Synthesis and Biological Evaluation of Novel Biased Mu-Opioid Receptor Agonists.

This study explored the potential of a series of PZM21 analogues for pain treatment. Specifically, the hydroxyphenyl ring of PZM21 was replaced with a naphthyl ring, the thienyl ring was substituted with either a phenyl ring or furan rings, and the essential dimethylamine and urea groups were retained. These compounds aimed to enhance safety and minimize the adverse effects associated with opioid drugs. The research findings suggest that compound 6a does not induce ß-arrestin recruitment at low-nanomolar concentrations but exhibits significant analgesic effects in established mouse models. Compared to morphine, 6a shows advantages in alleviating respiratory depression and minimizing physical dependence. Molecular docking studies underscore the pivotal role of the D147 amino acid residue in the analgesic mechanism of 6a. Consequently, 6a is a compelling candidate for the development of safer opioid analgesics and warrants further attention.

The differential influence of PZM21, a nonrewarding µ-opioid receptor agonist with G protein bias, on behavioural despair and fear response in mice.

A growing body of evidence points out the involvement of the µ-opioid receptors in the modulation of stress-related behaviour. It has been suggested that µ-opioid receptor agonists may attenuate behavioural despair following animals' exposure to an acute, inescapable stressor. Moreover, morphine was shown to ameliorate fear memories caused by a traumatic experience. As typical µ-opioid receptor agonists entail a risk of serious side effects and addiction, novel, possibly safer and less addictive agonists of this receptor are currently under investigation. One of them, PZM21, preferentially acting via the G protein signalling pathway, was previously shown to be analgesic, but less addictive than morphine. Here, we aimed to further test this ligand in stress-related behavioural paradigms in mice. The study has shown that, unlike morphine, PZM21 does not decrease immobility in the forced swimming and tail suspension tests. On the other hand, we observed that both mice treated with PZM21 and those receiving morphine presented a slight attenuation of freezing across the consecutive fear memory retrievals in the fear conditioning test. Therefore, our study implies that at the range of tested doses, PZM21, a nonrewarding representative of G protein-biased µ-opioid receptor agonists, may interfere with fear memory consolidation while having no beneficial effects on behavioural despair in mice.

Opioid signaling and design of analgesics.

Clinical treatment of acute to severe pain relies on the use of opioids. While their potency is significant, there are considerable side effects that can negatively affect patients. Their rise in usage has correlated with the current opioid epidemic in the United States, which has led to more than 70,000 deaths per year (Volkow and Blanco, 2021). Opioid-related drug development aims to make target compounds that show strong potency but with diminished side effects. Research into pharmaceuticals that could act as potential alternatives to current pains medications has relied on mechanistic insights of opioid receptors, a class of G-protein coupled receptors (GPCRs), and biased agonism, a common phenomenon among pharmaceutical compounds where downstream effects can be altered at the same receptor via different agonists. Opioids function typically by binding to an active site on the extracellular portion of opioid receptors. Once activated, the opioid receptor initiates a G-protein signaling pathway and/or the ß-arrestin2 pathway. The proposed concept for the development of safe analgesics around mu and kappa opioid receptor subtypes has focused on not recruiting ß-arrestin2 (biased agonism) and/or having low efficacy at the receptor (partial agonism). By altering chemical motifs on a common scaffold, chemists can take advantage of biased agonism as well as create compounds with low intrinsic efficacy for the desired treatments. This review will focus on ligands with bias profile, signaling aspects of the receptor and probe into the structural basis of receptor that leads to bias and/or partial agonism.

Discovery and Structural Explorations of G-Protein Biased µ-Opioid Receptor Agonists.

Compounds that activate only the G-protein signalling pathway represent an effective strategy for making safer opioids. In the present study, we report the design, synthesis and evaluation of two classes of novel PZM21 derivatives containing the benzothiophene ring and biphenyl ring group respectively as biased µ-opioid receptor (µOR) agonists. The new compound SWG-LX-33 showed potent µOR agonist activity and produced µOR-dependent analgesia. SWG-LX-33 does not activate the ß-arrestin-2 signalling pathway in vitro even at high concentrations. Computational docking demonstrated the amino acid residue ASN150 to be critical for the weak efficacy and potency of µOR agonists in arrestin recruitment.

Recent Molecular Insights into Agonist-specific Binding to the Mu-Opioid Receptor.

Opioid agonists produce their analgesic effects primarily by acting at the µ-opioid receptor (µOR). µOR agonists with different efficacies exert diverse molecular changes in the µOR which dictate the faith of the receptor's signaling pathway and possibly it's the degree of desensitization. Since the development of the active conformations of the µOR, growing data have been published in relation to ligand-specific changes in µOR activation. In this regard, this review summarizes recent data regarding the most studied opioid agonists in in silico µOR activation, including how these ligands are recognized by the µOR, how their binding signal is transmitted toward the intracellular parts of the µOR, and finally, what type of large-scale movements do these changes trigger in the µOR's domains.

