Targeting the putative allosteric site J of cannabinoid receptor type 2 with peptide modulators: From mechanistic investigation to candidate identification.

Abstract

The cannabinoid receptor type 2 (CB2) is a promising therapeutic target for neuroinflammatory diseases, whereas conventional orthosteric agonists suffer from poor selectivity and metabolic instability. Allosteric modulation, particularly by peptide-based modulators, offers a new strategy to overcome these limitations, yet the molecular mechanisms underlying CB2 allosteric regulation and efficient discovery tools remain lacking. In this study, we computationally investigated the role of the CB2 extracellular putative site J as a predicted binding site for peptide modulators. Using molecular docking and dynamics simulations, we obtained computational insights into the dynamic hydrogen-bonding networks through which the known peptide allosteric modulators Pepcan-12 and osteogenic growth peptide (OGP) are predicted to selectively bind to putative site J in the active-state receptor, with key predicted interacting residues including Ser180, Asn188, Thr272 and Met22. Based on this computational model, we established a flexible docking screening framework targeting putative site J and identified five promising sequences from 56 peptides, among which Pep-5 exhibited the best performance. In vitro functional assays demonstrated that Pep-5 concentration-dependently inhibited cAMP accumulation (EC₅₀ = 6.0 μM) in a manner dependent on CB2 expression. Furthermore, Pep-5 increased the cAMP inhibitory effect of the orthosteric agonist WIN55,212-2, an observation consistent with potential positive allosteric modulation. Through an integrated strategy of "computational mechanistic exploration-rational design-functional validation", this study proposes the putative site J as a potential allosteric site for peptide-based modulation of CB2 and provides candidate peptide Pep-5, offering a new theoretical basis for developing CB2-targeted allosteric drugs.