Computational Characterization of Nabilone-Induced Disruption of the CB2-HER2 Receptor Complex in HER2+ Breast Cancer
Laboratory of Drug Sciences (LRSM), Mohammed VI Faculty of Medicine Casablanca, Mohammed VI University of Sciences and Health (UM6SS), Morocco
Mohammed VI Center for Research and Innovation (CM6RI), Rabat, Morocco
Research Laboratory of Microbiology, Infectious Diseases, Allergology and Pathogen Surveillance (LARMIAS), Mohammed VI Faculty of Medicine Casablanca, Mohammed VI University of Sciences and Health (UM6SS), Morocco
Mohammed VI University of Sciences and Health (UM6SS), Casablanca, Morocco
Biotechnology Lab (MedBiotech), Bioinova Research Center, Medical and Pharmacy School, Mohammed V University, Rabat, Morocco
Abbou Hanane, Mohammed VI Center for Research and Innovation, Mohammed El Jazouli Street, Rabat 10112, Morocco. Email: habbou@um6ss.ma; hanane.abbou95@gmail.comAbstract
Human epidermal growth factor receptor 2-positive (HER2+) breast cancer, accounting for 15% to 20% of cases, is often resistant to treatment. Delta-9-tetrahydrocannabinol (THC) disrupts HER2-cannabinoid receptor (2CB2) receptor complexes and inhibits HER2 activation. This study evaluates whether Nabilone, a synthetic cannabinoid, can similarly disrupt HER2-CB2 interactions. A CB2-HER2 complex model was generated via protein-protein docking. Three 1-µs molecular dynamics simulations (CB2-HER2, CB2-HER2-THC, CB2-HER2-Nabilone) were performed using the Schrodinger Desmond with membrane embedding and solvent. Structural stability (root mean square deviation [RMSD] and root mean square fluctuation [RMSF]), binding free energy (molecular mechanics/generalized born surface area [MM/GBSA]), and intracellular/extracellular distances between receptors were analyzed. Intermolecular interactions were assessed using the MAPIYA server. Nabilone induced comparable structural instability to THC, with increased RMSD and RMSF. The MM/GBSA analysis showed Nabilone increased the binding free energy between CB2 and HER2, indicating stronger disruption. Intracellular and extracellular distances between CB2 and HER2 increased, especially intracellularly, with Nabilone. Intermolecular interaction analysis revealed that Nabilone decreased the number of contacts, particularly hydrophobic interactions, between CB2 and HER2. Our in silico model predicts that Nabilone may disrupt the HER2-CB2 complex, suggesting a hypothesis that it could serve as a potential therapeutic agent. These computational findings warrant urgent experimental validation.
Introduction
Breast cancer (BC) is a heterogeneous disease subdivided into hormone receptor-positive, human epidermal growth factor receptor 2-positive (HER2+), and triple-negative subtypes, guiding treatment decisions. The HER2, amplified or overexpressed in 15% to 20% of BC cases, is an oncogene that promotes proliferation, angiogenesis, and metastasis through dimerization with other erythroblastic oncogene B (ErbB) receptors.1bibr2-11779322261449332-3 Overexpression of this receptor is associated with higher rates of disease recurrence, brain metastasis, and mortality. 4
Despite the efficacy of HER2-targeted therapies, many patients show innate or acquired resistance due to mutations, receptor variants, alternative pathway activation, or inhibited cell death.5bibr6-11779322261449332bibr7-11779322261449332bibr8-11779322261449332bibr9-11779322261449332bibr10-11779322261449332bibr11-11779322261449332bibr12-11779322261449332bibr13-11779322261449332bibr14-11779322261449332bibr15-11779322261449332bibr16-11779322261449332bibr17-11779322261449332-18
Cannabinoids have produced antitumor responses in preclinical models of cancer, including HER2+ BC, via binding and activating cannabinoid receptors, CB1 and CB2, both G-protein coupled receptors (GPCRs).19bibr20-11779322261449332bibr21-11779322261449332-22 The CB1 mediates the psychoactive effects of cannabis, while CB2 is primarily expressed in the immune system but is upregulated in cancers, including HER2+ BC, where about 90% of tumors overexpress CB2.23,24 The research conducted so far in preclinical models of HER2+ BC points to CB2 as the main target of cannabinoid antitumor action.25,26
The CB2-HER2 receptor dimers on cancer cell surfaces correlate with poor prognosis; however, disrupting these heterodimers with the CB2 agonist, delta-9-tetrahydrocannabinol (THC), hampers HER2 activation and reduces HER2+ BC cell viability by destabilizing transmembrane interactions between CB2 transmembrane helix 5 (TM5) and HER2 transmembrane helix (TM) and activating CB2. 27
Nabilone, a synthetic analog of THC, was Food and Drug Administration (FDA)-approved in 1985 as a relief treatment for chemotherapy-related side effects, such as vomiting and nausea. 28 In this computational study, we use a bioinformatics approach to predict the capacity of Nabilone to alter the physical interaction between HER2 and CB2. This drug repurposing strategy29,30 aims to generate a strong, data-driven hypothesis for a new therapeutic avenue, which could potentially bypass HER2-related therapy resistance. All findings presented are in silico and require future experimental validation.
