Human MC1R Stable Cell Line - HEK293

Melanoma is a deadly skin cancer. Despite recent breakthroughs with BRAF-V600E and PD-1 inhibitors showing significant clinical efficacy, melanoma can ultimately develop resistance to these targeted therapies. To address this resistance problem, researchers designed and synthesized a ligand-drug conjugate that couples a cytotoxic drug with low resistance potential to the melanocortin 1 receptor (MC1R) agonist melanocortin-II (MT-II), thereby targeting melanoma cells that overexpress MC1R. In vitro experiments showed that the drug-MT-II conjugate maintained strong binding to MC1R and achieved selective drug delivery to A375 melanoma cells through its MT-II portion. Furthermore, using camptothecin as the cytotoxic drug, camptothecin-MT-II (compound 1) effectively inhibited the growth of A375 melanoma cells with an IC50 value of 16 nM. By conferring melanoma cell selectivity through its MT-II portion, this drug-MT-II conjugate approach allows for a wider selection of cytotoxic drugs, which may be key to overcoming melanoma drug resistance.

To evaluate the binding affinity of compounds 1 and 2 to MC1R, researchers performed competitive binding experiments using human MC1R overexpressing HEK293 cells and ¹²⁵I-labeled NDP-α-MSH as a competitive ligand (Figure 1, Table 1). The Ki values for CPT-MT-II and fluorescein-labeled MT-II binding to MC1R were determined to be 57 ± 7 nM and 172 ± 20 nM, respectively, while the Ki value for MT-II was 1.5 ± 0.2 nM. Although the binding affinity was lower than that of MT-II, compounds 1 and 2 were able to completely displace 125I-NDP-α-MSH in the micromolar concentration range. Since MT-II is not selective for MC1R and can also bind to and activate other melanocortin receptor subtypes, the researchers further conducted competitive binding experiments using HEK293 cells overexpressing MC3R, MC4R, or MC5R. The results showed that CPT-MT-II exhibited some selectivity for MC1R, with its binding affinity to MC1R being approximately 3-6 times higher than its binding affinity to MC3R, MC4R, and MC5R (Table 1).

Figure 1. Competitive binding assay results of MT-II, CPT-MT-II 1 and fluorescein-MT-II 2 binding to MC1R in competition with ¹²⁵I labeled NDP-α-MSH. ( Zhou Y, et al., 2020)

Table 1. K_i Values of MT-II, CPT-MT-II and Fluorescein-MT-II Binding to Melanocortin Receptors.