TRV130 partial agonism and capacity to induce anti-nociceptive tolerance revealed through reducing available µ-opioid receptor number.

BACKGROUND AND PURPOSE: ß-Arrestin2 recruitment to µ-receptors may contribute to the development of opioid side effects. This possibility led to the development of TRV130 and PZM21, opioids reportedly biased against ß-arrestin2 recruitment in favour of G-protein signalling. However, low efficacy ß-arrestin2 recruitment by TRV130 and PZM21 may simply reflect partial agonism overlooked due to overexpression of µ-receptors. EXPERIMENTAL APPROACH: Efficacies and apparent potencies of DAMGO, morphine, PZM21 and TRV130 as stimulators of ß-arrestin2 recruitment and inhibitors of cAMP accumulation were assessed in CHO cells stably expressing µ-receptors. Receptor availability was depleted through prior exposure of cells to the irreversible antagonist, ß-FNA. We also examined whether µ-receptor availability influences TRV130 anti-nociception and/or tolerance using the tail withdrawal assay in wild-type C57BL/6 and µ+/- mice. KEY RESULTS: Morphine, PZM21 and TRV130 were partial agonists in the ß-arrestin2 recruitment assay. Only TRV130 exhibited partial agonism in the cAMP assay. Exposure to ß-FNA to reduce µ-receptor availability further limited the efficacy of TRV130 and revealed morphine and PZM21 to be partial agonists. Despite having partial efficacy in vitro, TRV130 caused potent anti-nociception (ED50 : 0.33 mg·kg-1 ) in wild-type mice, without tolerance after daily administration for 10 days. TRV130 caused similar anti-nociception in µ+/- mice, with marked tolerance on day 4 of injections. CONCLUSION AND IMPLICATIONS: Our findings emphasise the importance of receptor reserve when characterising µ-receptor agonists. Reduced receptor availability reveals that TRV130 is a partial agonist capable of tolerance, despite having limited efficacy for ß-arrestin2 recruitment to the µ-receptor.

Probing biased activation of mu-opioid receptor by the biased agonist PZM21 using all atom molecular dynamics simulation.

Morphine is a commonly used opioid drug to treat acute pain by binding to the mu-opioid receptor (MOR), but its effective analgesic efficacy via triggering of the heterotrimeric Gi protein pathway is accompanied by a series of adverse side effects via triggering of the ß-arrestin pathway. Recently, PZM21, a recently developed MOR biased agonist, shows preferentially activating the G protein pathway over ß-arrestin pathway. However, there is no high-resolution receptor structure in complex with PZM21 and its action mechanism remains elusive. In this study, PZM21 and Morphine were docked to the active human MOR-1 homology structure and then subjected to the molecular dynamics (MD) simulations in two different situations (i.e., one situation includes the crystal waters but another does not). Detailed comparisons between the two systems were made to characterize the differences in protein-ligand interactions, protein secondary and tertiary structures and dynamics networks. PZM21 could strongly interact with Y3287.43 of TM7, besides the residues (Asp1493.32 and Tyr1503.33) of TM3. The two systems' network paths to the intracellular end of TM6 were roughly similar but the paths to the end of TM7 were different. The PZM21-bound MOR's intracellular ends of TM5-7 bent outward more along with the distance changes of the three key molecular switches (ionic lock, transmission and Tyr toggle) and the distance increase of some conserved inter-helical residue pairs. The larger intracellular opening of the receptor could potentially facilitate G protein binding.

Biased Opioid Ligands: Revolution or Evolution?

Opioid are the most powerful analgesics ever but their use is still limited by deleterious side effects such as tolerance, dependence, and respiratory depression that could eventually lead to a fatal overdose. The opioid crisis, mainly occurring in north America, stimulates research on finding new opioid ligands with reduced side effects. Among them, biased ligands are likely the most promising compounds. We will review some of the latest discovered biased opioid ligands and see if they were able to fulfill these expectations.

Molecular Modeling of µ Opioid Receptor Ligands with Various Functional Properties: PZM21, SR-17018, Morphine, and Fentanyl-Simulated Interaction Patterns Confronted with Experimental Data.