Methods
The starting 3-dimensional (3D) structures were selected based on their high resolution and structural relevance to the human protein sequences. The human CB2 receptor structure 5ZTY 31 was chosen because it represents a high-resolution (2.8 Å) crystal structure of the human receptor. While it is an antagonist-bound (inactive) state, it provides the most reliable template for modeling the orthosteric binding site and the critical transmembrane helix 5 (TM5) required for heterodimerization studies. For HER2, the nuclear magnetic resonance (NMR)-derived structure 2N2A was selected because it specifically captures the human HER2 TM domain in a dimeric state. As the TM domain is the primary site of interaction with CB2 TM5, 2N2A provides a validated structural basis for modeling the receptor-receptor interface within the lipid bilayer.
The 5ZTY PDB was prepared by reverting mutations using the Schrodinger Maestro module 32 and remodeling loops using SwissModel. 33 The quality of the resulting model was evaluated using PROCHECK.34,35 The stereochemical quality and structural integrity were further validated using the MolProbity server. 36 The model’s all-atom clashscore and rotamer consistency were evaluated to ensure physical viability.
The HER2 structure 2N2A 37 and the remodeled CB2 were prepared using the Protein Preparation wizard in the Schrodinger Maestro module. The 3D structures of Nabilone and THC were obtained from PubChem 38 and prepared using LigPrep. 39
Protein-protein docking between CB2 TM5 (from 189 to 214 aa) and the HER2 TM (from 653 to 675 aa) domain using HADDOCK 2.440,41 and then the binding affinity between the 2 proteins were assessed using PRODIGY,42,43 and the quality of the complex was assessed using the Ramachandran plot generated from PROCHECK.34,35 The ligands were docked into the CB2 orthosteric site within the CB2-HER2 complex employing Induced Fit Docking (IFD). 44
The molecular dynamics (MD) simulations were performed using the Schrödinger Desmond 32 with the optimized potentials for liquid 4 (OPLS4) force field and the TIP3P water model. The receptor-ligand complexes were embedded in a POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) lipid bilayer, aligned according to OPM (Orientations of Proteins in Membranes) coordinates (Supplementary 1). 45 The system was solvated in an orthorhombic box with a 10-Å buffer, neutralized with Cl− counterions, and maintained at a physiological salt concentration of 0.15 M NaCl. Before the production run, the system underwent a multistage equilibration protocol: (1) Brownian dynamics NVT simulation at 10 K (50 ps) with restraints on solute heavy atoms; (2) NVT and NPgT simulations at 100 K; (3) heating from 100 K to 300 K over 150 ps; and (4) a final relaxation step at 300 K. Three independent 1000 ns (1 µs) production simulations were conducted for the CB2-HER2, CB2-HER2-THC, and CB2-HER2-Nabilone complexes using fixed seed parameters to ensure reproducibility.
