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. 2016 May 24;113(21):6041-6.
doi: 10.1073/pnas.1521825113. Epub 2016 May 9.

Collybolide is a novel biased agonist of κ-opioid receptors with potent antipruritic activity

Achla Gupta  1 , Ivone Gomes  1 , Erin N Bobeck  1 , Amanda K Fakira  1 , Nicholas P Massaro  2 , Indrajeet Sharma  2 , Adrien Cavé  3 , Heidi E Hamm  3 , Joseph Parello  4 , Lakshmi A Devi  5
Affiliations

Collybolide is a novel biased agonist of κ-opioid receptors with potent antipruritic activity

Achla Gupta et al. Proc Natl Acad Sci U S A. .

Abstract

Among the opioid receptors, the κ-opioid receptor (κOR) has been gaining considerable attention as a potential therapeutic target for the treatment of complex CNS disorders including depression, visceral pain, and cocaine addiction. With an interest in discovering novel ligands targeting κOR, we searched natural products for unusual scaffolds and identified collybolide (Colly), a nonnitrogenous sesquiterpene from the mushroom Collybia maculata. This compound has a furyl-δ-lactone core similar to that of Salvinorin A (Sal A), another natural product from the plant Salvia divinorum Characterization of the molecular pharmacological properties reveals that Colly, like Sal A, is a highly potent and selective κOR agonist. However, the two compounds differ in certain signaling and behavioral properties. Colly exhibits 10- to 50-fold higher potency in activating the mitogen-activated protein kinase pathway compared with Sal A. Taken with the fact that the two compounds are equipotent for inhibiting adenylyl cyclase activity, these results suggest that Colly behaves as a biased agonist of κOR. Behavioral studies also support the biased agonistic activity of Colly in that it exhibits ∼10-fold higher potency in blocking non-histamine-mediated itch compared with Sal A, and this difference is not seen in pain attenuation by these two compounds. These results represent a rare example of functional selectivity by two natural products that act on the same receptor. The biased agonistic activity, along with an easily modifiable structure compared with Sal A, makes Colly an ideal candidate for the development of novel therapeutics targeting κOR with reduced side effects.

