Background and purpose: Biased GPCR ligands are able to engage with their target receptor in a manner that preferentially activates distinct downstream signalling and offers potential for next generation therapeutics. However, accurate quantification of ligand bias in vitro is complex, and current best practice is not amenable for testing large numbers of compound. We have therefore sought to apply ligand bias theory to an industrial scale screening campaign for the identification of new biased μ receptor agonists.
Experimental approach: μ receptor assays with appropriate dynamic range were developed for both Gαi -dependent signalling and β-arrestin2 recruitment. Δlog(Emax /EC50 ) analysis was validated as an alternative for the operational model of agonism in calculating pathway bias towards Gαi -dependent signalling. The analysis was applied to a high throughput screen to characterize the prevalence and nature of pathway bias among a diverse set of compounds with μ receptor agonist activity.
Key results: A high throughput screening campaign yielded 440 hits with greater than 10-fold bias relative to DAMGO. To validate these results, we quantified pathway bias of a subset of hits using the operational model of agonism. The high degree of correlation across these biased hits confirmed that Δlog(Emax /EC50 ) was a suitable method for identifying genuine biased ligands within a large collection of diverse compounds.
Conclusions and implications: This work demonstrates that using Δlog(Emax /EC50 ), drug discovery can apply the concept of biased ligand quantification on a large scale and accelerate the deliberate discovery of novel therapeutics acting via this complex pharmacology.
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The CB2 cannabinoid receptor (CB2) remains a tantalizing, but unrealized therapeutic target. CB2 receptor ligands belong to varied structural classes and display extreme functional selectivity. Here, we have screened diverse CB2 receptor ligands at canonical (inhibition of adenylyl cyclase) and noncanonical (arrestin recruitment) pathways. The nonclassic cannabinoid (-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol (CP55940) was the most potent agonist for both pathways, while the classic cannabinoid ligand (6aR,10aR)-3-(1,1-Dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran JWH133) was the most efficacious agonist among all the ligands profiled in cyclase assays. In the cyclase assay, other classic cannabinoids showed little [(-)-trans-Δ(9)-tetrahydrocannabinol and (-)-(6aR,7,10,10aR)-tetrahydro-6,6,9-trimethyl-3-(1-methyl-1-phenylethyl)-6H-dibenzo[b,d]pyran-1-ol] (KM233) to no efficacy [(6aR,10aR)-1-methoxy-6,6,9-trimethyl-3-(2-methyloctan-2-yl)-6a,7,10,10a-tetrahydrobenzo[c]chromene(L759633) and (6aR,10aR)-3-(1,1-dimethylheptyl)-6a,7,8,9,10,10a-hexahydro-1-methoxy-6,6-dimethyl-9-methylene-6H-dibenzo[b,d]pyran]L759656. Most aminoalkylindoles, including [(3R)-2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenyl-methanone, monomethanesulfonate (WIN55212-2), were moderate efficacy agonists. The cannabilactone 3-(1,1-dimethyl-heptyl)-1-hydroxy-9-methoxy-benzo(c)chromen-6-one (AM1710) was equiefficacious to CP55940 to inhibit adenylyl cyclase, albeit with lower potency. In the arrestin recruitment assays, all classic cannabinoid ligands failed to recruit arrestins, indicating a bias toward G-protein coupling for this class of compound. All aminoalkylindoles tested, except for WIN55212-2 and (1-pentyl-1H-indol-3-yl)(2,2,3,3-tetramethylcyclopropyl)-methanone (UR144), failed to recruit arrestin. WIN55212-2 was a low efficacy agonist for arrestin recruitment, while UR144 was arrestin biased with no significant inhibition of cyclase. Endocannabinoids were G-protein biased with no arrestin recruitment. The diarylpyrazole antagonist 5-(4-chloro-3-methylphenyl)-1-[(4-methylphenyl)methyl]-N-[(1S,2S,4R)-1,3,3-trimethylbicyclo[2.2.1]hept-2-yl]-1H-pyrazole-3-carboxamide (SR144258) was an inverse agonist in cyclase and arrestin recruitment assays while the aminoalkylindole 6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)methanone (AM630) and carboxamide N-(1,3-benzodioxol-5-ylmethyl)-1,2-dihydro-7-methoxy-2-oxo-8-(pentyloxy)-3-quinolinecarboxamide (JTE907) were inverse agonists in cyclase but low efficacy agonists in arrestin recruitment assays. Thus, CB2 receptor ligands display strong and varied functional selectivity at both pathways. Therefore, extreme care must be exercised when using these compounds to infer the role of CB2 receptors in vivo. View publication