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Ion Binding Site As a Mechanism for Positive Allosteric Modulation of the Mu-Opioid Receptor

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Ion Binding Site As a Mechanism for Positive Allosteric Modulation of the Mu-Opioid Receptor + Disruption of the Na ion binding site as a mechanism for positive allosteric modulation of the mu-opioid receptor Kathryn E. Livingston and John R. Traynor1 Department of Pharmacology, University of Michigan, Ann Arbor, MI 48109 Edited by Leslie Lars Iversen, University of Oxford, Oxford, United Kingdom, and approved November 13, 2014 (received for review August 6, 2014) Positive allosteric modulation of the mu-opioid receptor (MOPr), of in vitro and in vivo literature describing allostery at GPCRs the site of action of all clinically used opioids, represents a poten- (9–11). In contrast, apart from our initial description of mu-PAMs, tial approach for the management of pain. We recently reported very little is known about allostery at MOPr. on positive allosteric modulators of MOPr (mu-PAMs), a class A G Allosteric modulators exhibit probe dependence, meaning they protein coupled receptor (GPCR). This study was designed to show disparate effects depending on the agonist bound to the examine the mechanism of allostery by comparing the degree to orthosteric site (12). A striking example of this is LY2033298, which opioid ligand structure governs modulation. To do this we a PAM of the muscarinic acetylcholine receptors M2 and M4. examined the interaction of the mu-PAM, BMS-986122, with LY2033298 increases the affinity of the agonist oxotremorine, a chemically diverse range of MOPr orthosteric ligands. Generally, while having no effect on the binding of the agonists pilocarpine for full agonists BMS-986122 enhanced the binding affinity and and McN-A-343 (13). Currently, it is not known if all opioid potency to activate G protein with no alteration in the maximal agonists are equally sensitive to the PAM effect of BMS-986122, effect. In contrast, lower efficacy agonists including morphine nor the mechanism underlying the allosteric modulation. Our were insensitive to alterations in binding affinity and showed little initial characterization showed that BMS-986122 causes a shift 2 4 to no change in potency to stimulate G protein. Instead, there was in the potency of the agonist DAMGO ([D-Ala , N-MePhe , an increase in maximal G protein stimulation. Antagonists were Gly-ol]-enkephalin), but increases the maximal stimulation of G unresponsive to the modulatory effects of BMS-986122. Sodium is protein by morphine (1). Opioid ligands are extremely diverse, a known endogenous allosteric modulator of MOPr and alters ranging from the 31-amino acid endogenous peptide β-endorphin to orthosteric agonist affinity and efficacy. The sensitivity of an small alkaloids like morphine. Therefore, this study sought to an- orthosteric ligand to BMS-986122 was strongly correlated with its swer two questions: (i) does BMS-986122 show probe dependence sensitivity to NaCl. In addition, BMS-986122 decreased the ability for the orthosteric ligand? And (ii) if probe dependence is seen, of NaCl to modulate agonist binding in an allosteric fashion. what is the mechanistic basis for this? Overall, BMS-986122 displayed marked probe dependence that To address these questions we examined the effect of BMS- was based upon the efficacy of the orthosteric ligand and can be 986122 on the MOPr properties of a wide range of opioid ligands PHARMACOLOGY explained using the Monod–Wyman–Changeux two-state model + from endogenous peptides to small molecules (Fig. S2). The of allostery. Furthermore, disruption of the Na ion binding site results reveal that the PAM effects of BMS-986122 are de- may represent a common mechanism for allosteric modulation of pendent on the efficacy of the orthosteric ligand and not on the class A GPCRs. structure per se. We find a strong correlation between the pos- + itive action of BMS-986122 and the negative action of Na ions endogenous opioids | opiates | GTPgammaS | ligand binding | receptor states Significance he mu-opioid receptor (MOPr) is the site of action of all Tclinically used opioid drugs. MOPr is a class A G protein- Morphine and related compounds are the gold standard for the coupled receptor (GPCR) that activates heterotrimeric Gi/o management of pain. Such drugs bind to the orthosteric site on proteins. Clinically used opioid agonists bind to the orthosteric the mu-opioid receptor (MOPr), a G protein-coupled receptor site on MOPr and although they are efficacious at causing pain (GPCR). We have proposed that targeting an allosteric site on relief, have a number of unwanted side effects resulting from MOPr could result in improved pain management and have direct MOPr activation. We have recently discovered and pre- reported positive allosteric modulators (PAMs) of MOPr. High- + sented a preliminary characterization of positive allosteric modu- resolution X-ray structures have