Structural dynamics were analyzed using root mean square deviation (RMSD) and root mean square fluctuation (RMSF). The molecular mechanics/generalized born surface area (MM/GBSA) binding free energies of receptor-receptor (MM/GBSA-PP) and receptor-ligand complexes (MM/GBSA-PL) were calculated with HawkDock 46 and Prime MM/GBSA. 47 It is worth mentioning that the MM/GBSA method implemented in the HawkDock server is calculated as the sum of the van der Waals interaction energy, the electrostatic interaction energy, the polar solvation energy, and the non-polar solvation energy estimated from the solvent-accessible surface area. This method does not explicitly calculate the conformational entropy change upon binding. Consequently, the resulting values are enthalpy-dominated and typically exhibit larger negative magnitudes compared to experimental absolute binding affinities.
Intermolecular interactions were quantified using MAPIYA, analyzing final MD frames. Intracellular (Gly29-Trp214) and extracellular (Ser10-Leu196) distances between CB2 and HER2 were monitored during simulations. Residue numbering is based on the used Protein Data Banks (PDBs).
Results
The Ramachandran plot of the re-engineered CB2 receptor (Figure 1B) indicates a high-quality structure, as 92.3% of the residues are located within the favorable regions, 6.6% within the allowed regions, 1.1% within the generously allowed regions, and 0% within the disallowed regions, supporting its use to model the CB2-HER2 complex (Figure 1A). The structural integrity of the re-engineered CB2 receptor was further validated using the MolProbity server. The model achieved a MolProbity score of 1.45, placing it in the 96th percentile relative to all structures in the database. The all-atom clashscore was 2.48 (99th percentile), indicating minimal steric overlaps.
The docking protocol conducted using the HADDOCK 2.4 40 , 41 produced multiple conformations; the best-scoring model had 90.5% of residues in the most favored Ramachandran regions and 9.2% within the additionally allowed regions. The PRODIGY42,43 analysis estimated a binding affinity of −7.1 kcal/mol between CB2 and HER2, involving diverse interaction types (Table 1 and Figure 2).
| Protein-protein complex | CB2-HER2 |
|---|---|
| ΔG (kcal/mol) | −7.1 |
| Kd (M) at °C | 6.00E-06 |
| ICs charged-charged | 2 |
| ICs charged-polar | 4 |
| ICs charged-apolar | 12 |
| ICs polar-polar | 3 |
| ICs polar-apolar | 14 |
| ICs apolar-apolar | 31 |
| NIS charged (%) | 13.64 |
| NIS apolar (%) | 58.44 |
Protein-ligand docking with the Schrodinger IFD provided multiple poses for THC and Nabilone in the CB2 orthosteric site. The top-ranked poses exhibited binding affinities of −9.95 kcal/mol for THC and −9.88 kcal/mol for Nabilone, serving as starting points for MD simulations (Supplementary 2). These results align with established experimental pharmacology results, as a benchmark kinetic study reported that both ligands exhibit high nanomolar affinity for the CB2 receptor, with THC showing a Ki of approximately 8.6 nM and Nabilone a Ki of 14.1 nM. While the thermodynamic affinities are similar, the kinetic profiles distinguish these agonists significantly: Nabilone exhibits a residence time (26.6 min) nearly 13-fold longer than that of THC (2.1 min). 48
The structural analysis of the CB2-HER2 complex without any ligands provides a baseline for understanding the effects of THC and Nabilone. The RMSD values averaged 3.24 Å, indicating moderate structural stability. The RMSF analysis highlighted residues such as ASP189 and TRP214 (present in the interaction surface between CB2 and HER2), with values of 0.89 Å and 0.96 Å, respectively, suggesting these residues are relatively stable (Figure 3). A focused RMSF analysis, where we focus on each protein separately, shows that CB2 averaged 1.28 Å, while HER2 averaged 2.08 Å, indicating that the CB2 receptor is more stable than HER2 (Supplementary 3). The MM/GBSA-PP values for CB2-HER2 were −91.65 kcal/mol, showing strong binding between the 2 receptors (Table 2). The intermolecular interactions indicate 69 contacts between CB2 and HER2, among which the hydrophobic interaction displays the most abundant interaction type, accounting for 36 out of 69 interactions (Figure 4). In addition, the calculated distance between the extracellular region of HER2-TM and CB2-TM5 (10.03 Å) and the intracellular region of the same helices (4.89 Å) demonstrated a relatively stable distance, emphasizing the stable interaction between the 2 proteins (Figure 5).
