradine. Bio-Beads SM-2 (Bio-Rad) were added at ~200 mg/mL to remove detergent. The reconstitution reaction was rotated overnight at 4\u0026deg;C. The following morning, the Bio-beads were removed and the sample was collected for size-exclusion chromatography.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMonoclonal antibody digestion and Fab purification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e1F4 monoclonal antibody (mAB) against the \u0026alpha;\u003csub\u003e1\u003c/sub\u003esubunit of the GABA\u003csub\u003eA\u003c/sub\u003e receptor (IgG2b, \u0026kappa;) was raised using standard methods (Monoclonal Core, Vaccine and Gene Therapy Institute, Oregon Health \u0026amp; Science University). Fab fragments were purified through papain cleavage. For 2 h at 37\u0026deg;C, 0.5 mg/mL of mAb was incubated with papain in a 1:30 ratio (w/w) in 50 mM NaPO\u003csub\u003e4\u003c/sub\u003e, pH 7.0, 1 mM EDTA and 10 mM cysteine. The reaction was quenched by incubation with 30 mM iodoacetamide at 25\u0026deg;C for 10 min. Fab was purified through anion exchange chromatography using a HiTrap Q HP (GE Healthcare) column in 10 mM Tris, pH 8.0 with a NaCl gradient elution.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCryo-EM sample preparation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe reconstituted receptors were mixed with 1F4 Fab in a 3:1 (w/w) ratio and incubated on ice for 15 min. The sample was concentrated to 0.5-1 mL and injected into a Superose 6 Increase 10/300 GL column (GE Healthcare) which had previously been equilibrated with TBS buffer supplemented with 2 mM GABA and 100 \u0026micro;M \u003cem\u003eS\u003c/em\u003e-nidradine. Peak fractions were analyzed by fluorescence-detection size-exclusion chromatography, using tryptophan fluorescence. Target fractions were pooled and concentrated to ~2 mg/mL then another 100 \u0026micro;M of \u003cem\u003eS\u003c/em\u003e-nidradine was spiked in to a final concentration of 200 \u0026micro;M. The sample was supplemented with 0.5 mM fluorinated Fos-Choline-8 (Anatrace) to induce random orientations immediately prior to freezing grids. Copper R2/1 200 mesh 2 nm continuous carbon grids (Quantifoil) were glow-discharged (PELCO easiGlow) for 30 seconds at 10 mA before addition of 3 \u0026mu;L of sample followed by plunge-freezing in liquid ethane using a Vitrobot Mark IV (FEI). The grids were blotted for 3 seconds with a blot force of -15 at 100% humidity and 4\u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCryo-EM data collection and processing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCryo-EM data was collected at the University of California, San Diego Cryo-EM facility for 24 h on a 300 kV Titan Krios Microscope (FEI) equipped with a Falcon 4 direct electron detector and Selectris X energy filter. The total exposure was 50 e\u003csup\u003e\u0026minus;\u003c/sup\u003e \u0026Aring;\u003csup\u003e\u0026minus;2\u003c/sup\u003e and the defocus range was set to -1.2 \u0026micro;m to -2.0 \u0026micro;m. The dataset was processed using CryoSPARC v4.6.2 and v.4.7. The images were motion and gain corrected using patch motion correction. Patch CTF was used for contrast transfer function estimation. Particles were selected and 2x Fourier binned before three rounds of 2D classification. The selected particles were then re-extracted at full size which were then aligned with non-uniform refinement. Two focused 3D classification jobs were run with focus masks on the two \u0026beta;-\u0026alpha; TMD interfaces. Classes with well-ordered TMDs were combined and duplicates removed. These particles were then aligned through another non-uniform refinement. This resulted in the final unsharpened map at a resolution of 2.9 \u0026Aring;, which was further sharped through a DeepEMhancer job.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eModel building, refinement, and validation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn initial model was built for the \u003cem\u003eS\u003c/em\u003e-nidradine structure using ModelAngelo.\u003csup\u003e76\u003c/sup\u003e This model was then docked into the \u003cem\u003eS\u003c/em\u003e-nidradine experimental map and was manually adjusted in Coot,\u003csup\u003e77\u003c/sup\u003e with the GABA\u003csub\u003eA\u003c/sub\u003e receptor in complex with 1F4 fab, GABA and etomidate (PDB ID: 6X3V) as reference. The model was subjected to global real space and B-factor refinement with stereochemistry restraints in Phenix.\u003csup\u003e78\u003c/sup\u003e Geometry restraints for \u003cem\u003eS\u003c/em\u003e-nidradine were generated using the Grade Web Server (Smart, O.S., Sharff A., Holstein, J., Womack, T.O., Flensburg, C., Keller, P., Paciorek, W., Vonrhein, C. and Bricogne G. (2021) Grade2 version 1.7.0. Cambridge, United Kingdom: Global Phasing Ltd). Model quality was checked with Phenix and Molprobity.\u003csup\u003e79\u003c/sup\u003e Sequences for sequence alignments were downloaded from the UniProt database and alignments were made with Clustal Omega.\u003csup\u003e80\u003c/sup\u003e Hole2 was used to generate pore radius profiles.