Biasing of G -signaling with Small Molecules as a Novel Therapeutic Approach to Improve Opioid-mediated Antinociception

Abstract

Opioid analgesics are widely used as a treatment option for pain management and relief. However, the misuse of opioid analgesics has contributed to the current opioid epidemic in the United States. The National Institute of Drug Abuse (NIDA) reports that in 2021, more than 106,000 deaths were attributed to opioid overdoses, and an estimated 16,000 involved a prescription opioid. Prescribed opioids such as morphine, codeine, oxycodone, and fentanyl are primarily used in the clinic to treat pain or during medical procedures. Nevertheless, the rewarding and reinforcing effects of opioids have led to patient misuse of prescribed opioids and increased their illicit use and distribution. Patients who take prescribed opioids report adverse effects like constipation, nausea, dizziness, itchiness, respiratory depression, and development of tolerance to the analgesic effects. Scientists in the drug discovery field aim to identify molecular targets that could separate the therapeutic effects of opioid analgesics from the detrimental side effects and improve pharmacological strategies to relieve pain. This thesis explores the application of targeting G protein βγ subunit signaling as a novel therapeutic approach to increase opioid-induced analgesia and decreases the development of opioid tolerance. To bias Gβγ-signaling, we used gallein, a small molecule that binds to the Gβγ subunit downstream of opioid receptors. We proposed that when gallein is bound, Gβγ promotes pro-analgesic signaling but cannot interact with signaling pathways that oppose the analgesic response. First, we investigated activation of phospholipase Cβ (PLCβ) signaling downstream of the µ-opioid receptor (MOR). We hypothesized that PLCβ signaling opposes the opioid analgesic response and that activation of PLCβ requires Gβγ signaling downstream of MOR and coincident Gαq signaling. We assessed this model in cellular, ex vivo, and in vivo assays. Using a fluorescent biosensor, we tested the coactivation of PLCβ by MOR and Gαq-coupled receptors in HEK-293 cells. Then, MOR-dependent inhibition of neurotransmission was tested in GABA-ergic synapses in the mouse periaqueductal grey (PAG) in presence of a Gβγ-inhibitor (gallein) or Gαq-inhibitor. And lastly, we evaluated the effects of gallein and Gαq inhibitor treatment on morphine-dependent antinociception. Our results show that coincident activation of MOR and Gαq-coupled receptors produces synergistic activation of PLCβ in HEK-293 cells. In ex vivo and in vivo experiments, treatment with either gallein or a Gαq-inhibitor increased DAMGO-mediated inhibition of GABA-release and increased morphine-mediated antinociception. We continued exploring the therapeutic potential of biasing Gβγ-signaling and its application to chronic morphine treatment in vivo. We hypothesized that biasing Gβγ-signaling with gallein could prevent activation of regulatory signaling pathways that result in opioid tolerance. We tested gallein using two paradigms for treatment: administration during the development of opioid tolerance and administration after tolerance is developed. Our results showed that gallein cotreatment during repeated administration of morphine decreased opioid tolerance development, and gallein treatment in an opioid-tolerant state enhanced the potency of morphine. Additionally, our data showed that PLCβ is necessary for gallein’s potentiating effects in an opioid-tolerant state but not in preventing the development of tolerance. Overall, we propose that biasing Gβγ-signaling could translate into a novel therapeutic approach that improves the analgesic effects of prescription opioids and aids in preventing opioid tolerance. These studies demonstrate that small molecules that target Gβγ-signaling could reduce the need for large opioid doses to treat pain and, therefore, benefit the opioid epidemic.