| Complex | VDW (kcal/mol) | ELE (kcal/mol) | GB (kcal/mol) | SA (kcal/mol) | Total (kcal/mol) |
|---|---|---|---|---|---|
| CB2-HER2 | −130.9 | 465.25 | −409.86 | −16.13 | −91.65 |
| C2-HER2-THC | −81.62 | 398.82 | −362.1 | −9.91 | −54.8 |
| CB2-HER2-Nabilone | −80.77 | 404.25 | −364 | −9.88 | −50.39 |
Thus, the second MD simulation, of the CB2-HER2-THC complex, was carried out to validate the ability of the complex to simulate the activity of THC against the CB2-HER2 complex, which is interfering with the physical interaction between the 2 receptors. 27 Upon the introduction of THC to the CB2-HER2 system, notable changes in structural stability and flexibility were observed. The RMSD values increased, with an average of 3.79 Å, a maximum of 5.50 Å, and a minimum of 1.60 Å. This indicates that the THC-bound system is globally less stable compared to the CB2-HER2 complex. The RMSF analysis highlights a significant increase in the flexibility of several residues in the CB2 region that binds to HER2, such as ILE206 (RMSF: 0.90 Å) and TYR207 (RMSF: 1.12 Å). These fluctuations suggest that THC binding induces conformational changes that enhance flexibility (Figure 3). The HER2 TM region also exhibited increased flexibility, particularly in the residues PHE671 (RMSF: 2.26 Å) and LEU674 (RMSF: 2.59 Å). The focused RMSF analysis showed that CB2 (RMSF avg = 1.49 Å) keeps the tendency to stay less flexible than HER2 (RMSF avg = 2.13 Å), but it is more flexible than it was in the absence of ligands (Supplementary 3). The MM/GBSA-PP results exhibit a binding free energy of −54.80 kcal/mol, indicating a noticeably weakened interaction between CB2 and HER2 in the presence of THC (Table 2). The MM/GBSA-PL analysis shows a strong binding energy of −66.10 kcal/mol between CB2 and THC, indicating a stable and strong interaction throughout the simulation (Table 3). Analysis of the interaction profile (Supplementary 4) reveals that THC is firmly anchored by a consistent hydrogen bond with THR114 and robust hydrophobic contacts with PHE183. Beyond this stabilization, THC also engages the toggle switch TRP258 through specific hydrophobic interactions, suggesting that the switch is broken, and therefore, the CB2 receptor is activated, as seen in the conformational state of TRP258, in comparison to both the active and inactive states of CB2 (Supplementary 6). 49 The intermolecular contacts data generated by MAPIYA reveal a significant reduction in the total interactions between CB2 and HER2, dropping from 69 in the unliganded complex to 39 in the presence of THC. Hydrophobic interactions, the most prominent contact type, decreased from 35 to 26 occurrences (Figure 4). The PHE202 (CB2) redirected its interaction from VAL658 to ILE661 (HER2), altering the extracellular alignment of the 2 receptors. The central core residues ILE206 and LEU213 (CB2) maintained specific contacts with VAL665 and VAL669 (HER2), but they lost their stabilizing interactions with VAL658 and GLY672. This suggests that while THC weakens the dimer by pruning peripheral contacts, it preserves a residual hydrophobic interaction. On the contrary, the average extracellular and intracellular distances between the 2 receptors were 11.50 Å and 8.10 Å, respectively, both of which indicate a clear elevation of the distance between the 2 proteins, which correlates with the lower number of interactions between them after THC binding (Figure 5).
