Identification of 1,3,8-Triazaspiro[4.5]Decane-2,4-Dione Derivatives as a Novel δ Opioid Receptor-Selective Agonist Chemotype.

δ opioid receptors (DORs) hold potential as a target for neurologic and psychiatric disorders, yet no DOR agonist has proven efficacious in critical phase II clinical trials. The exact reasons for the failure to produce quality drug candidates for the DOR are unclear. However, it is known that certain DOR agonists can induce seizures and exhibit tachyphylaxis. Several studies have suggested that those adverse effects are more prevalent in delta agonists that share the (+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC80)/4-[(αR*)-α-((2S*,5R*)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-hydroxybenzyl]-N,N-diethylbenzamide chemotype. There is a need to find novel lead candidates for drug development that have improved pharmacological properties to differentiate them from the current failed delta agonists. Our objective in this study was to identify novel DOR agonists. We used a ß-arrestin assay to screen a small G-protein coupled receptors (GPCR)-focused chemical library. We identified a novel chemotype of DOR agonists that appears to bind to the orthosteric site based of docking and molecular dynamic simulation. The most potent agonist hit compound is selective for the DOR over a panel of 167 other GPCRs, is slightly biased toward G-protein signaling and has anti-allodynic efficacy in a complete Freund's adjuvant model of inflammatory pain in C57BL/6 male and female mice. The newly discovered chemotype contrasts with molecules like SNC80 that are highly efficacious ß-arrestin recruiters and may suggest this novel class of DOR agonists could be expanded on to develop a clinical candidate drug. SIGNIFICANCE STATEMENT: δ opioid receptors are a clinical target for various neurological disorders, including migraine and chronic pain. Many of the clinically tested delta opioid agonists share a single chemotype, which carries risks during drug development. Through a small-scale high-throughput screening assay, this study identified a novel δ opioid receptor agonist chemotype, which may serve as alternative for the current analgesic clinical candidates.

ClickArr: a novel, high-throughput assay for evaluating ß-arrestin isoform recruitment.

Background: Modern methods for quantifying signaling bias at G protein-coupled receptors (GPCRs) rely on using a single ß-arrestin isoform. However, it is increasingly appreciated that the two ß-arrestin isoforms have unique roles, requiring the ability to assess ß-arrestin isoform preference. Thus, methods are needed to efficiently screen the recruitment of both ß-arrestin isoforms as they compete for a target GPCR in cells. Methods: We used molecular cloning to develop fusion proteins of the δ-opioid receptor (δOR), ß-arrestin 1, and ß-arrestin 2 to fragments of click beetle green and click beetle red luciferases. In this assay architecture, recruitment of either ß-arrestin 1 or 2 to the δOR generates a spectrally distinct bioluminescent signal, allowing us to co-transfect all three constructs into cells prior to agonist challenge. Results: We demonstrate that our new assay, named "ClickArr," is a live-cell assay that simultaneously reports the recruitment of both ß-arrestin isoforms as they compete for interaction with the δOR. We further find that the partial δOR agonist TAN67 has a significant efficacy bias for ß-arrestin 2 over ß-arrestin 1 when recruitment is normalized to the reference agonist leu-enkephalin. We confirm that ClickArr reports this bias when run either as a high-throughput endpoint or high-throughput kinetic assay, and cross-validate this result using the PathHunter assay, an orthogonal commercial assay for reporting ß-arrestin recruitment to the δOR. Conclusion: Our results suggest that agonist:GPCR complexes can have relative ß-arrestin isoform bias, a novel signaling bias that may potentially open up a new dimension for drug development.

Modified Akuamma Alkaloids with Increased Potency at the Mu-opioid Receptor.

Akuammine (1) and pseudoakuammigine (2) are indole alkaloids found in the seeds of the akuamma tree (Picralima nitida). Both alkaloids are weak agonists of the mu opioid receptor (µOR); however, they produce minimal effects in animal models of antinociception. To probe the interactions of 1 and 2 at the opioid receptors, we have prepared a collection of 22 semisynthetic derivatives. Evaluation of this collection at the µOR and kappa opioid receptor (κOR) revealed structural-activity relationship trends and derivatives with improved potency at the µOR. Most notably, the introduction of a phenethyl moiety to the N1 of 2 produces a 70-fold increase in potency and a 7-fold increase in selectivity for the µOR. The in vitro potency of this compound resulted in increased efficacy in the tail-flick and hot-plate assays of antinociception. The improved potency of these derivatives highlights the promise of exploring natural product scaffolds to probe the opioid receptors.

Opportunities and Challenges for In Silico Drug Discovery at Delta Opioid Receptors.

