ML-18 is a non-peptide bombesin receptor subtype-3 (BRS-3) antagonist with an IC50 of 4.8 [mu]M.

BRS3:4.8 μM.

ML-18, at a concentration of 16 µM, reversibly inhibits BA1 at 10 nM from increasing cytosolic Ca2+ levels and at 100 nM from inducing tyrosine phosphorylation of EGFR and ERK in lung cancer cells, using FURA2-AM-loaded lung cancer cells for the former evaluation. This compound also suppresses lung cancer cell proliferation. Furthermore, ML-18 demonstrates specific inhibition of 125I-BA1 (DTyr-Gln-Trp-Ala-Val-βAla-His-Phe-Nle-NH2)BB6-14 binding to BRS-3-expressing NCI-H1299 lung cancer cells, presenting an IC50 value of 4.8 µM. It exhibits reduced affinity towards GRPR and NMBR with IC50 values of 16 µM and over 100 µM, respectively.

The cells are incubated in SIT buffer containing 0.25% bovine serum albumin and 250 μg/mL bacitracin and 125I-BA1 (100,000 cpm) is added, as well as various concentrations of unlabelled competitor (ML-18). After incubation at 37°C for 30 min, free 125I-BA1 is removed by washing 3 times in buffer and the cells which contain bound 125I-BA1 is dissolved in 0.2 N NaOH and counted in a gamma counter. The IC50 is calculated for each unlabeled competitor.

ML-18 is dissolved in DMSO at a concentration of 10 mM prior to use. Cell viability is measured using the MTT assay. NCI-H727 or NCI-H1299 cells transfected with BRS-3 are treated with ML-18 (0, 4.8, 16, 48 μM) or gefitinib added. After 2 days, 15 μL of 0.1 % MTT solution added. After 4 h, 150 μL of DMSO is added. After 16 h, the optical density at 570 nm is determined.