| Complex | ΔG average (kcal/mol) | ΔG standard deviation (kcal/mol) | ΔG range (kcal/mol) |
|---|---|---|---|
| C2-HER2-THC | −66.10 | 25.80 | −86.6536 to 0.1773 |
| CB2-HER2-Nabilone | −72.76 | 32.14 | −97.7323 to 0.1622 |
The presence of Nabilone in the CB2-HER2 complex resulted in notable structural changes. The RMSD values averaged 3.81 Å, with a maximum of 5.10 Å and a minimum of 2.08 Å, indicating a level of instability similar to THC. The RMSF values showed moderate increases in flexibility, with an average of 1.38 Å, a maximum of 8.65 Å, and a minimum of 0.50 Å. Residues present in the interaction surface, such as ILE206 (RMSF = 0.732 Å) and TYR207 (RMSF = 0.75 Å) exhibited less flexibility than the THC-bound system, yet slightly elevated than the CB2-HER2 complex (Figure 3). As for HER2, the RMSF values (RMSF avg = 2.56 Å) are noticeably higher than HER2 RMSF in the presence of THC, as shown in the focused RMSF analysis (Supplementary 3). Our initial observation is that Nabilone can destabilize the CB2-HER2 complex but has minimal effect on the flexibility of the CB2 receptor (RMSF avg = 1.23 Å), in contrast to the high flexibility it induced in HER2.
The MM/GBSA-PP value was higher than both the unliganded complex and THC-bound complex (−50.39 kcal/mol), which indicates that Nabilone weakens the CB2-HER2 interaction compared to THC (Table 2). Nabilone exhibits a distinct interaction profile, anchored by a unique water bridge with SER165, a hydrogen bond with THR114, and the toggle switch residue TRP258 is engaged in a hydrophobic interaction with Nabilone for a significantly longer fraction of the simulation time, compared to THC. Crucially, our analysis reveals that Nabilone induces a unique rotational state of this ‘toggle switch’ that is distinct from both the classical active (PDB: 6KPC) and inactive (PDB: 5ZTY) conformations. While the residue successfully shifts away from the inactive lock, it stabilizes in an alternative orientation, suggesting a potential ligand-specific activation mode. To our knowledge, this is the first study to provide an atomistic resolution of these unique structural dynamics induced by Nabilone at the CB2 receptor (Supplementary 6). The presence of Nabilone in the CB2-HER2 complex resulted in a reduced number of intermolecular interactions, down to 45 (compared to 69 in the unliganded complex). Although this total is slightly higher than in the THC-bound system (39), the nature of these contacts reveals a critical destabilization: hydrophobic interactions dropped to 22 occurrences (vs 26 with THC), while weaker van der Waals forces dropped to 14 (vs 10 with THC) (Figure 4). Nabilone induced the complete loss of the interaction in the N-terminal region, specifically between PHE202 (CB2) and VAL658 (HER2), effectively unzipping the extracellular interface. Furthermore, the central and intracellular locking mechanisms were compromised, as ILE206 and LEU213 (CB2) lost their stabilizing contacts with ILE661 and VAL665 (HER2), respectively. Since hydrophobic interactions are significantly stronger than van der Waals forces, 50 this specific loss of high-energy contacts explains why the cumulative interaction force is lower, as evidenced by the reduced MM/GBSA-PP value. The MM/GBSA-PL analysis shows that Nabilone has a free binding energy of −72.76 kcal/mol, which proves that Nabilone interacts better than THC with CB2 (Table 3). Finally, the distance between HER2 and CB2 was pronounced, as the distances between the extracellular and intracellular components of the 2 proteins were 10.68 Å and 8.28 Å (Figure 5). These distances, which are higher than those of the non-bounded complex and comparable to those of the THC-bound complex, further show Nabilone’s potential as a HER2+ BC inhibitor.
Discussion
The overexpression of HER2 and its dimerization with other cellular receptors is a well-established driver of aggressive tumor proliferation and therapy resistance in BC. 51 Recent oncological research has highlighted the CB2 receptor as a promising therapeutic target, given its significant upregulation in HER2+ tumors and its role in forming oncogenic CB2-HER2 heterodimers. While the disruption of these complexes by the phytocannabinoid THC has been experimentally shown to induce antitumor responses, 27 the precise atomistic mechanisms of this disruption, as well as the potential of synthetic analogs to replicate or improve upon this effect, have remained unexplored. To our knowledge, this study provides the first computational evidence detailing the structural dynamics of the CB2-HER2 heterodimer and hypothesizes the superior capacity of the FDA-approved synthetic cannabinoid, Nabilone, to disrupt this oncogenic complex.
