Discovery of positive allosteric modulators and silent allosteric modulators of the μ-opioid receptor Neil T. Burforda, Mary J. Clarkb, Tom S. Wehrmanc, Samuel W. Gerritza, Martyn Banksa, Jonathan O’Connella, John R. Traynorb,1, and Andrew Alta,1 aMolecular Sciences and Candidate Optimization, Bristol-Myers Squibb Company, Wallingford, CT 06492; bDepartment of Pharmacology, University of Michigan Medical School, Ann Arbor, MI 48109; and cDiscoveRx Corporation, Fremont, CA 94538 Edited by Leslie Lars Iversen, University of Oxford, Oxford, United Kingdom, and approved May 13, 2013 (received for review January 11, 2013) μ-Opioid receptors are among the most studied G protein-coupled To overcome these side effects, studies have focused on de- receptors because of the therapeutic value of agonists, such as mor- veloping ligands with defined selectivity profiles between the various phine, that are used to treat chronic pain. However, these drugs opioid receptors (μ, κ, δ), or partial agonists (which have reduced have significant side effects, such as respiratory suppression, consti- efficacy compared with full agonists), or agonists used together in pation, allodynia, tolerance, and dependence, as well as abuse po- combination therapy (7, 8). However, these diverse approaches tential. Efforts to fine tune pain control while alleviating the side have a single commonality in that they target the orthosteric effects of drugs, both physiological and psychological, have led to (endogenous) agonist-binding site of the receptor. A different the development of a wide variety of structurally diverse agonist approach that has been used successfully with other GPCRs is the μ discovery and development of allosteric ligands, which can have ligands for the -opioid receptor, as well as compounds that target fi κ-andδ-opioid receptors. In recent years, the identification of allo- speci c advantages over their orthosteric counterparts. steric ligands for some G protein-coupled receptors has provided Allosteric Ligands breakthroughs in obtaining receptor subtype-selectivity that can re- duce the overall side effect profiles of a potential drug. However, Allosteric ligands for a GPCR bind to a site on the receptor that is distinct from the site that binds the orthosteric (or endoge- positive allosteric modulators (PAMs) can also have the specificad- nous) agonist (9). An allosteric modulator can exhibit a range of vantage of only modulating the activity of the receptor when the activities at the target protein. Positive allosteric modulators orthosteric agonist occupies the receptor, thus maintaining spatial (PAMs) may have no intrinsic efficacy but, when they bind to the PHARMACOLOGY and temporal control of receptor signaling in vivo. This second ad- receptor, enhance the binding affinity and/or efficacy of the vantage of allosteric modulators may yield breakthroughs in opioid orthosteric agonist. Negative allosteric modulators (NAMs) have receptor research and could lead to drugs with improved side-effect no intrinsic agonist efficacy but, when they bind to the receptor, profiles or fewer tolerance and dependence issues compared with inhibit the binding affinity and/or efficacy of the orthosteric ag- orthosteric opioid receptor agonists. Here, we describe the discovery onist. Silent allosteric modulators (SAMs), also known as neutral and characterization of μ-opioid receptor PAMs and silent allosteric allosteric ligands, bind to the receptor but have no effect on modulators, identified from high-throughput screening using a β- orthosteric agonist affinity or efficacy. However, SAMs can act as arrestin–recruitment assay. competitive antagonists at the same allosteric site, blocking PAM or NAM activity. Although not particularly useful from a thera- analgesia | DAMGO | enkephalins | endorphins | endomorphin peutic standpoint, SAMs can be effective tools to show that presumed PAM or NAM effects are receptor-mediated. Finally, allosteric agonists can bind and produce direct agonist activation Significance of Opioid Receptors of the receptor even in the absence of orthosteric agonist. protein-coupled receptors (GPCRs) are plasma membrane- Because allosteric sites are less evolutionarily conserved than spanning proteins that transduce signals via heterotrimeric orthosteric agonist-binding sites, allosteric ligands have the po- G tential to exhibit greater selectivity between subtypes of GPCRs G proteins on the inner surface of the plasma membrane, leading in the same family compared with orthosteric ligands. This has to intracellular signaling cascades involved in many aspects of been demonstrated for some GPCRs, including metabotropic cellular function (1). The cell surface location, tissue distribu- glutamate receptors, adenosine receptors, and muscarinic re- tion, and diversity of these GPCRs make them ideal targets for ceptors (10–14). Although highly selective orthosteric agonist drug intervention. Indeed, about 30% of marketed drugs target ligands exist for opioid receptors, opioid receptor PAMs could specific GPCR activity (1, 2). potentially provide benefits