CB2 cannabinoid receptor agonist enantiomers HU-433 and HU-308: An inverse relationship between binding affinity and biological potency

Activation of the CB2 receptor is apparently an endogenous protective mechanism. Thus, it restrains inflammation and protects the skeleton against age-related bone loss. However, the endogenous cannabinoids, as well as Δ⁹-tetrahydrocannabinol, the main plant psychoactive constituent, activate both cannabinoid receptors, CB1 and CB2. HU-308 was among the first synthetic, selective CB2 agonists. HU-308 is antiosteoporotic and antiinflammatory. Here we show that the HU-308 enantiomer, designated HU-433, is 3–4 orders of magnitude more potent in osteoblast proliferation and osteoclast differentiation culture systems, as well as in mouse models, for the rescue of ovariectomy-induced bone loss and ear inflammation. HU-433 retains the HU-308 specificity for CB2, as shown by its failure to bind to the CB1 cannabinoid receptor, and has no activity in CB2-deficient cells and animals. Surprisingly, the CB2 binding affinity of HU-433 in terms of ³H]CP55,940 displacement and its effect on ³⁵S]GTPγS accumulation is substantially lower compared with HU-308. A molecular-modeling analysis suggests that HU-433 and -308 have two different binding conformations within CB2, with one of them possibly responsible for the affinity difference, involving ³⁵S]GTPγS and cAMP synthesis. Hence, different ligands may have different orientations relative to the same binding site. This situation questions the usefulness of universal radioligands for comparative binding studies. Moreover, orientation-targeted ligands have promising potential for the pharmacological activation of distinct processes.The CB2 cannabinoid receptor functions as an endogenous protective entity (1). Thus, it is an important regulator of bone mass and inflammation. It represents a therapeutic target that avoids the undesired psychotropic effects caused by CB1 receptor activation. CB2 is expressed in osteoblasts, the bone-forming cells, and in osteoclasts, the bone-resorbing cells (2). Monocytes/macrophages, B cells, certain T-cell subtypes, and mast cells also express CB2 (3–7). In the skeleton, activation of CB2 favors bone formation over resorption, thus protecting the skeleton against age-related bone loss. Inflammatory responses are restrained by CB2 agonists in several instances such as hepatic ischemia-reperfusion injury (8), uveitis (9), and contact dermatitis (10). With their terpene and resorcinol-derived moieties, some synthetic CB2 agonists, such as HU-308 (10), JWH-133 (11), and HU-910 (12), structurally resemble the phytocannabinoids Δ⁹-tetrahydrocannabinol and cannabidiol. Other, non-phytocannabinoid-type agonists have been also reported (13). HU-308 was one of the first fully characterized, highly selective, and highly efficacious cannabinoid type-2 agonist (10). It has three chiral centers, namely, at carbon atoms in positions 3, 4, and 6 (Scheme 1). HU-308 has a 3R, 4S, 6S configuration; that of its enantiomer (HU-433) is 3S, 4R, 6R.Open in a separate windowScheme 1.Structures of HU-433 and -308.In most experiments, the optimal HU-308 mitogenic activity in osteoblasts, as well as its antiosteoclastogenic effect, was obtained by using concentrations in the nanomolar range (2, 14). Surprisingly, testing a new HU-308 batch, we noticed a 3- to 4-magnitude decrease in the dose that triggers optimal proliferative response in osteoblasts, reasoning that this preparation contained a significant amount of the HU-308 enantiomer, presumably responsible for the enhanced activity. We report here that this enantiomer, designated HU-433, was synthesized and compared with HU-308, and indeed showed markedly enhanced bone-anabolic and antiinflammatory activities, but inferior CB2 receptor binding affinity. Although both enantiomers seem to target the same binding pocket, two different orientations relative to the binding site are possible, leading to different behavior of the two enantiomers due to their different occupancy of these orientations. This observation appears to reflect a situation in which relatively small differences in possible binding conformations of the ligands within the receptor—referred to as poses—lead to significant diminution of receptor-binding properties and a marked increase in biological activity.