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Background and Purpose: Orthosteric agonists and positive allosteric modulators (PAMs) of the α7 nicotinic ACh receptor (nAChR) represent novel therapeutic approaches for pain modulation. Moreover, compounds with dual function as allosteric agonists and PAMs, known as ago-PAMs, add further regulation of receptor function.Experimental Approach: Initial studies examined the α7 ago-PAM, GAT107, in the formalin, complete Freund's adjuvant (CFA), LPS inflammatory pain models, the chronic constriction injury neuropathic pain model and the tail flick and hot plate acute thermal nociceptive assays. Additional studies examined the locus of action of GAT107 and immunohistochemical markers in the dorsal horn of the spinal cord in the CFA model.Key Results: Complementary pharmacological and genetic approaches confirmed that the dose-dependent antinociceptive effects of GAT107 were mediated through α7 nAChR. However, GAT107 was inactive in the tail flick and hot plate assays. In addition, GAT107 blocked conditioned place aversion elicited by acetic acid injection. Furthermore, intrathecal, but not intraplantar, injections of GAT107 reversed nociception in the CFA model, suggesting a spinal component of action. Immunohistochemical evaluation revealed an increase in the expression of astrocyte-specific glial fibrillary acidic protein and phosphorylated p38MAPK within the spinal cords of mice treated with CFA, which was attenuated by intrathecal GAT107 treatment. Importantly, GAT107 did not elicit motor impairment and continued to produce antinociceptive effects after subchronic administration in both phases of the formalin test.Conclusions and Implications: Collectively, these results provide the first proof of principle that α7 ago-PAMs represent an effective pharmacological strategy for treating inflammatory and neuropathic pain. [ABSTRACT FROM AUTHOR]

Undesirable side effects associated with orthosteric agonists/antagonists of cannabinoid 1 receptor (CB1R), a tractable target for treating several pathologies affecting humans, have greatly limited their translational potential. Recent discovery of CB1R negative allosteric modulators (NAMs) has renewed interest in CB1R by offering a potentially safer therapeutic avenue. To elucidate the CB1R allosteric binding motif and thereby facilitate rational drug discovery, we report the synthesis and biochemical characterization of first covalent ligands designed to bind irreversibly to the CB1R allosteric site. Either an electrophilic or a photoactivatable group was introduced at key positions of two classical CB1R NAMs: Org27569 (1) and PSNCBAM-1 (2). Among these, 20 (GAT100) emerged as the most potent NAM in functional assays, did not exhibit inverse agonism, and behaved as a robust positive allosteric modulator of binding of orthosteric agonist CP55,940. This novel covalent probe can serve as a useful tool for characterizing CB1R allosteric ligand-binding motifs. [ABSTRACT FROM AUTHOR]

Anexpeditious microwave-assisted synthesis of 4BP-TQS, its enantiomericseparation, and their functional evaluation is reported. Electrophysiologicalcharacterization in Xenopusoocytesrevealed that activity exclusively resided in the (+)-enantiomer 1b(GAT107) and (−)-enantiomer 1adidnot affect its activity when coapplied. X-ray crystallography studiesrevealed the absolute stereochemistry of 1bto be 3aR,4S,9bS. 1brepresents the most potent ago-PAM of α7 nAChRs availableto date and is considered for further in vivo evaluation. [ABSTRACT FROM AUTHOR]

Supplemental oxygen therapy with supraphysiological concentrations of oxygen (hyperoxia; >21% O2) is a life-saving intervention for patients experiencing respiratory distress. However, prolonged exposure to hyperoxia can compromise bacterial clearance processes, due to oxidative stress-mediated impairment of macrophages, contributing to the increased susceptibility to pulmonary infections. This study reports that the activation of the α7 nicotinic acetylcholine receptor (α7nAChR) with the delete allosteric agonistic-positive allosteric modulator, GAT107, decreases the bacterial burden in mouse lungs by improving hyperoxia-induced lung redox imbalance. The incubation of RAW 264.7 cells with GAT107 (3.3 µM) rescues hyperoxia-compromised phagocytic functions in cultured macrophages, RAW 264.7 cells, and primary bone marrow-derived macrophages. Similarly, GAT107 (3.3 µM) also attenuated oxidative stress in hyperoxia-exposed macrophages, which prevents oxidation and hyper-polymerization of phagosome filamentous actin (F-actin) from oxidation. Furthermore, GAT107 (3.3 µM) increases the (1) activity of superoxide dismutase 1; (2) activation of Nrf2 and (3) the expression of heme oxygenase-1 (HO-1) in macrophages exposed to hyperoxia. Overall, these data suggest that the novel α7nAChR compound, GAT107, could be used to improve host defense functions in patients, such as those with COVID-19, who are exposed to prolonged periods of hyperoxia. [ABSTRACT FROM AUTHOR]

