Structural basis for the functions of dopamine receptors, neurotransmitter transporters, and sigma 1 receptor

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Illicitly manufactured fentanyl is driving the current opioid crisis, and various fentanyl analogs are appearing in recreational drug markets worldwide. To assess the potential health risks posed by fentanyl analogs, it is necessary to understand structure-activity relationships for these compounds. Here we compared the pharmacology of two structurally related fentanyl analogs implicated in opioid overdose: cyclopropylfentanyl and valerylfentanyl. Cyclopropylfentanyl has a three-carbon ring attached to the carbonyl group on the fentanyl scaffold, whereas valerylfentanyl has a four-carbon chain at the same position. In vitro assays examining mu-opioid receptor (MOR) coupling to G proteins in CHO cells showed that cyclopropylfentanyl is a full agonist (EC(50)=8.6nM, %Emax=113%), with potency and efficacy similar to fentanyl (EC(50)=10.3nM, %Emax=113%). By contrast, valerylfentanyl is a partial agonist at MOR (EC(50)=179.8nM, %Emax=60%). Similar results were found in assays assessing MOR-mediated beta-arrestin recruitment in HEK cells. In vivo studies in male CD-1 mice demonstrated that both fentanyl analogs induce naloxone-reversible antinociception and respiratory suppression, but cyclopropylfentanyl is 100-times more potent as an antinociceptive agent (ED(50)=0.04mg/kg, s. c.) than valerylfentanyl (ED(50)=4.0mg/kg, s. c.). Molecular simulation results revealed that the alkyl chain of valerylfentanyl cannot be well accommodated by the active state of MOR and may transition the receptor toward an inactive state, converting the fentanyl scaffold to a partial agonist. Taken together, our results suggest that cyclopropylfentanyl presents much greater risk of adverse effects when compared to valerylfentanyl. Moreover, the summed findings may provide clues to the design of therapeutic opioids with reduced adverse side effects. While fentanyl analogs have historically dominated the NSO market, a shift towards non-fentanyl compounds is now occurring. We examined the neuropharmacology of structurally distinct non-fentanyl NSOs, including U-47700, isotonitazene, brorphine, and N-desethyl isotonitazene, as compared to morphine and fentanyl. Compounds were tested in vitro using opioid receptor binding assays in rat brain tissue and by monitoring forskolin-stimulated cAMP accumulation in cells expressing the human MOR. Compounds were administered subcutaneously to male Sprague-Dawley rats, and hot plate antinociception, catalepsy score, and body temperature changes were measured. Receptor binding results revealed high MOR selectivity for all compounds, with MOR affinities comparable to those of morphine and fentanyl (i.e., nM). All drugs acted as full-efficacy MOR agonists in the cAMP assay, but nitazene analogs had greater functional potencies (i.e., pM) compared to the other drugs (i.e., nM). When administered to rats, all compounds induced opioid-like antinociception, catalepsy, and body temperature changes, but nitazenes were the most potent. Similar to fentanyl, the nitazenes had faster onset and decline of in vivo effects when compared to morphine. In vivo potencies to induce antinociception and catalepsy (i.e., ED(50)s) correlated with in vitro functional potencies (i.e., EC(50)s) but not binding affinities (i.e., K(i)s) at MOR. Collectively, our findings indicate that non-fentanyl NSOs pose grave danger to those individuals who use opioids. Continued vigilance is needed to identify and characterize synthetic opioids as they emerge in clandestine drug markets.