Collybolide is a novel biased agonist of κ-opioid receptors with potent antipruritic activity Achla Guptaa, Ivone Gomesa, Erin N. Bobecka, Amanda K. Fakiraa, Nicholas P. Massarob, Indrajeet Sharmab, Adrien Cavéc, Heidi E. Hammc, Joseph Parelloc,d,1, and Lakshmi A. Devia,1 aDepartment of Pharmacology & Systems Therapeutics, Icahn School of Medicine at Mount Sinai, New York, NY 10029; bDepartment of Chemistry and Biochemistry, University of Oklahoma, Norman, OK 73019; cDepartment of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232; and dDepartment of Chemistry, Vanderbilt University, Nashville, TN 37232 Edited by Solomon H. Snyder, Johns Hopkins University School of Medicine, Baltimore, MD, and approved April 5, 2016 (received for review November 6, 2015) Among the opioid receptors, the κ-opioid receptor (κOR) has been that could provide novel treatment strategies for a variety of dis- gaining considerable attention as a potential therapeutic target orders where κOR involvement has been implicated (16, 17). for the treatment of complex CNS disorders including depression, Previously identified synthetic κOR ligands were mostly ni- visceral pain, and cocaine addiction. With an interest in discover- trogenous compounds with a tertiary amine group (16). However, ing novel ligands targeting κOR, we searched natural products for about a decade ago, Salvinorin A (Sal A), a nonnitrogenous unusual scaffolds and identified collybolide (Colly), a nonnitroge- diterpene extracted from the Mexican mint Salvia divinorum and nous sesquiterpene from the mushroom Collybia maculata. This characterized by the presence of a furyl-δ-lactone motif (Fig. 1), compound has a furyl-δ-lactone core similar to that of Salvinorin was identified as a potent and highly selective κOR agonist (18). A (Sal A), another natural product from the plant Salvia divinorum. The structural novelty of Sal A led to extensive structure–activity Characterization of the molecular pharmacological properties re- relationship (SAR) studies through hemisynthetic approaches veals that Colly, like Sal A, is a highly potent and selective κOR (19). The furyl-δ-lactone group of Sal A (Fig. 1; motif highlighted agonist. However, the two compounds differ in certain signaling in red) is present in other members of the terpene families (20) and behavioral properties. Colly exhibits 10- to 50-fold higher po- and appears as a highly oxygenated motif in the diterpene scaffold tency in activating the mitogen-activated protein kinase pathway that is involved in ligand binding to κOR in conjunction with other NEUROSCIENCE compared with Sal A. Taken with the fact that the two compounds oxygenated groups (esters) in the molecule. are equipotent for inhibiting adenylyl cyclase activity, these re- In this study, we focused on the sesquiterpene Colly and its sults suggest that Colly behaves as a biased agonist of κOR. Be- diastereoisomer 9-epi-Colly, extracted from the fungus Collybia havioral studies also support the biased agonistic activity of Colly maculata, because they represent the first examples, to our in that it exhibits ∼10-fold higher potency in blocking non–hista- knowledge, of sesquiterpene structures with the furyl-δ-lactone mine-mediated itch compared with Sal A, and this difference is not motif (21). For molecular pharmacological characterization, we seen in pain attenuation by these two compounds. These results used heterologous cells individually expressing the three human represent a rare example of functional selectivity by two natural opioid receptor types and show that Colly and 9-epi-Colly function products that act on the same receptor. The biased agonistic ac- as selective and potent κOR agonists. Furthermore, we show that, tivity, along with an easily modifiable structure compared with Sal although Colly and Sal A activate some signal transduction A, makes Colly an ideal candidate for the development of novel pathways to the same extent and have comparable behavior effects therapeutics targeting κOR with reduced side effects. in some assays, they exhibit substantial differences in other path- ways and behavioral assays suggesting biased agonism. Finally, G-protein–coupled receptors | natural compounds | salvinorin A | taken with our finding that Colly exhibits potent antipruritic dynorphin | antinociception Significance pioid receptors, members of family A of G-protein–coupled Oreceptors (GPCRs), and their ligands have been extensively In recent years, the κ-opioid receptor (κOR) has become an studied for their involvement in analgesia and sedation (1, 2). attractive therapeutic target for the treatment of a number of κ Among the different subtypes of opioid receptors, the opioid disorders including depression, visceral pain, and drug addic- receptor (κOR) displays a repertoire of physiological actions dis- tion. A search for natural products with novel scaffolds tar- tinct from μ (μOR) and δ (δOR) receptors (3). κOR