POSITIVE ALLOSTERIC MODULATORS OF THE DELTA-OPIOID RECEPTOR

Abstract

Described are the discovery, synthesis and pharmacological characterization of δ-opioid receptor-selective positive allosteric modulators (δ PAMs). These δ PAMs may increase the affinity and/or efficacy of the orthosteric agonists leu-enkephalin and SNC80, as measured by β-arrestin recruitment and adenylyl cyclase inhibition. The compounds may be useful pharmacological tools to probe the molecular pharmacology of the δ receptor and to explore the therapeutic potential of δ PAMs in diseases such as chronic pain and depression.

Claims

1. A method of screening to identify delta-opioid receptor positive allosteric modulators comprising the steps of: (a) adding a positive allosteric modulator test compound and a low concentration of a delta-selective orthosteric agonist to cells; (b) measuring the effect of said delta-selective orthosteric agonist and said test compound on said cells; and (c) identifying said test compound as being a positive allosteric modulator as evidenced by a decrease in the positive allosteric agonist activity of said test compound.

2. The method according to claim 1, wherein the low concentration of a delta-selective orthosteric agonist is selected from the group consisting of: (a) less than or equal to about the calculated EC80 in said cells; (b) less than or equal to about the calculated EC70 in said cells; (c) less than or equal to about the calculated EC60 in said cells; (d) less than or equal to about the calculated EC50 in said cells; (e) less than or equal to about the calculated EC40 in said cells; (f) less than or equal to about the calculated EC30 in said cells; (g) less than or equal to about the calculated EC20 in said cells; (h) less than or equal to about the calculated EC10 in said cells.

3. A method of screening to identify delta-opioid receptor negative allosteric modulators comprising the steps of: (i) adding a negative allosteric modulator test compound and a high concentration of a delta-selective orthosteric agonist to cells; (ii) measuring the effect of said delta-selective orthosteric agonist and said test compound on said cells; and (iii) identifying said test compound as being a negative allosteric modulator as evidenced by a decrease in the negative allosteric agonist activity of said test compound.

4. The method according to claim 3, wherein the low concentration of a delta-selective orthosteric agonist is selected from the group consisting of: (a) greater than or equal to about the calculated EC10 in said cells; (b) greater than or equal to about the calculated EC20 in said cells; (c) greater than or equal to about the calculated EC30 in said cells; (d) greater than or equal to about the calculated EC40 in said cells; (e) greater than or equal to about the calculated EC50 in said cells; (f) greater than or equal to about the calculated EC60 in said cells; (g) greater than or equal to about the calculated EC70 in said cells; (h) greater than or equal to about the calculated EC80 in said cells; (i) greater than or equal to about the calculated EC90 in said cells; and (j) greater than or equal to about the calculated EC100 in said cells.

5. A method of treating pain in a patient in need thereof comprising administering to the patient a compound which is a positive allosteric modulator for the delta-opioid receptor.

6. A method of treating pain in a patient in need thereof comprising administering to the patient a compound which is a positive allosteric modulator for the delta-opioid receptor in combination with another compound which is an orthosteric agonist for the delta-opioid receptor.

7. The method of claim 6 wherein the compound is selective for delta-opioid receptors over mu-opioid receptors

8. The method of claim 7 wherein the compound which is a positive allosteric modulator for the delta-opioid receptor and is selective for delta-opioid receptors over mu-opioid receptors

9. The method of claim 7 wherein the compound is effective to provide augmentation of at least one delta-opioid receptor function selected from G protein activation, inhibition of adenylyl cyclase activity, or b-arrestin recruitment.

10. The method of claim 8 wherein the compound which is a positive allosteric modulator for the mu-opioid receptor and is effective to provide augmentation of at least one delta-opioid receptor function selected from G protein activation, inhibition of adenylyl cyclase activity, or b-arrestin recruitment.

11. A method of modulating the delta-opioid receptor comprising contacting the receptor with a compound that is effective to provide an increase in the receptor function in the presence of orthosteric exogenous or endogenous agonist.

12. The method of claim 11 wherein the increase in receptor function is observed in maximal effect, potency, or both.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0060] FIG. 1 β-arrestin recruitment response to COMPOUND A in agonist mode (in the absence of orthosteric agonist) and in PAM mode (in the presence of an EC.sub.20 of orthosteric agonist) in PathHunter cells expressing δ receptors (CHO-OPRD1) and μ receptors (CHO-OPRM1). For CHO-OPRD1 cells the orthosteric agonist was leu-enkephalin and for CHO-OPRM1 cells the orthosteric agonist was endomorphin-I. In PAM mode, The EC.sub.20 response of orthosteric agonist was normalized to 0%. 100% represents the response to a maximally effective concentration of orthosteric agonist. Data are the mean±sem, n=4.

Table 1

[0061] Structure activity relationship of the δ-PAM chemotype in PathHunter CHO-OPRD1 and CHO-OPRM1 cells. No activity was observed in agonist mode (in the absence of orthosteric agonist (data not shown)). In PAM mode (in the presence of an EC.sub.20 of leu-enkephalin for OPRD1 cells, or an EC.sub.20 of endomorphin-I for OPRM1 cells), robust responses were observed (similar in E.sub.max to the full orthosteric agonist) with the mean EC.sub.50s reported in the table (n=3).

[0062] FIG. 2 Effect of increasing concentrations of COMPOUND A on leu-enkephalin concentration response curves in □-arrestin recruitment (A), and in inhibition of forskolin-stimulated cAMP accumulation (B), in CHO-OPRD1 cells. Data is the mean±sem, n=4. Data were fitted to the operational model of allosterism (see Table 2).

Table 2

[0063] Allosteric parameters for COMPOUND A at the δ receptor. Values for affinity, efficacy, and allosteric cooperativity for orthosteric ligands and COMPOUND A are derived from the operational model of allosterism. Two different orthosteric agonists were used (Leu-enkephalin and SNC80), in β-arrestin recruitment and cAMP inhibition assays. In the model τ.sub.A and τ.sub.B represent the efficacy of the orthosteric agonist and allosteric modulator, respectively; pK.sub.A and pK.sub.B represent the binding affinity of the orthosteric agonist and the allosteric modulator, respectively, to the free receptor; and αβ represents the composite allosteric cooperativity factor. Data represent the mean±sem of 3 to 7 expts.

[0064] * pK.sub.A is fixed to its equilibrium binding affinity as ligand is a full agonist in all endpoints tested

[0065] ** the pK.sub.A of Leu-enk in endpoints where they are partial agonists was obtained from fitting their CRC to the Operational model of agonism to obtain a functional affinity in each endpoint tested.