identified a Na binding site + lators of MOPr (mu-PAMs) and are currently pursuing the idea on multiple GPCRs and have shown how bound Na stabilizes that mu-PAMs could be a viable way to manage pain (1, 2). The inactive receptor states. Here we demonstrate that PAM ac- ligand BMS-986122 (Fig. S1) represents the most active mu-PAM tivity at MOPr allosterically disrupts Na+ binding, thereby + currently identified. It was discovered in a high-throughput screen forming an active-state receptor. The Na binding site is highly for its ability to enhance the recruitment of β-arrestin to MOPr conserved across class A GPCRs so this may represent a funda- by the agonist endomorphin-1. Although having little agonist mental mechanism of allosteric modulation. activity on its own, this modulator has the ability to enhance the affinity, potency, and/or maximal response of MOPr agonists. In A preliminary report of some of the findings presented in this paper was given at the 45th annual meeting of the International Narcotics Research Conference, July 13–18, the same systems, BMS-986122 has no activity when the delta 2014, Montreal, Canada. opioid receptor (DOPr) is expressed, indicating the importance Author contributions: K.E.L. and J.R.T. designed research; K.E.L. performed research; of MOPr for BMS-986122 activity. K.E.L. and J.R.T. analyzed data; and K.E.L. and J.R.T. wrote the paper. The study of allosteric modulation of GPCRs has recently gained The authors declare no conflict of interest. momentum (3) and represents a relatively unexplored avenue for This article is a PNAS Direct Submission. drug development (4, 5). Allosteric modulators have been dis- 1To whom correspondence should be addressed. Email: [email protected]. covered for several GPCRs including the muscarinic, cannabinoid, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. and metabotropic glutamate receptors (6–8) with a growing body 1073/pnas.1415013111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1415013111 PNAS | December 23, 2014 | vol. 111 | no. 51 | 18369–18374 Downloaded by guest on September 25, 2021 to inhibit agonist binding. Moreover, we also show that BMS- Table 1. Binding affinity of MOPr ligands in the presence or + 986122 allosterically inhibits the ability of Na ions to reduce absence of BMS-986122 agonist binding. The PAM effect of BMS-986122 can conse- + Ligand Ki(Veh), nM Ki(BMS), nM Ki(Veh)/Ki(BMS) quently be explained by an inhibition of the ability of Na ions to stabilize the inactive state of the receptor, thereby allowing the Peptides receptor to shift to an active conformation. Thus, the mechanism β-Endorphin 194 ± 13 47 ± 8*** 4.1 ± ± of positive allosteric modulation can be simply explained by the Endomorphin-1 104 32 17 8* 6.1 ± ± two-state Monod–Wyman–Changeux model of allosterism (14). Leu-Enk 664 67 100 15** 6.6 Met-Enk 423 ± 133 63 ± 16* 6.7 Results Small molecules ± ± We first investigated the effects of a maximally effective con- Buprenorphine 0.5 0.1 0.4 0.2 1.3 ± ± centration (10 μM; ref. 1) of BMS-986122 on the MOPr activity Etorphine 2.4 0.3 1.5 0.8 1.6 ± ± of a range of endogenous opioid peptides (Fig. S2A). Using cell Fentanyl 222 34 89 25* 2.5 Loperamide 215 ± 54 14 ± 2* 15.4 membrane homogenates prepared from C6 glioma cells stably (RS)-Methadone 1076 ± 85 100 ± 4*** 10.7 expressing MOPr (C6MOPr) (15), we performed radioligand ± ± 3 (R)-Methadone 382 10 36 10*** 10.6 competition binding assays using H-diprenorphine (DPN, an ± ± γ (S)-Methadone 6,358 2,065 896 35* 7.1 opioid antagonist) in the presence of GTP S and NaCl to gen- Morphine 163 ± 18 143 ± 42 1.1 erate an inactive receptor state known to predominate in native Naloxone 2.5 ± 0.7 3.4 ± 1.0 0.7 membranes (16, 17). As shown in Fig. 1A and Table 1, BMS- 986122 caused an approximate sixfold enhancement in the Affinity (Ki values) were determined by competitive displacement of 3 affinity of both methionine-enkephalin (Met-Enk) and leucine- H-DPN binding from C6MOPr cell membranes in a buffer containing 10 μM γ enkephalin (Leu-Enk). A similar increase in affinity was seen for GTP S and 100 mM NaCl as described in Methods, in the absence or presence of 10 μM BMS-986122. *P < 0.05, **P < 0.01, ***P < 0.001 compared with control the smaller putative endogenous peptide endomorphin-1 (18). In (vehicle) data by Student t test. Data shown are means ± SEM of three to five independent experiments each in duplicate. addition to enhancing its affinity to bind MOPr, BMS-986122 caused a leftward shift in the concentration-response for Met- Enk to activate G protein, with no alteration in the maximal response (Emax), as measured by GTPγ35S binding in membrane homogenates (Fig. 1C and Table 2); an effect also seen with Leu-Enk and endomorphin-1 (Table 2). The endogenous opioid β-endorphin, a much larger 31-amino acid peptide, was also modulated by BMS-986122 with rightward shifts in both the af- finity (fourfold; Table 1) and potency (sixfold; Table 2) to acti- vate G protein.
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