over existing medications. PAMs, Opioid receptors are members of the class A family of GPCRs. unlike allosteric agonists, may have no effect when they bind to Four opioid receptor types exist [μ, δ, κ, and opioid receptor-like the receptor in the absence of orthosteric agonist. Therefore, the 1 (ORL1)], which share about 60% amino acid identity (mainly modulation occurs only when an orthosteric agonist is bound to in the transmembrane domains) and signal through the Gi/o the receptor. In vivo, this leads to preservation of the temporal family of heterotrimeric G proteins, resulting in inhibition of and spatial characteristics of cell signaling; this is important, adenylyl cyclase, modulation of ion channel activity, and tran- especially for signaling in the complex neuronal networks in the scriptional changes in the cell (2). Opioid receptors (and many brain and enteric nervous system. Additionally, by preserving the other GPCRs) can also signal through non-G protein-mediated pathways, one of which is initiated by β-arrestin recruitment to the receptor. β-Arrestin is involved in receptor desensitization and Author contributions: N.T.B., M.J.C., T.S.W., S.W.G., M.B., J.O., J.R.T., and A.A. designed research; N.T.B. and M.J.C. performed research; T.S.W., S.W.G., and J.R.T. contributed new internalization/recycling (3, 4). reagents/analytic tools; N.T.B., M.J.C., S.W.G., J.R.T., and A.A. analyzed data; and N.T.B., Opioid receptors are key targets in the management of pain T.S.W., J.R.T., and A.A. wrote the paper. and morphine and its derivatives induce pain relief by acting as The authors declare no conflict of interest. μ agonists at opioid receptors, especially the -opioid receptor (5, 6). This article is a PNAS Direct Submission. Opioid receptors have been extensively studied because of the need 1 To whom correspondence may be addressed. E-mail: [email protected] or andrew. for better pain control while trying to reduce or eliminate adverse [email protected]. side effects. These side effects include tolerance, respiratory sup- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. pression, constipation, allodynia, and dependence (3, 7). 1073/pnas.1300393110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1300393110 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 temporal aspects of native receptor signaling, PAMs may avoid (agonist-detection mode), but both compounds significantly aug- receptor down-regulation and other compensatory mechanisms mented the β-arrestin–recruitment response produced by a low that are triggered by sustained receptor activation produced by concentration of endomorphin-I (PAM-detection mode) (Fig. 1 C exogenous orthosteric agonists. Therefore, one can speculate and D). In PAM-detection mode, BMS-986121 increased that opioid receptor PAMs could produce less tolerance and β-arrestin recruitment by 20 nM endomorphin-I to a maximal dependence than exogenous orthosteric agonists. Here, we de- effect (Emax) of 76% [95% confidence interval (CI): 69–83%] of scribe the discovery and characterization of μ-opioid receptor the response evoked by a maximally effective (1 μM) concen- PAMs and SAMs. A high-throughput screen (HTS) was de- tration of endomorphin-I, with an EC50 of 1.0 μM (95% CI: 0.7– veloped and executed using a β-arrestin–recruitment assay. 1.6 μM). BMS-986122 produced a similar PAM-detection mode μ-Selective PAMs resulting from the HTS were shown to be active response, increasing the effect of the low concentration of in β-arrestin–recruitment assays and in G protein-mediated sig- endomorphin-I to 83% (95% CI: 78–89%) of the maximal 35 μ – naling assays (inhibition of adenylyl cyclase activity and [ S] endomorphin-I response with an EC50 of 3.0 M (95% CI: 1.9 GTPγS binding). These studies describe the existence of μ-selec- 3.9 μM). tive PAMs and SAMs, implicating positive allostery as a potential We have no direct evidence that these compounds bind to the avenue for the discovery of tightly regulated pain therapeutics. μ-opioid receptor as opposed to an associated protein. To approach this problem, the compounds were examined in a similar assay in Results and Discussion U2OS PathHunter cells expressing PK-tagged δ-opioid receptors fi Identification of μ-Opioid Receptor PAMs. Potential opioid receptor (U2OS-OPRD1). Neither compound had any signi cant effect ligands were identified from an HTS campaign using the Path- in the absence (agonist-detection mode) or the presence (PAM-detection mode) of an ∼EC10 (0.4 nM) of the δ agonist Hunter enzyme complementation assay technology (DiscoveRx) C D (15). In this system, an N-terminal deletion mutant of β-galacto- leu-enkephalin (Fig. 1 and ). Therefore, the compounds ap- β pear to be selective for μ- over δ-opioid receptors, which would sidase is fused to the C terminus of stably expressed -arrestin 2 in μ human osteosarcoma (U2OS) cells. A mutated amino-terminal support a direct interaction with receptors. However, we fragment of β-galactosidase, termed ProLink/enzyme donor (PK), cannot rule out that the compounds might be SAMs at the μ δ-opioid receptor.