The direct blockade of CB 1 cannabinoid receptors produces therapeutic effects as well as adverse side-effects that limit their clinical potential. CB 1 negative allosteric modulators (NAMs) represent an indirect approach to decrease the affinity and/or efficacy of orthosteric cannabinoid ligands or endocannabinoids at CB 1. We recently reported that GAT358, a CB 1 -NAM, blocked opioid-induced mesocorticolimbic dopamine release and reward via a CB 1 -allosteric mechanism of action. Whether a CB 1 -NAM dampens opioid-mediated therapeutic effects such as analgesia or alters other unwanted opioid side-effects remain unknown. Here, we characterized the effects of GAT358 on nociceptive behaviors in the presence and absence of morphine in male rats. We examined the impact of GAT358 on formalin-evoked pain behavior and Fos protein expression, a marker of neuronal activation, in the lumbar spinal cord. We also assessed the impact of GAT358 on morphine-induced slowing of colonic transit, tolerance, and withdrawal behaviors in male mice. GAT358 attenuated morphine antinociceptive tolerance without blocking acute antinociception and reduced morphine-induced slowing of colonic motility without impacting fecal boli production. GAT358 also produced antinociception in the presence and absence of morphine in the formalin model of inflammatory nociception and reduced the number of formalin-evoked Fos protein-like immunoreactive cells in the lumbar spinal cord. Finally, GAT358 mitigated the somatic signs of naloxone-precipitated, but not spontaneous, opioid withdrawal following chronic morphine dosing. Our results support the therapeutic potential of CB 1 -NAMs as novel drug candidates aimed at preserving opioid-mediated analgesia while preventing their unwanted side-effects. Our studies also uncover previously unrecognized antinociceptive properties associated with an arrestin-biased CB 1 -NAM. [Display omitted] • CB 1 negative allosteric modulator (NAM) GAT358 attenuated morphine tolerance. • GAT358 reduced morphine-induced slowing of colonic motility but not fecal production. • GAT358 was antinociceptive for formalin pain alone and when combined with morphine. • GAT358 reduced formalin-evoked Fos protein expression in the lumbar spinal cord. • GAT358 mitigated naloxone precipitated withdrawal after chronic morphine dosing. [ABSTRACT FROM AUTHOR]

Blockade of cannabinoid type 1 (CB 1)-receptor signaling decreases the rewarding properties of many drugs of abuse and has been proposed as an anti-addiction strategy. However, psychiatric side-effects limit the clinical potential of orthosteric CB 1 antagonists. Negative allosteric modulators (NAMs) represent a novel and indirect approach to attenuate CB 1 signaling by decreasing affinity and/or efficacy of CB 1 ligands. We hypothesized that a CB 1 -NAM would block opioid reward while avoiding the unwanted effects of orthosteric CB 1 antagonists. GAT358, a CB 1 -NAM, failed to elicit cardinal signs of direct CB 1 activation or inactivation when administered by itself. GAT358 decreased catalepsy and hypothermia but not antinociception produced by the orthosteric CB 1 agonist CP55,940, suggesting that a CB 1 -NAM blocked cardinal signs of CB 1 activation. Next, GAT358 was evaluated using in vivo assays of opioid-induced dopamine release and reward in male rodents. In the nucleus accumbens shell, a key component of the mesocorticolimbic reward pathway, morphine increased electrically-evoked dopamine efflux and this effect was blocked by a dose of GAT358 that lacked intrinsic effects on evoked dopamine efflux. Moreover, GAT358 blocked morphine-induced reward in a conditioned place preference (CPP) assay without producing reward or aversion alone. GAT358-induced blockade of morphine CPP was also occluded by GAT229, a CB 1 positive allosteric modulator (CB 1 -PAM), and absent in CB 1 -knockout mice. Finally, GAT358 also reduced oral oxycodone (but not water) consumption in a two-bottle choice paradigm. Our results support the therapeutic potential of CB 1 -NAMs as novel drug candidates aimed at preventing opioid reward and treating opioid abuse while avoiding unwanted side-effects. [Display omitted] • CB 1 negative allosteric modulator GAT358 does not produce cannabimimetic effects. • GAT358 decreases CB 1 agonist-induced catalepsy, hypothermia but not antinociception. • GAT358 eliminates morphine-induced phasic dopamine release in the NAc shell. • GAT358 blocks conditioned place preference to morphine via a CB 1 mechanism. • GAT358 reduces oral oxycodone but not water intake in a two-bottle choice assay. [ABSTRACT FROM AUTHOR]