The delta opioid receptor is a Gi-protein-coupled receptor (GPCR) with a broad expression pattern both in the central nervous system and the body. The receptor has been investigated as a potential target for a multitude of significant diseases including migraine, alcohol use disorder, ischemia, and neurodegenerative diseases. Despite multiple attempts, delta opioid receptor-selective molecules have not been translated into the clinic. Yet, the therapeutic promise of the delta opioid receptor remains and thus there is a need to identify novel delta opioid receptor ligands to be optimized and selected for clinical trials. Here, we highlight recent developments involving the delta opioid receptor, the closely related mu and kappa opioid receptors, and in the broader area of the GPCR drug discovery research. We focus on the validity and utility of the available delta opioid receptor structures. We also discuss the increased ability to perform ultra-large-scale docking studies on GPCRs, the rise in high-resolution cryo-EM structures, and the increased prevalence of machine learning and artificial intelligence in drug discovery. Overall, we pose that there are multiple opportunities to enable in silico drug discovery at the delta opioid receptor to identify novel delta opioid modulators potentially with unique pharmacological properties, such as biased signaling.

Identification of a Novel Delta Opioid Receptor Agonist Chemotype with Potential Negative Allosteric Modulator Capabilities.

The δ-opioid receptor (δOR) holds great potential as a therapeutic target. Yet, clinical drug development, which has focused on δOR agonists that mimic the potent and selective tool compound SNC80 have largely failed. It has increasingly become apparent that the SNC80 scaffold carries with it potent and efficacious ß-arrestin recruitment. Here, we screened a relatively small (5120 molecules) physical drug library to identify δOR agonists that underrecruit ß-arrestin, as it has been suggested that compounds that efficaciously recruit ß-arrestin are proconvulsant. The screen identified a hit compound and further characterization using cellular binding and signaling assays revealed that this molecule (R995045, compound 1) exhibited ten-fold selectivity over µ- and κ-opioid receptors. Compound 1 represents a novel chemotype at the δOR. A subsequent characterization of fourteen analogs of compound 1, however did not identify a more potent δOR agonist. Computational modeling and in vitro characterization of compound 1 in the presence of the endogenous agonist leu-enkephalin suggest compound 1 may also bind allosterically and negatively modulate the potency of Leu-enkephalin to inhibit cAMP, acting as a 'NAM-agonist' in this assay. The potential physiological utility of such a class of compounds will need to be assessed in future in vivo assays.

Modulating ß-arrestin 2 recruitment at the δ- and µ-opioid receptors using peptidomimetic ligands.

µ-Opioid receptor agonists provide potent and effective acute analgesia; however, their therapeutic window narrows considerably upon repeated administration, such as required for treating chronic pain. In contrast, bifunctional µ/δ opioid agonists, such as the endogenous enkephalins, have potential for treating both acute and chronic pain. However, enkephalins recruit ß-arrestins, which correlate with certain adverse effects at µ- and δ-opioid receptors. Herein, we identify the C-terminus of Tyr-ψ[(Z)CF[double bond, length as m-dash]CH]-Gly-Leu-enkephalin, a stable enkephalin derivative, as a key site to regulate bias of both δ- and µ-opioid receptors. Using in vitro assays, substitution of the Leu5 carboxylate with amides (NHEt, NMe2, NCyPr) reduced ß-arrestin recruitment efficacy through both the δ-opioid and µ-opioid, while retaining affinity and cAMP potency. For this series, computational studies suggest key ligand-receptor interactions that might influence bias. These findings should enable the discovery of a range of tool compounds with previously unexplored biased µ/δ opioid agonist pharmacological profiles.

Engineering Quick- and Long-acting Naloxone Delivery Systems for Treating Opioid Overdose.

PURPOSE: Opioids have been the main factor for drug overdose deaths in the United States. Current naloxone delivery systems are effective in mitigating the opioid effects only for hours. Naloxone-loaded poly(lactide-co-glycolide) (PLGA) microparticles were prepared as quick- and long-acting naloxone delivery systems to extend the naloxone effect as an opioid antidote. METHODS: The naloxone-PLGA microparticles were made using an emulsification solvent extraction approach with different formulation and processing parameters. Two PLGA polymers with the lactide:glycolide (L:G) ratios of 50:50 and 75:25 were used, and the drug loading was varied from 21% to 51%. Two different microparticles of different sizes with the average diameters of 23 µm and 50 µm were produced using two homogenization-sieving conditions. All the microparticles were critically characterized, and three of them were evaluated with ß-arrestin recruitment assays. RESULTS: The naloxone encapsulation efficiency (EE) was in the range of 70-85%. The EE was enhanced when the theoretical naloxone loading was increased from 30% to 60%, the L:G ratio was changed from 50:50 to 75:25, and the average size of the particles was reduced from 50 µm to 23 µm. The in vitro naloxone release duration ranged from 4 to 35 days. Reducing the average size of the microparticles from 50 µm to 23 µm helped eliminate the lag phase and obtain the steady-state drug release profile. The cellular pharmacodynamics of three selected formulations were evaluated by applying DAMGO, a synthetic opioid peptide agonist to a µ-opioid receptor, to recruit ß-arrestin 2. CONCLUSIONS: Naloxone released from the three selected formulations could inhibit DAMGO-induced ß-arrestin 2 recruitment. This indicates that the proposed naloxone delivery system is adequate for opioid reversal during the naloxone release duration.