\u003csup\u003e81\u003c/sup\u003e UCSF ChimeraX\u003csup\u003e82\u003c/sup\u003e was used to generate structural figures. Densities consistent with some phospholipids were present in the unsharpened cryo-EM density map. While these densities were not particularly strong, we built them into the model based on consistency with earlier models.\u003csup\u003e83\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eElectrophysiology of the \u0026alpha;1\u0026beta;2\u0026gamma;2 GABA\u003csub\u003eA\u003c/sub\u003e receptor\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhole-cell voltage-clamp electrophysiology recordings were collected from adherent HEK293S GnTI\u003csup\u003e-\u003c/sup\u003e cells. Recordings for WT receptors were collected from stable cell lines created with a Sleeping Beauty transposon system. For the mutant receptors, adherent HEK293S GnTI- cells were transiently transfected with a tri-cistronic pEZT construct encoding the mutant receptors and a GFP protein in pEZT for selection. After protein expression was induced, the cells were incubated at 30\u0026deg;C for 48-72 h. On the day of recording, cells were plated onto 35 mm dishes and washed with bath solution (140 mM NaCl, 4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM glucose and 10 mM HEPES, pH 7.4). Borosilicate pipettes were pulled and polished to an initial resistance of 2.5-3.5 M\u0026Omega;. Pipettes were filled with pipette solution (110 mM CsF, 10 mM CsCl, 10 mM NaCl, 10 mM HEPES pH 7.2 and 10 mM EGTA). Cells were clamped at -75mV. Whole cell currents were recorded with an Axopatch 200B amplifier, sampled at 10 kHz, and low pass filtered at 2 kHz using a Digidata 1440 A (Molecular Devices), and analyzed with Clampfit 11 software (Molecular Devices). Solutions were exchanged using a gravity-driven RSC-200 rapid solution changer (Bio-Logic). Ligand solutions were prepared in bath solution.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis of the GABA\u003csub\u003eA\u003c/sub\u003e receptor electrophysiology data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analysis was performed using GraphPad Prism 10.6.0 software (GraphPad Software, Inc., La Jolla, CA). Data are expressed as mean valuesstandard error of the mean of at least four recordings from independent cells. Two-tailed Welch\u0026rsquo;s t-tests were used. A \u003cem\u003ep\u003c/em\u003e-value of \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eGeneral procedure for synthesis of oxazolecarboxamides and isoxazolecarboxamides\u003c/em\u003e\u003cem\u003eand spectral data of synthesized intermediates see Supplementary Materials.\u003c/em\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAUTHORS AND AFFILIATIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Pharmaceutical Chemistry, University of California, San Francisco, CA, 94158, USA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMatthew N. McCarroll, Xilin Gu, Elizabeth Sisko, Lain X. Pierce, Ralph Zhang, Ricardo Da Luz, Jason K. Sello, Cole Helsell, Douglas Myers-Turnbull, Brian K. Shoichet\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Chemistry, Brown University, Providence, RI, 02912, USA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmanda Dombroski\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Anesthesia and Perioperative Care University of California, San Francisco, California 94143, USA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJarret A.P. Weinrich, Michael P. Bokoch\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Anatomy, University of California, San Francisco, California 94143, USA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJarret A.P. Weinrich, Allan I. Basbaum, Madison Jewell, Karnika Bhardwaj, Sian Rodriguez-Rosado,\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Neurobiology, University of California, San Diego, CA, 92093, USA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRyan E. Hibbs, Alyssa Marinas, Jinfeng Teng, Megan J. Larmore\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitute for Neurodegenerative Diseases, University of California, San Francisco, California 94143, USA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDave Kokel, Jack C. Taylor, Amanda Carbajal, Reid Kinser\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitute for Molecule Bioscience, University of Queensland, St Lucia QLD 4072, Australia\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJennifer Deuis, Asa Andersson, Irina Vetter\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBlavatnik Institute of Neurobiology, Harvard Medical School, Boston, MA, 02115, USA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBruce Palmer Bean\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, M.N.N., D.K., J.K.S., J.A.P.W.; Methodology M.N.M., J.A.P.W., R.E.H., A.H.M., M.L., Jf.T.; Software, M.N.M., D.M.T., C.H., J.A.P.W., R.K.; Formal Analysis, M.N.M., A.H.M., Jf.T., J.A.P.W., R.K.; Investigation, M.N.M, J.A.P.W., X.G., A.D., A.H.M., E.S., Jf.T., J.C.T., L.X.P., C.A., A.C., R.D.L., R.Z., M.J., K.B., S.R.R., J.D., A.A.; Resources, M.N.M, D.K., J.K.S., R.E.H, I.V., B.P.B., A.I.B.; Data Curation, M.N.M, D.M.T., C.H., A.H.M., Jf.T.; Writing - Original Draft, M.N.M., J.A.P.W., A.H.M.; Writing - Reviewing and Editing, M.N.M, R.E.H., J.A.P.W., M.B., A.I.B., A.H.M., J.K.S.; Visualization, M.N.M., J.A.P.W., A.H.M., Jf.T.; Supervision, M.N.M., R.E.H., D.K., A.I.B., B.P.B., I.V., B.K.S., J.K.S.; Project Administration, M.N.M., A.I.B., R.E.H., J.K.S.; Funding Acquisition, M.N.M, D.K., A.I.B., B.K.S., B.P.B., I.V., M.B., R.E.H., J.K.S.