Molecular modeling approaches are an indispensable part of the drug design process. They not only support the process of searching for new ligands of a given receptor, but they also play an important role in explaining particular activity pathways of a compound. In this study, a comprehensive molecular modeling protocol was developed to explain the observed activity profiles of selected µ opioid receptor agents: two G protein-biased µ opioid receptor agonists(PZM21 and SR-17018), unbiased morphine, and the ß-arrestin-2-biased agonist,fentanyl. The study involved docking and molecular dynamics simulations carried out for three crystal structures of the target at a microsecond scale, followed by the statistical analysis of ligand-protein contacts. The interaction frequency between the modeled compounds and the subsequent residues of a protein during the simulation was also correlated with the output of in vitro and in vivo tests, resulting in the set of amino acids with the highest Pearson correlation coefficient values. Such indicated positions may serve as a guide for designing new G protein-biased ligands of the µ opioid receptor.

Molecular Dynamics Simulations to Investigate How PZM21 Affects the Conformational State of the µ-Opioid Receptor Upon Activation.

Opioid analgesics such as morphine have indispensable roles in analgesia. However, morphine use can elicit side effects such as respiratory depression and constipation. It has been reported that G protein-biased agonists as substitutes for classic opioid agonists can alleviate (or even eliminate) these side effects. The compounds PZM21 and TRV130 could be such alternatives. Nevertheless, there are controversies regarding the efficacy and G protein-biased ability of PZM21. To demonstrate a rationale for the reduced biasing agonism of PZM21 compared with that of TRV130 at the molecular level, we undertook a long-term molecular dynamics simulation of the µ-opioid receptor (MOR) upon the binding of three ligands: morphine, TRV130, and PZM21. We found that the delayed movement of the W2936.48 (Ballesteros-Weinstein numbering) side chain was a factor determining the dose-dependent agonism of PZM21. Differences in conformational changes of W3187.35, Y3267.43, and Y3367.53 in PZM21 and TRV130 explained the observed differences in bias between these ligands. The extent of water movements across the receptor channel was correlated with analgesic effects. Taken together, these data suggest that the observed differences in conformational changes of the studied MOR-ligand complexes point to the low-potency and lower bias effects of PZM21 compared with the other two ligands, and they lay the foundation for the development of G protein-biased agonists.

Discovery of Biased Mu-Opioid Receptor Agonists for the Treatment of Pain.

G protein-biased mu-opioid receptor (MOR) agonists have been developed as promising new potent analgesic drugs with fewer adverse side effects than standard MOR agonists. PZM21 represents a unique chemotype unrelated to known opioids, which makes it a desirable lead for modification to find analgesics with new chemical entities. In the present study, we synthesized and tested novel PZM21 derivatives as potent biased MOR agonists by introducing a benzodioxolane group to replace the hydroxybenzene of PZM21. The new compounds displayed more potent analgesic activities in vivo and greater bias toward G protein signaling in vitro than did PZM21. These results suggest that the benzodioxolane group is essential for the maintenance of bias. Compounds 7 i ((S)-1-(3-(benzo[d][1,3]dioxol-4-yl)-2-(dimethylamino)propyl)-3-phenethylurea) and 7 j ((S)-1-(3-(benzo[d][1,3]dioxol-4-yl)-2-(dimethylamino)propyl)-3-benzylurea) could serve as new leads for further modifications to find novel biased MOR agonists with greater G protein signaling potency and less ß-arrestin-2 recruitment.

Synthesis and Evaluation of Novel Biased µ-Opioid-Receptor (µOR) Agonists.

'Biased' ligands of G protein-coupled receptors (GPCRs) represent a type of promising analgesic with reduced on-target side effects. PZM21, a potent µ-opioid-receptor (µOR)-biased agonist with a new chemical scaffold compared to classic opioids, has been identified as a therapeutic lead molecule for treating pain. In the current study, novel PZM21 analogues were synthesized and evaluated for their in vitro and in vivo efficacy. Novel compound 7a and PZM21 demonstrated undetectable ß-arrestin-2 recruitment, however, their analgesic effects need to be further confirmed. Compounds 7b, 7d, and 7g were stronger analgesics than PZM21 in both the mouse formalin injection assay and the writhing test. Compound 7d was the most potent analogue, requiring a dose that was 1/16th to 1/4th of that of PZM21 for its analgesic activity in the two assays, respectively. Therefore, compound 7d could serve as a lead to develop new biased µOR agonists for treating pain.

Synthesis of Enantiopure PZM21, A Novel Biased Agonist of the Mu-Opioid Receptor.

PZM21 (1) was recently reported as a biased agonist of the mu-opioid receptor (MOR) with improved antinociceptive effects but reduced side effects than traditional opioid-based analgesics. The original synthesis of PZM21 with the desired (S,S) configuration required the separation of diastereomeric mixture in the final step using chiral HPLC. We have designed a concise synthesis of 1 in the enantiomeric pure form starting with commercially available L-alanine and via a chiral aziridine as a key intermediate. The final product 1 as the (S,S) diastereomer was obtained in 7 steps in 22.5% yield from L-alanine. This synthetic strategy could be readily applied to the development of PZM21 analogs at the thiophenyl position.