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

非法制造的芬太尼正在推动当前的阿片类药物危机，各种芬太尼类似物正在出现在全球娱乐性药物市场上。为了评估芬太尼类似物带来的潜在健康风险，有必要了解这些化合物的结构-活性关系。在这里，我们比较了两种结构相关的芬太尼类似物在阿片类药物过量：环丙基芬太尼和戊酰芬太尼的药理学。环丙基芬太尼具有连接到芬太尼支架上的羰基的三碳环，而戊酰基芬太尼在相同位置具有四碳链。检查CHO细胞中μ阿片受体（莫尔）与G蛋白偶联的体外测定显示，环丙基芬太尼是完全激动剂（EC（50）= 8.6 nM，%Emax=113%），其效力和功效与芬太尼（EC（50）= 10.3 nM，%Emax=113%）相似。相比之下，戊酰芬太尼在莫尔比下是部分激动剂（EC（50）=179.8nM，%Emax=60%）。在评估HEK细胞中MOR介导的β-抑制蛋白募集的测定中发现了类似的结果。在雄性CD-1小鼠中的体内研究表明，两种芬太尼类似物均诱导纳洛酮可逆的抗伤害感受和呼吸抑制，但环丙基芬太尼作为抗伤害感受剂的效力是其100倍（艾德（50）= 0.04 mg/kg，s. c.）戊酰芬太尼（艾德（50）=4.0mg/kg，s. c.）。分子模拟结果表明，戊酰芬太尼的烷基链不能很好地适应莫尔的活性状态，并可能向非活性状态的受体转变，将芬太尼支架转化为部分激动剂。综上所述，我们的结果表明，环丙基芬太尼与戊基芬太尼相比，具有更大的不良反应风险。此外，总结的发现可能为设计具有减少不良副作用的治疗性阿片类药物提供线索。 虽然芬太尼类似物在历史上主导了NSO市场，但现在正在向非芬太尼化合物转变。我们研究了结构不同的非芬太尼NSO的神经药理学，包括U-47700，isotonitazene，溴啡，和N-去乙基isotonitazene，相比吗啡和芬太尼。使用大鼠脑组织中的阿片样物质受体结合测定和通过监测毛喉素刺激的cAMP在表达人莫尔的细胞中的积累，在体外测试化合物。向雄性Sprague-Dawley大鼠皮下施用化合物，并测量热板抗伤害感受、僵住症评分和体温变化。受体结合结果显示对所有化合物的高莫尔选择性，其中莫尔亲和力与吗啡和芬太尼的亲和力相当（即，nM）。在cAMP测定中，所有药物均作为全功效莫尔激动剂，但硝氮类似物具有更大的功能效力（即，pM）与其他药物相比（即，nM）。当给予大鼠时，所有化合物均诱导阿片样抗伤害感受、僵住症和体温变化，但硝氮类化合物最有效。与芬太尼类似，与吗啡相比，硝氮类药物的体内效应起效和下降更快。诱导抗伤害感受和僵住症的体内效力（即，艾德（50）s）与体外功能效力（即，EC（50）s）但不具有结合亲和力（即，K（i）s）。总的来说，我们的研究结果表明，非芬太尼NSO对使用阿片类药物的人构成严重危险。需要继续保持警惕，以便在合成类阿片出现在秘密毒品市场时对其进行识别和定性。

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.