Cannabitwinol, a Dimeric Phytocannabinoid from Hemp, Cannabis sativa L., Is a Selective Thermo-TRP Modulator

Abstract

Cannabitwinol (CBDD, 3), the second member of a new class of dimeric phytocannabinoids in which two units are connected by a methylene bridge, was isolated from a hemp (Cannabis sativa L.) industrial extract. The structural characterization of cannabitwinol, complicated by broadening of 1H NMR signals and lack of expected 2D NMR correlations at room temperature, was fully carried out in methanol-d 4 at −30 °C. All the attempts to prepare CBDD by reaction of CBD with formaldehyde or its iminium analogue (Eschenmoser salt) failed, suggesting that this sterically congested dimer is the result of enzymatic reactions on the corresponding monomeric acids. Analysis of the cannabitwinol profile of transient receptor potential (TRP) modulation evidenced the impact of dimerization, revealing a selectivity for channels activated by a decrease of temperature (TRPM8 and TRPA1) and the lack of significant affinity for those activated by an increase of temperature (e.g., TRPV1). The putative binding modes of cannabitwinol with TRPA1 and TRPM8 were investigated in detail by a molecular docking study using the homology models of both channels.

An Analysis of the Putative CBD Binding Site in the Ionotropic Cannabinoid Receptors

“…TRPV1 shares the highest sequence identity within the putative CBD binding site as described above at 79%, second only to TRPV2 itself. Additionally, CBD is reported to have the highest efficacy of the vanilloid subfamily at TRPV1 (∼78%) (Chianese et al, 2020).…”

“…Additionally, L537 shows a rotameric change, going from trans in the apo structure to g+ in the CBD-bound structure which is reported to allow accommodation for the aromatic ring of CBD. With two CBDbound cryo-EM structures identifying CBD in the same general location (Pumroy et al, 2019) and a reported efficacy of ∼67% at TRPV2 (Chianese et al, 2020), exploring this region should provide insight on the binding mode of CBD.…”

“…While both hydrophobic, the shorter chain of the valine might affect the extent of the lipophilic interactions within TRPV3 ( Figure 2B). While the efficacy of CBD at TRPV3 is generally considered poor, it still retains a submicromolar potency of around 0.51 µM (Chianese et al, 2020).…”

“…Initially, it appears that there are only moderate differences in the putative CBD binding site in TRPV4 and TRPV2 due to the 68% sequence homology of this region. However, CBD is the least efficacious at TRPV4 with a mere 15% efficacy (Chianese et al, 2020). The aspect that likely has the largest effect on CBD binding is the structurally different helical arrangement that TRPV4 takes in comparison to its family members.…”

“…Additionally, TRPA1 plays an important role in neuropathic and inflammatory pain through the mediation of bradykinin-evoked and mechanical hyperalgesia (Jordt et al, 2004). CBD has been shown to act as an agonist at TRPA1 with an efficacy of 108% compared to its usual agonist of allylisothiocyanate (100 µM) (Chianese et al, 2020), and while the putative CBD binding site is structurally comparable to that of the TRPVs, there is low sequence homology within the binding site (∼30%). Similar to the previous TRP channels discussed, TRPA1 maintains a leucine in the same position as in TRPV2 (L870 and L537, respectively).…”