Optimization of novel thioesters as a therapeutic strategy for combating opioid overdoses and abuse

摘要 合成阿片类药物的可获得性和极端效力加剧了美国目前的阿片类药物危机 芬太尼、卡芬太尼和舒芬太尼。这份U-01提案的主要目标是优化我们的硫代铅酯 候选的D-半胱氨酸乙酯(JMS-19)作为一种可行的治疗剂，可诱导快速和持续的逆转 有效的合成阿片类药物引起的阿片类药物引起的呼吸抑制(OIRD，死亡的主要原因) 芬太尼、舒芬太尼和卡芬太尼，在阿片成瘾患者中不会引起戒断症状。这 这项工作将涉及一系列硅胶、体外、体外和体内技术，这些技术将首先优化化学物质。 JMS-19的结构(即产生一种更有效和更化学稳定性的衍生物)，然后确定 先导化合物的药效学、药动学和安全性，为 寻求FDA批准对人体进行研究。药物开发的一个示例流程是从D-CySee到D-Cysee 半胱氨酸甲酯(我们预测的效力大约是D-半胱氨酸的100倍)对S-亚硝基的作用 (SnO)-D-半胱氨酸甲酯(我们已确定SnO-D-半胱氨酸乙酯约为1,000 对S-乙基-D-半胱氨酸甲酯和S-亚磺酸-D-半胱氨酸甲酯的抑制作用 (我们预测它将与SnO-衍生物同等效力，但在化学上更稳定，更具抗性 到酶降解)。我们将提供令人信服的证据，证明JMS-19在没有引发任何后果的情况下逆转了OIRD 使用吗啡或芬太尼治疗的大鼠的任何戒断症状，并将提供关键数据，这强烈地 提示JMS-19和相关硫酯的主要作用机制是通过结合和 失活β-arrestins 1和2。这些分子关键靶点将使我们能够最大限度地优化JMS-19 在硅胶和体外对阿片类药物在自由活动的雄性和雌性大鼠中的最终测试效果。这 将结合分子动力学模拟，优化JMS-19‘S膜的通透性和结合 活性β-arrestins 1和2。这些计算结果将通过实验得到证实 在最终的药效学数据将被共享之前的氢-重离子交换质谱仪 项目的其余部分。体内研究将包括：(1)测试D-半胱氨酸和优化的结构类似物 对照芬太尼、卡芬太尼和舒芬太尼，详细确定测试硫酯是如何逆转阴性的 阿片类药物对自由活动呼吸时机、机械性能及动脉血气化学的影响 雄性和雌性大鼠，以及(2)芬太尼和甲基苯丙胺的组合，通常是致命的(呼吸机- 镇静剂)在人类中的结合。最后，我们将对至少一种药物进行药代动力学研究 优化的D-半胱氨酸衍生物，以建立这些化合物在没有和 芬太尼的存在，以便将硫酯的疗效与OIRD的药理逆转联系起来。 总而言之，我们有信心我们的项目将生产出一系列具有强大能力的活性硫酯 促使强效合成阿片类药物引起的OIRD迅速和持续逆转。

Abstract The current opioid crisis in the US is fueled by the availability and extreme potency of the synthetic opioids fentanyl, carfentanil and sufentanil. The major objective of this U-01 proposal is to optimize our lead thiolester candidate, D-cysteine ethyl ester (JMS-19) as a viable therapeutic agent to elicit a rapid and sustained reversal of opioid-induced respiratory depression (OIRD, a major cause of death) elicited by the potent synthetic opioids fentanyl, sufentanil and carfentanil, without eliciting withdrawal symptoms in opioid-addicted subjects. This work will involve a series of in silico, in vitro, ex vivo, and in vivo techniques that will first optimize the chemical structure of JMS-19 (i.e., produce a derivative with greater efficacy and chemical stability) and then determine the pharmacodynamics, pharmacokinetics, and safety for the lead compound, laying the groundwork for seeking FDA approval for studies in humans. An example flow of drug development will be D-CYSee to D- cysteine methyl ester (that we predict will be approximately 100 times more potent than D-CYSee) to S-nitroso (SNO)-D-cysteine methyl ester (we have established that SNO-D-cysteine ethyl ester is approximately 1,000 times more potent than D-CYSee) to S-ethyl-D-cysteine methyl ester and S-sulfinic-D-cysteine methyl ester (which we predict will be equally potent to the SNO-derivatives but much more chemically stable and resistant to enzymatic degradation). We will provide compelling evidence that JMS-19 reverses OIRD without eliciting any withdrawal symptoms in rats treated with morphine or fentanyl, and will provide key data, which strongly suggest that the primary mechanism of action for JMS-19 and the related thiolesters is by binding to and inactivating β-arrestins 1 and 2. These molecular key targets will allow us to optimize JMS-19 for maximum efficacy in silico and in vitro, for ultimate testing against the opioids in freely-moving male and female rats. This will combine molecular dynamics simulations which will optimize JMS-19's membrane permeability and binding to active β-arrestins 1 and 2. These computational results will be confirmed experimentally by means of hydrogen deuterium exchange mass spectrometry before the final pharmacodynamics data will be shared with the rest of the project. The in vivo studies will involve (1) testing D-CYSee and optimized structural analogues against fentanyl, carfentanil and sufentanil to determine in detail how out test thiolesters reverse the negative effects of the opioid on ventilatory timing and mechanics and arterial blood-gas chemistry in freely-moving male and female rats, and (2) combinations of fentanyl and methamphetamine, and often deadly (ventilatory- depressant) combination in humans. Finally, we will perform pharmacokinetic studies on at least one of the optimized D-CYSee derivatives to establish the temporal distribution of these compounds in the absence and presence of fentanyl in order to relate the efficacy of the thiolesters with the pharmacological reversal of OIRD. In summary, we are confident that our project will produce a series of active thiolesters with potent abilities to elicit rapid and sustained reversal of the OIRD elicited by potent synthetic opioids.