BA 1 (DTyr-Gln-Trp-Ala-Val- Ala-His-Phe-Nle-NH2) is a potent BRS-3 agonist (IC50 = 2.52 nM) and a NMBR and GRPR agonist (IC50 values are 0.26 and 1.55 nM respectively). It can stimulate NCI-H1299 lung cancer cell proliferation in vitro and enhance glucose transport in obese and diabetic primary myocytes.
BRS-3 binds synthetic, [D-Tyr6, β-Ala11, Phe13, Nle14]BB(6-14), abbreviated BA 1, with high affinity. Besides, BA 1 binds with high affinity to the NMBR and GRPR causing their activation. BRS-3 does not bind BB, GRP or NMB with high affinity whereas the NMBR prefers NMB relative to BB or GRP and the GRPR prefers BB or GRP relative to NMB. So BA 1 represents a universal agonist for the BB family of receptors.
ML-18 is a steroselective BRS-3 antagonist. After addition of BA 1 to lung cancer cells the cytosolic Ca2+ transiently increased within seconds. And the increase in cytosolic Ca2+ caused by BA1 was blocked by the (S) isomer ML-18 but not the (R) isomer EMY-98. The addition of 16 μM ML-18 blocked the increase in cytosolic Ca2+ caused by 10nM BA 1. However, there was a moderate and strong Ca2+ response if 100nM and 1000nM BA 1 was subsequently added, respectively.
It remains to be determined whether the BBR antagonists increase the potency of gefitinib in vivo. BA 1 increased NCI-H727 colony number, using the clonogenic assay which takes 2 weeks. The increase in colony number caused by BA1 is inhibited by ML-18 and/or gefitinib but not EMY-98. These results indicate that BA1 may increase colony number in an EGFR-dependent manner.
Pharmacokinetics and metabolism
BRS-3 regulates EGFR and ERK tyrosine phosphorylation in lung cancer cells. Experimental result shows that addition of 0.1 M BA1 addition to NCI-H1299 cells transfected with BRS-3 increases FAK (125 kDa) tyrosine phosphorylation after two min. BA 1 significantly increased FAK tyrosine phosphorylation by 10.9 times. Besides, the increase in FAK tyrosine phosphorylation caused by BA 1 was significantly inhibited 16 but not 1.6 or 0.16 M ML-18. The total FAK was not altered by ML-18 as a control. And the addition of BA1 to NCI-H1299 cells transfected with BRS-3 increased ERK (44 and 42 kDa) tyrosine phosphorylation. BA 1 remarkably increased ERK tyrosine phosphorylation by 2.9 times. And the increased in ERK tyrosine phosphorylation caused by BA1 was significantly inhibited by 16 but not 1.6 or 0.16 M ML-18. Addition of BA 1 to NCI-H1299 cells transfected with BRS-3 increased EGFR (170 kDa) tyrosine phosphorylation. BA 1 increased significantly EGFR tyrosine phosphorylation by 5.9 times. Similarly, the increase in EGFR tyrosine phosphorylation caused by BA 1 was significantly inhibited by 16 but not 1.6 or 0.16 M ML-18.
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