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCORRESPONDING AUTHORS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Matthew N. McCarroll ([email protected]) Ryan E. Hibbs ([email protected]), Allan I. Basbaum ([email protected]), and Jason K. Sello\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e([email protected]).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Louie Ramos for exceptional animal care and husbandry. ELISA plate reader analysis was performed at the UCSF center for advanced technology, supported by UCSF PBBR, RRP IMIA, and NIH 1S10OD028511-01 grants. This work was supported by the Defense Advanced Research Projects Agency (DARPA) grant: W911NF-24-2-0109 (MNM, JKS, AIB, REH, BKS MPB) and the US National Institutes of Health (NIH) grants: DP1DA058350 (MNM), AI123400-03 (JKS), R01AA022583 (DK), F31NS145564 and T32GM007752 (AHM), and R01DA047325 (REH). This work was also supported by the Chan Zuckerberg Initiative (JKS), an advised fund of the Silicon Valley Community Foundation and Ono pharmaceuticals (JKS).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCOMPETING INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe University of California San Francisco and the Chan Zuckerberg Biohub have filed a patent application for compounds related to the new anesthetics with analgesic potential. Drs. McCarroll, Sello, Kokel, Basbaum, and Weinrich are named as co-inventors. Other authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll motion index time series, behavioral screening data, and cryo-EM maps and models are available for peer review upon request. Cryo-EM map and model will be deposited in the electron microscopy data bank and protein data bank, respectively, upon acceptance of the manuscript for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCODE AVAILABILITY\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll code is available upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSUPPORTING INFORMATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe supporting information includes Supplementary Figures showing selected chemical structures, mouse pharmacokinetics, stereoisomer behavioral analysis, electrophysiology representative traces, electrophysiology at mutant GABA\u003csub\u003eA\u003c/sub\u003e receptors, Cryo-EM data processing workflow, cortisol level analysis, PDSP data and voltage gated sodium channel electrophysiology. Also included are Supplementary Tables with chemical names, SMILES, SAR information, and extended methods and spectral Analysis of oxazoles and isoxazoles synthesis.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMcQueen, K. A. K. Anesthesia and the global burden of surgical disease. \u003cem\u003eInt. Anesthesiol. Clin.\u003c/em\u003e \u003cstrong\u003e48\u003c/strong\u003e, 91\u0026ndash;107 (2010).\u003c/li\u003e\n\u003cli\u003eGu, W.-J., Wang, F., Tang, L. \u0026amp; Liu, J.-C. Single-dose etomidate does not increase mortality in patients with sepsis: a systematic review and meta-analysis of randomized controlled trials and observational studies. \u003cem\u003eChest\u003c/em\u003e \u003cstrong\u003e147\u003c/strong\u003e, 335\u0026ndash;346 (2015).\u003c/li\u003e\n\u003cli\u003eBruder, E. A., Ball, I. M., Ridi, S., Pickett, W. \u0026amp; Hohl, C. 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Using a bespoke 96-well-plate format platform enabling high-throughput imaging of larval zebrafish behavior, we screened 12,000 compounds and identified an isoxazole chemotype that phenocopies the intravenous anesthetics etomidate and propofol. Its optimization via medicinal chemistry yielded a novel anesthetic that we call nidradine. This anesthetic is efficacious in both zebrafish and mice and lacks the problematic adrenal suppression characteristic of etomidate. Mechanistic studies via electrophysiology and structural biology revealed that nidradine, like etomidate and propofol, is a positive allosteric modulator of the GABA\u003csub\u003eA\u003c/sub\u003e receptor. Remarkably, behavioral assays in mice demonstrate that nidradine differs from most general anesthetics in that it also produces analgesia.\u003c/p\u003e","manuscriptTitle":"Phenotypic discovery of GABAA receptor-mediated general anesthetics with analgesic activity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-13 10:27:47","doi":"10.21203/rs.3.rs-8341587/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"21d4c5f7-7b3c-4d27-8557-8eb50fde924e","owner":[],"postedDate":"January 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":61040088,"name":"Biological sciences/Drug discovery/Drug screening/Phenotypic screening"},{"id":61040089,"name":"Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy"}],"tags":[],"updatedAt":"2026-03-06T08:31:06+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-13 10:27:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8341587","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8341587","identity":"rs-8341587","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}