Discovery of potential small molecule inhibitors against β-hCG: an <i>in silico</i> study with <i>in vitro</i> validation

Previous <i>in vitro</i> and <i>in vivo</i> studies have shown an association between BRCA1-defective cancers and the expression of β-hCG, where accelerated tumor progression has been observed in the presence of β-hCG due to its binding to and phosphorylation of TGFβR-II. Given the absence of reported small molecule inhibitors against β-hCG, the present study endeavours to identify the interacting residues of β-hCG with TGFβR-II. Through virtual screening, molecular docking and dynamic simulation studies, the investigation identified six potential small molecule inhibitors, namely, cefotetan, 2',7'-dichlorofluorescein (DCF), 2',7'-difluorofluorescein (DFF), F6658-4634, F0922-0590 and F0385-0029. Notably, all inhibitors exhibited interacting residues on β-hCG that coincided with the site to which it binds to TGFβR-II. Further <i>in vitro</i> MTT assays showed a reduction in proliferation in various cell lines, including the cell line developed in our laboratory, which was induced to have BRCA1 promoter hypermethylation using modified CRISPR technology. Binding studies such as Microscale Thermophoresis (MST) and Isothermal Titration Calorimetry (ITC) exhibited the binding of cefotetan to hCG, which could be indicative of the selective cytotoxic effect of cefotetan in β-hCG secreting breast cancer cell lines. The results indicate selective inhibition by these potential inhibitors in BRCA1-defective breast cancer cell lines, with cefotetan being the best among all with an IC₅₀ of 32 μM. This is the first study to report the anticancer activity of cefotetan, an FDA-approved drug used to treat bacterial infections, and this drug could be repurposed as a targeted therapeutic against β-hCG expressing cancers. Findings of this study hold significant implications for developing potential therapeutic interventions, which target β-hCG in the context of BRCA1-defective breast cancers, aligning with the broader goal of advancing precision medicine in oncology.