Our findings align with and expand upon the foundational work by Blasco-Benito et al, 27 which established that THC exerts its antitumor efficacy by breaking the physical interaction between CB2 TM5 and HER2 TM, subsequently activating CB2. Our in silico baseline models confirm this phenomenon. In our simulations, the unliganded CB2-HER2 complex exhibited a highly stable interface driven primarily by 36 hydrophobic interactions, resulting in a strong binding free energy (MM/GBSA-PP of −91.65 kcal/mol). Upon the introduction of THC, this interaction network was significantly compromised; the total number of contacts dropped from 69 to 39, and the binding free energy weakened to −54.80 kcal/mol. This computational validation of existing in vitro and in vivo data underscores the reliability of our MD simulation protocol.
The primary novelty of our study lies in the comparative analysis of Nabilone. 52 While Nabilone induced a level of global structural instability (RMSD = 3.81 Å) comparable to THC, it achieved a more profound destabilization of the dimer interface. Nabilone specifically pruned high-energy hydrophobic contacts, dropping them to 22 occurrences compared to THC’s 26. Nabilone induced the complete loss of interaction in the N-terminal region between PHE202 (CB2) and VAL658 (HER2), effectively unzipping the extracellular interface of the heterodimer. The resulting MM/GBSA-PP value (−50.39 kcal/mol) indicates that Nabilone weakens the CB2-HER2 interaction even more effectively than THC.
This enhanced disruptive capacity correlates strongly with the distinct binding profiles of the 2 ligands. While previous benchmark kinetic studies reported that THC and Nabilone share similar thermodynamic affinities in the nanomolar range, Nabilone exhibits a residence time (26.6 min) nearly 13-fold longer than that of THC (2.1 min). 48 Our MM/GBSA-PL calculations strongly support these kinetic findings, revealing a superior binding free energy for Nabilone (−72.76 kcal/mol) compared to THC (−66.10 kcal/mol). This prolonged, highly stable occupation of the orthosteric site by Nabilone likely translates into the sustained mechanical strain observed at the heterodimer interface.
From a clinical translation perspective, these findings hold significant weight for drug repurposing.29,30 Nabilone is currently utilized in clinical settings exclusively for the palliative management of chemotherapy-induced nausea and vomiting.53,54 However, our data suggest that it possesses targeted, mechanism-based antineoplastic potential. By actively dismantling the CB2-HER2 heterodimer and inducing a unique state of CB2 activation, Nabilone could circumvent common resistance pathways associated with traditional HER2-targeted monoclonal antibodies or kinase inhibitors.55,56
The combination of the results obtained from our MD simulation analyses, MM/GBSA analyses, and intermolecular analyses supports the initial hypothesis, which is the ability of Nabilone to induce a disruption and reduction of the interactions between CB2 and HER2 receptors, upon binding to the CB2 orthosteric binding site. Beyond the physical destabilization of the interface, this dissociation likely triggers downstream signaling changes that inhibit tumor progression. Network pharmacology analysis identifies the cannabinoid receptor 2 gene (CNR2) as a master regulatory hub whose signaling structurally converges on multiple oncogenic pathways. 57 Specifically, CB2 activation in HER2-driven models has been shown to suppress the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) axis, a pathway crucial for cell survival and growth, leading to reduced Akt activation and subsequent apoptosis. 58 Furthermore, CB2 signaling acts as a molecular brake on mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) machinery by hindering the activation of ERK1/2, thereby impairing the proliferation signals required for tumor growth. 59 These findings provide a biological rationale for our in silico model, suggesting that Nabilone-induced CB2-HER2 dissociation effectively switches off these core pro-survival nodes.
Conclusion
Our results indicate that Nabilone effectively disrupts the oncogenic CB2-HER2 complex, weakening the heterodimer interface through a mechanism of structural instability similar to THC but with superior binding affinity to CB2. While these findings rely on in silico predictions, limited by simulation timescales and simplified membrane models, they highlight a distinct opportunity for repurposing Nabilone from symptom management to active cancer therapy. We conclude that these data provide a robust theoretical framework that justifies urgent experimental validation in living systems to confirm the therapeutic potential of disrupting CB2-HER2 signaling.
Supplemental Material
Acknowledgements
The authors thank Mr Abdellah ABBOU for providing the workstation that made the computational biology calculations possible.