Keywords: G-protein–coupled receptors; antinociception; dynorphin; natural compounds; salvinorin A.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Structures of salvinorin A, collybolide, and 9-epi-collybolide. Chemical structures of the naturally occurring Sal A (from the Mexican mint S. divinorum) and of collybolide and 9-epi-collybolide (from the fungus C. maculata) shown with the common furyl-δ-lactone motif highlighted in red. The absolute configurations of collybolide (2R,4R,5S,6R,7S,9R) and 9-epi-collybolide (2R,4R,5S,6R,7S,9S) were determined by the Bijvoet’s method using the oxygen atoms as anomalous scattering centers (SI Materials and Methods).
Fig. 2.
Fig. 2.
Displacement of radiolabeled ligand binding to human κOR. Membranes (200 μg) from HEK-293 cells expressing hκOR were incubated with [3H]Naloxone (Nal), [3H]Diprenorphine (Dip), or [3H]U69,493 (U69) (3 nM) in 50 mM Tris⋅Cl, pH 7.8, containing 1 mM EGTA, 5 mM MgCl2, and protease inhibitor mixture in the absence or presence of Colly (Col), 9-epi-Colly (9-epi-Col), or Sal A (10−13–10−5 M). Values at 10−13 M cold ligand were taken as 100%. Data represent mean ± SEM; n = 6.
Fig. S1.
Fig. S1.
Displacement of radiolabeled ligand binding to human κOR. (A) HEK-293 cells (2 × 105) expressing human κOR were incubated with [3H]Naloxone, [3H]Diprenorphine, or [3H]U69,493 (3 nM) in 50 mM Tris⋅Cl buffer, pH 7.8, containing 1 mM EGTA, 5 mM MgCl2, and protease inhibitor mixture in presence of either collybolide (Col), 9-epi-collybolide (9-epi-Col), or Sal A (Sal) (10−13–10−5 M). (B–D) Membranes (200 μg) from HEK-293 cells expressing hκOR were incubated with [3H]Naloxone (3 nM) in 50 mM Tris⋅Cl buffer, pH 7.8, containing protease inhibitor mixture (B), 50 mM Tris⋅Cl, pH 7.4, containing 100 mM NaCl, 5 mM MgCl2, 0.2 mM EGTA, and protease inhibitor mixture (C), or 50 mM Tris⋅Cl buffer, pH 7.8, containing 1 mM EGTA, 5 mM MgCl2, 1 μM GTPγS, and protease inhibitor mixture (D) in the presence of collybolide (10−13–10−5 M). Values obtained at 10−13 M ligand were taken as 100%. Data represent mean ± SEM; n = 6.
Fig. 3.
Fig. 3.
Collybolides bind to human κOR but not μOR or δOR. Membranes (200 μg) from HEK-293 cells expressing hκOR, hδOR, or hμOR were incubated with [3H]Naloxone (3 nM) in 50 mM Tris⋅Cl, pH 7.8, containing 1 mM EGTA, 5 mM MgCl2, and protease inhibitor mixture in the presence of collybolide (A) or 9-epi-collybolide (B) (10−13–10−5 M). Data represent mean ± SEM; n = 6. (C) Cerebral cortex membranes (100 μg) from WT or κOR KO mice (κOR k/o) were incubated with [3H]Naloxone (3 nM) in the presence of collybolide (10−13–10−5 M). Data represent mean ± SEM; n = 3 individual animals. Values at 10−13 M cold ligand were taken as 100%. n.d., not detected.
Fig. S2.
Fig. S2.
Signaling properties of collybolides at human μOR or δOR. (A and B) Membranes (20 μg) from HEK-293 cells expressing hμOR (A) or hδOR (B) were subjected to a [35S]GTPγS binding assay in the presence of either Colly (Col) or 9-epi-Colly (9-epi-Col) (10−13–10−5 M). * represents values obtained at 10−5 M. (C and D) HEK-293 cells expressing hμOR (C) or hδOR (D) were treated for 3 min at 37 °C with either Col or 9-epi-Col (10−13–10−5 M) and phosphorylated ERK1/2 levels measured as described in Materials and Methods. Values (A–D) obtained at 10−13 M ligand were taken as 100%. (E and F) HEK-293 cells expressing hμOR (E) or hδOR (F) were treated for 3 min at 37 °C with either 1 μM DAMGO ± 10 μM Col or 9-epi-Col (E) or 1 μM deltorphin II (Delt II) ± 10 μM Col or 9-epi-Col (F) and phosphorylated ERK1/2 levels measured as described in Materials and Methods. Data represent mean ± SEM; n = 3.
Fig. S3.
Fig. S3.
Selectivity of collybolide for κOR. Membranes from the brain of WT and from κOR KO mice were treated with [3H]Naloxone (3 nM) in the absence or presence of 10 μM of either collybolide (Col), Sal A, U69,593 (U69), or naloxone (Nal). Data represent mean ± SEM; n = 3.
Fig. 4.
Fig. 4.
