Decoding the Molecular Mechanisms of a Kappa Opioid Receptor Selective Negative Allosteric Modulator

About This Grant

Project Summary The widespread prescription and illicit use of opioids over the past several decades has created a public health epidemic known as the "opioid crisis." Synthetic opioids acting as Mu Opioid receptor (µOR) agonists have demonstrated effectiveness as pain management therapeutics yet have high addiction and overdose potential in addition to fatal side effects which have driven this ongoing crisis. Towards mitigating this crisis, naloxone is the primary therapeutic used to reverse opioid overdose, yet its use can precipitate withdrawal symptoms. Kappa Opioid receptor (κOR) has emerged as a promising target within the opioid receptor family, as selective antagonists have shown to counter emotional states and behaviors associated with withdrawal in preclinical studies. However, the utilization of κOR-targeted therapeutics has been limited by gaps in our understanding of receptor signaling and inhibition, resulting in unintended effects and unsuccessful clinical trials. As such allosteric modulators have gained attention due to their potential to provide a more fine-tuned way to modulate κOR signaling. As allosteric modulators occupy binding pockets that are distinct from the highly conserved orthosteric site, negative allosteric modulators (NAMs) can be drastically more selective than conventional antagonists. Furthermore, NAMs, by definition, enable residual endogenous signaling, which significantly broadens their therapeutic window. Until recently, no κOR-selective NAMs have been characterized. This proposal seeks to address these gaps by investigating the molecular mechanisms of a recently discovered NAM at κOR. By elucidating the structural and functional basis of κOR modulation via negative allosteric modulation, this research will contribute to the framework towards enhancing the clinical utility of κOR antagonists. To explore this fundamental aspect of κOR inhibition, we will delve into the molecular aspects of negative allosteric modulators (NAMs) in the context of our recent findings. Our first ever cryoEM structures of a novel conformational state of an inhibitor bound receptor κOR: G protein complex with inverse agonists JDTic, GB18 and norBNI highlight that inhibition is driven by highly complex mechanisms including the allosteric modulation of receptor-G protein affinities or even G protein nucleotide exchange which can result from stabilizing different states along the receptor activation pathway. Similar to orthosteric antagonists, NAMs may also stabilize distinct receptor states along the receptor activation pathway, promoting the inhibitory effects of orthosteric ligands. Taken together, this highlights the importance of investigating novel conformational states to provide a more complete picture of opioid receptor signaling and pharmacology. Using cryo-electron microscopy (cryoEM), in vitro, and ex vivo techniques to investigate inhibition via a κOR-selective NAM we will test the hypothesis that signaling inhibition arises from structural alterations that modulate G-protein dynamics. Through an extensive 1) Pharmacological Characterization of a κOR-selective NAM and 2) Structural Analysis of NAM-Induced Conformational States and Their Impact on Downstream Signaling we will link NAM-induced conformational changes at the atomic level to distinct pharmacological profiles. The results of this study will support the development of more effective therapeutic strategies at κOR and across the opioid receptor family.