activation geting κOR has been intensive. Here, we report the discovery leads to antinociception without the side effects associated with of a natural product (Colly) from the fungus Collybia maculata μOR activation such as physical dependence, respiratory de- as a novel scaffold that contains a furyl-δ-lactone core structure pression, or inhibition of gastrointestinal transit (4, 5). In addition, similar to that of Salvinorin A, another natural product isolated κOR agonists have antipruritic effects and can block the effects of from the mint Salvia divinorum. We show that Colly functions psychostimulants (6–8). Thus, κOR agonists could be potential as a κOR agonist with antinociceptive and antipruritic activity. therapeutics to treat addiction, visceral pain, pruritus, or pain Interestingly, Colly exhibits biased agonistic activity, suggest- killers with low abuse potential (9–12). However, in vivo κOR ing that it could be used as a backbone for the generation of activation is associated with side effects like anxiety, stress, di- novel therapeutics targeting κOR with reduced side effects. uresis, dysphoria/aversion, and psychomimetic effects (6–8). Also, activation of the κOR system has been implicated in relapse to Author contributions: E.N.B., I.S., A.C., H.E.H., J.P., and L.A.D. designed research; A.G., I.G., κ E.N.B., A.K.F., and N.P.M. performed research; A.G., I.G., E.N.B., A.K.F., I.S., and L.A.D. drugs of abuse; this limits the use of OR agonists in the treatment analyzed data; and I.G., I.S., H.E.H., J.P., and L.A.D. wrote the paper. of drug addiction (13, 14). Studies correlating the dysphoric effects The authors declare no conflict of interest. of κOR agonists to the G-protein–independent activation of the κ This article is a PNAS Direct Submission. p38 MAPK pathway (8, 13, 15) suggest that identification of OR 1 – To whom correspondence may be addressed. Email: [email protected] or agonists biased to G-protein mediated signaling could lead to the [email protected]. development of therapeutics with reduced side effects. This sug- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. gestion has spurred the search for novel κOR ligands or scaffolds 1073/pnas.1521825113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1521825113 PNAS Early Edition | 1of6 Downloaded by guest on September 29, 2021 Collybolides Exhibit Selectivity to hκOR. We examined the selec- tivity of Collys for hκOR by comparing their ability to displace [3H]Naloxone binding to hμOR or hδORandtosignalthrough these receptors. We find that neither Colly nor 9-epi-Colly dis- place [3H]Naloxone binding to hμOR or hδOR (Fig. 3). Also, Collys do not elicit changes in [35S]GTPγS binding or ERK1/2 phosphorylation in cells expressing hμOR or hδOR (Fig. S2) nor do they block DAMGO (μOR agonist) or deltorphin II (δOR)- mediated increases in phospho ERK1/2 (Fig. S2). These results indicate that Collys exhibit selectivity toward hκOR. This selec- tivity is supported by the ability of Colly to displace [3H]Naloxone Fig. 1. Structures of salvinorin A, collybolide, and 9-epi-collybolide. Chemical binding to membranes from WT mice but not from mice lacking structures of the naturally occurring Sal A (from the Mexican mint S. divinorum) κOR (Fig. 3 and Fig. S3). and of collybolide and 9-epi-collybolide (from the fungus C. maculata) δ shown with the common furyl- -lactone motif highlighted in red. The ab- Collybolide Is a High Affinity Agonist of hκOR. Next we examined solute configurations of collybolide (2R,4R,5S,6R,7S,9R)and9-epi-col- 35 γ lybolide (2R,4R,5S,6R,7S,9S) were determined by the Bijvoet’smethod signaling by Colly using [ S]GTP S binding assays. Colly dose- 35 using the oxygen atoms as anomalous scattering centers (SI Materials dependently increases [ S]GTPγS binding with an EC50 of − and Methods). ∼10 9 M (Fig. 4A), a potency similar to that of 9-epi-Colly, Sal A, U69,593, and Dyn A8 (Fig. 4A and Table 1). Interestingly, 9-epi- Colly exhibits substantial lower efficacy suggesting partial ago- activities, these results demonstrate that Collys represent a novel nism (Fig. 4A). The increase in [35S]GTPγS binding mediated by class of κOR-selective agonists that could be used for further ex- both Collys is blocked by a κOR antagonist, nor-BNI (Fig. 4B). plorations of the molecular pharmacology and functioning of this 35 γ important therapeutic target. Moreover, Colly dose-dependently increases [ S]GTP S binding to WT but not κOR KO membranes (Fig. 4C), supporting Collys Results as κOR selective agonists. κ Collybolides Exhibit Structural Similarity to Sal A. Colly and 9-epi- Because OR activation leads to inhibition of adenylyl cyclase Colly share the common furyl-δ-lactone motif (i.e., a δ-lactone activity and consequently decreases in cAMP levels (22), we with its Cδ carbon substituted by the furyl group) present in Sal A examined effects of Colly on adenylyl cyclase activity. We find (Fig. 1).