Signaling by collybolides in membranes expressing hκOR. Membranes (20 μg) from HEK-293 cells expressing hκOR were subjected to a [35S]GTPγS binding assay in the presence of Colly (Col), 9-epi-Colly (9-epi-Col), or Sal A (10−13–10−5 M) (A) without or with preincubation (30 min) with nor-BNI (B). (C) [35S]GTPγS binding using cerebral cortex membranes from WT and κOR KO mice and Col (10−13–10−5 M). (D) Membranes (2 μg) from HEK-293 cells expressing hκOR were treated with Col or Sal A (10−13–10−5 M), and cAMP levels were measured. Values (A–D) at 10−13 M ligand were taken as 100%. (E) Receptor internalization in HEK-293 cells expressing hκOR treated with Col or Sal A (0–1 μM) for 30 min at 37 °C; percent internalized receptors were calculated as described in SI Materials and Methods. (F–H) HEK-293 cells expressing hκOR were treated for 3 min at 37 °C with either Col or Sal A (10−13–10−6 M) and phosphorylated levels of ERK1/2 (F), Akt at S473 (G), or Akt at T308 (H) measured. Data (A–H) represent mean ± SEM; n = 3–6. *Values obtained with 10 μM ligand. n.d., not detected.
Fig. S4.
Fig. S4.
Trafficking properties of human κOR. HEK-293 cells expressing hκOR were incubated at 4 °C for 1 h with 1:1,000 anti-HA antibody in media to label cell surface κOR, washed three times to remove unbound antibody, and then treated without or with 100 nM κOR ligands for different time intervals (0–120 min) (A) or with different concentrations (0–1 μM) of κOR ligands for 30 min (B) at 37 °C. Receptors present at the cell surface were determined as described in Materials and Methods. The percent internalized receptors was calculated by taking total cell surface receptors before agonist treatment for each individual experiment as 100% and subtracting percent surface receptors following agonist treatment. Data represent mean ± SEM; n = 6.
Fig. S5.
Fig. S5.
Signaling properties of collybolides. (A) Membranes (2 μg) from HEK-293 cells expressing hκOR were treated with 9-epi-Col (10−13–10−5 M) and cAMP levels measured as described in Materials and Methods. Values obtained at 10−13 M ligand were taken as 100%. (B) HEK-293 cells expressing hκOR were treated for different time intervals (30 s to 30 min) at 37 °C with vehicle, Col, or 9-epi-Col (100 nM) and phosphorylated ERK1/2 levels measured as described in Materials and Methods. (C) Representative Western blots for Col and Sal A data presented in Fig. 4F. (D) HEK-293 cells expressing hκOR were treated for 3 min at 37 °C with 9-epi-Col (10−13–10−6 M) and phosphorylated ERK1/2 levels measured as described in Materials and Methods. Representative blot is shown. (E) CHO cells expressing hκOR were treated for 3 min at 37 °C with either Col or 9-epi-Col (10−13–10−6 M) and phosphorylated ERK1/2 levels measured as described in Materials and Methods. Data (A–E) represent mean ± SEM; n = 3–6.
Fig. 5.
Fig. 5.
Behavioral effects of collybolide. (A) Mice were administered with vehicle (Veh), Sal A (Sal), or Colly (Col) (2 mg/kg), and antinociception was measured at different time intervals using the tail flick assay. Two-way ANOVA: time, F(2,42) = 1.486, P = 0.238; drug, F(2,21) = 4.199, P = 0.029; Bonferroni post hoc: *P < 0.05 vehicle vs. Sal A/Col. (B) Forced swim test with mice administered with Veh or Col (2 mg/kg). (C) Mice administered with Veh, Sal, Col (2 mg/kg), or nor-BNI (10 mg/kg) + Col were placed in the elevated plus maze, and time spent in the open arms was measured. (D) Mice were administered with Veh or Col (2 mg/kg) for 4 d, and place preference was measured. t test: *P < 0.05. (E) Mice were administered with chloroquine-phosphate (20 mg/kg) in the absence or presence of Sal or Col (2–2,000 μg/kg), and number of scratches was measured for 15 min. Veh vs. Col 2,000 μg/kg, unpaired t test: *P < 0.05. (F) Assessment of the effect of nor-BNI (10 mg/kg) on Col-mediated attenuation of itch. *P < 0.05; one-way ANOVA. (G) Mice were injected i.p. with Veh, Col (2 mg/kg), Sal A (2 mg/kg), or nor-BNI (10 mg/kg) + Col (2 mg/kg) 20 min before subjecting them to the open field test. Data (A–G) represent mean ± SEM; n = 7–10 mice per group.
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