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目前的阿片类药物危机，各种芬太尼类似物正出现在世界各地的娱乐毒品市场上。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，我们比较了与阿片类药物过量有关的两种结构相关的芬太尼类似物：环丙基芬太尼和戊基芬太尼的药理学。环丙基芬太尼在芬太尼支架上的羰基上有一个三碳环，而戊基芬太尼在相同的位置上有一个四碳链。体外实验表明，环丙基芬太尼是一种完全激动剂(EC(50)=8.6 nM，%Emax=113%)，其药效与芬太尼相似(EC(50)=10.3 nM，%Emax=113%)。反之，戊芬太尼在静息状态下为部分激动剂(EC(50)=179.8 nM，%Emax=60%)。在评估MOR介导的HEK细胞中的β-arrestin募集的试验中也发现了类似的结果。在雄性CD-1小鼠的体内研究表明，两种芬太尼类似物均可引起纳洛酮可逆的抗伤害作用和呼吸抑制，但环丙基芬太尼的抗伤害作用是后者的100倍(ED(50)=0.04 mg/kg，S，C.)高于丙戊基芬太尼(ED(50)=4.0 mg/kg，S，C.C.)。分子模拟结果表明，戊基芬太尼的烷基链不能很好地被MOR的活性状态所调节，并且可能使受体转变为非活性状态，从而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，环丙基芬太尼与丙戊基芬太尼相比，出现不良反应的风险要大得多。此外，总结的发现可能为设计减少副作用的治疗性阿片类药物提供线索。 虽然芬太尼类似物历史上一直主导着NSO市场，但现在正在发生向非芬太尼化合物的转变。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、异硝氮烯、溴吗啡和N-去甲基异硝氮烯，并与吗啡和芬太尼进行了比较。使用大鼠脑组织中的阿片受体结合分析和监测Forsklin刺激的表达人MOR的细胞中cAMP积累的方法，对化合物进行了体外测试。将化合物皮下注射给雄性SD大鼠，测量热板抗伤害性反应、过敏性麻痹评分和体温变化。受体结合结果显示，所有化合物都有很高的MOR选择性，MOR亲和力与吗啡和芬太尼(即NM)相当。在cAMP实验中，所有药物都作为全效MOR激动剂，但与其他药物(即NM)相比，硝氮烯类似物具有更大的功能潜力(即Pm)。当用在老鼠身上时，所有化合物都会引起阿片样抗伤害性感觉、过敏性癫痫和体温变化，但硝氮烯的作用最强。与芬太尼类似，与吗啡相比，硝基氮烯的体内效应起效和衰退更快。体内诱发抗伤害性和惊厥的能力(即ED(50)S)与体外功能能力(即EC(50)S)相关，但与MOR的结合亲和力(即K(I)S)无关。总体而言，我们的发现表明，非芬太尼NSO对那些使用阿片类药物的人构成严重危险。需要继续保持警惕，以便在合成阿片类药物出现在秘密药物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.