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

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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.