# Molecular Targets of Minor Cannabinoids in Breast Cancer: In Silico and In Vitro Studies
## 2 Results
### Molecular Docking Results
In fact, the protein-ligand complexes were clearly overlapping, demonstrating the stability of the complexes and the lack of significant conformational changes related to the ligand binding. Furthermore, the  
Figure 3: Molecular docking of the nine minor cannabinoids, **CBG** (**A,B**), **CBDV** (**C,D**), **CBN** (**E,F**), **CBDB** (**G,H**), **CBDA** (**I,J**), **CBDM** (**K,L**), **CBCA** (**M,N**), **CBGVA** (**O,P**), and **CBC** (**Q,R**), in aromatase. The images represent both 2D and 3D aromatase/cannabinoid interactions. Arornatase structure has the PDB code 3S79 and is represented in as cartoon. The main amino acid residues of the target, including Asp309, Phe221, Trp224, Arg115, Leu372, Leu477, Ser478, and Met374, as well as the heme group, are shown as gray sticks, while the cannabinoids are shown as different colorful sticks. Hydrogen bonds are represented as black dashed lines in the 3D representations and as purple arrows in the 2D maps. MD simulations indicate that the complex structures are in a stable state as the potential energy at the end of the simulation is lower than the initial conformations, indicating more stability of the system (Supplementary Figure S4). As demonstrated by RMSD plots, all the ligands were stable after 2-6 ns with an RMSD value of around 2 A, and the complex backbone acquired a stable trajectory beyond 2-6 ns with an RMSD value of 2.5-3 A (Supplementary Figure S4). The complete MD simulation study was carried out for 10 ns, during which the ligand lost some of the interactions established in the pre-MD docked pose (Figure 4), and established new ones, as evident from Figure 5.  
Figure 4: Molecular docking of the nine minor cannabinoids, **CBG** (**A**,**B**), **CBDV** (**C**,**D**), **CBN** (**E**,**F**), **CBDB** (**G**,**H**), **CBDA** (**I**,**J**), **CBDM** (**K**,**L**), **CBCA** (**M**,**N**), **CBGVA** (**O**,**P**), and **CBC** (**Q**,**R**), as well as **CBD** (**S**,**T**), in ER\(\alpha\). The images represent both 2D and 3D ER\(\alpha\)/cannabinoid interactions. ER\(\alpha\) structure was generated by the authors and is represented in as cartoon. The main amino acid residues of the target, including Asp351, Glu353, Arg394, Phe404, Met421, His 524, Leu525, Val534, and Trp383, are shown as gray sticks, while the cannabinoids are shown as different colorful sticks. Hydrogen bonds are represented as black dashed lines in the 3D representations and as purple arrows in the 2D maps. The results showed that, with the exception of **CBDM** (Figure 5F), for all the other cannabinoids the complex ER\(\alpha\)/cannabinoid acquires a position in which the cannabinoid slightly moves away from Asp351, a residue that is essential for the antagonistic activity, and gets buried in the bottom of the ER\(\alpha\) binding site, suggesting that these compounds may not antagonize the receptor (Figure 5). However, considering the 2D maps, **CBDM** (Figure 5L) and **CBD** (Figure 5T) are complexed in the vicinity of Asp351 (referred to as Asp47 in the 2D maps), suggesting that these cannabinoids might influence the antagonistic conformation of the receptor. In fact, in vitro studies have already demonstrated that **CBD** displays antagonistic activity on ER [44]. Nevertheless, the cannabinoids were still able to establish some important interactions for their stability, namely with Phe404 and Trp383. AR has been receiving a lot of attention for its role in ER\({}^{+}\) breast cancer development and, consequently, as a potential therapeutic target for this disease [24; 86]. According to the  
Figure 5: Molecular dynamics simulation on ER\(\alpha\) for the nine minor cannabinoids, **CBG** (**A,B**), **CBDV** (**C,D**), **CBN** (**E,F**), **CBDB** (**G,H**), **CBDA** (**I,J**), **CBDM** (**K,L**), **CBCA** (**M,N**), **CBGVA** (**O,P**), and **CBC** (**Q,R**), as well as **CBD** (**S,T**). A molecular dynamics simulation was performed around the conformation of the small molecules obtained from the docking analysis on ER\(\alpha\).