Cannabidiphorol (CBDP) acts as a negative allosteric modulator at two distinct sites of cannabinoid receptor 1

While Δ⁹-tetrahydrocannabinol (THC) offers significant pain relief, its psychoactive side effects limit its clinical utility. The discovery of cannabidiol (CBD) as a negative allosteric modulator (NAM) of cannabinoid receptor 1 (CB1R) establishes allosteric targeting as a viable framework for refining cannabinoid-based therapeutics. Cannabidiphorol (CBDP), a naturally occurring homolog of CBD identified from the FM2 chemovar of Cannabis sativa, is structurally similar to CBD but its pharmacology is largely unexplored. This study investigated the potential of CBDP as a NAM of CB1R. Site-directed mutagenesis, in vitro CB1R allosterism assays and molecular dynamics analyses revealed that CBDP acts as an NAM by stably engaging two distinct allosteric sites: the established ORG27569 site and an intracellular site located between transmembrane helices 1, 2, 6, 7, and helix 8. In vivo, tetrad assays and novel object recognition (NOR) testing in male C57BL/6NHsd mice suggested that CBDP co-administration with THC was associated with modest modulation of cognitive performance relative to THC alone, while THC's antinociceptive effects were preserved. These findings identify CBDP as a possible dual-site CB1R NAM and support future studies to evaluate its potential therapeutic and mechanistic utility.