# Molecular Targets of Minor Cannabinoids in Breast Cancer: In Silico and In Vitro Studies
## 3 Discussion
Additionally, despite the absence of in silico evidence of a relevant modulation of aromatase and ER\(\alpha\), **CBG** and **CBN** were included in our study, as some studies in different tumors, including triple-negative breast cancer, have pointed out their therapeutic potential as anti-cancer compounds [75; 76]. Regarding aromatase activity, we confirmed that **CBG** and **CBN** do not inhibit this enzyme. However, none of the other seven cannabinoids were able to greatly inhibit aromatase (only up to 12% inhibition). **CBD**, as expected and corroborating our previous work [65], presented high anti-aromatase activity. In fact, our previous molecular docking studies suggested the binding of **CBD** to key aromatase residues [65] and herein, **CBD** displayed the best docking score (\(-8.7\) kcal/mol), followed by **CBDV** (\(-7.6\) kcal/mol), which was found to inhibit aromatase by \(12\%\) at \(10\) uM. For aromatase inhibition, among the investigated cannabinoids, the presence of a carboxylic acid seems detrimental for activity (comparing, for example, **CBG** with **CBGVA**) and slight modifications in the hydrophobic tail of the cannabidiol type of metabolites influence the affinity for the enzyme, as evidenced when comparing **CBDV** (C\({}_{3}\)) with its homologues **CBDB** (C\({}_{4}\)) and **CBDM** (C\({}_{5}\)). In relation to ER\(\alpha\), different results were obtained for the nine studied phytocannabinoids. According to our transactivation results, cannabidiol derivatives **CBDV**, **CBDB**, and **CBDM** are ER\(\alpha\) agonists, with **CBDV** and **CBDB** presenting full agonist activity properties (i.e., \(75\)-fold increase over basal response [105]). The ER agonist activity verified for these cannabinoids is not in line with previous studies that, based on **THC** and **CBD** activities, indicated that cannabinoids do not exhibit ER agonist activity [62]. Although this agonist activity is not attractive for ER\({}^{+}\) breast cancer cases, it may be clinically relevant in other pathological conditions, such as neurodegenerative disorders, liver injuries, or cardiovascular diseases, where a beneficial action should not be ruled out [106; 107]. On the other hand, **CBN**, **CBDA**, **CBCA**, and **CBC** may potentially act as ER inverse agonists, which may be beneficial for ER\({}^{+}\) breast cancer cases since, by presenting opposite actions to ER agonists, they prevent the tumorigenic effects of ER\(\alpha\). These results correlate with in silico studies in which **CBDA** (\(-8.3\) kcal/mol) presented the highest affinity for the ER. For ER inverse agonism, cyclization, particularly chromene formation, was important for activity, as in the case of cannabichromene derivatives **CBC** and **CBCA**, and for chromane **CBN**. Moreover, except for the linear **CBGVA**, the carboxylic acid derivatives investigated (**CBCA**, **CBDA**) were active against this receptor. Nevertheless, the behavior exhibited by the phytocannabinoids changes under hormone influence. Contrary to **CBDV** that loses ER agonistic activity, **CBDB** and **CBDM** become ER antagonists in the presence of E\({}_{2}\). In the presence of T, **CBDA** maintains ER\(\alpha\) antagonist activity, while in the presence of E\({}_{2}\), this activity is abolished. These different behaviors can be explained by the binding affinities to ER\(\alpha\) in the presence of the different hormones. Moreover, the fixed E\({}_{2}\) concentration (\(91.8\) pM) used for the assessment of antagonistic activity induces the maximum activation of the receptor, which might be too high to detect activity of weaker antagonistic compounds. On the other hand, antagonistic activities in the presence of T (\(1\) nM) might be more notable due to the concentration of T used, which is not associated with maximum effects.