BRS-3 ( IC50 = 4.8 μM )
ML-18 blocks specific ¹²⁵I-BA1 (DTyr-Gln-Trp-Ala-Val-βAla-His-Phe-Nle-NH2)BB6-14 binding to NCI-H1299 lung cancer cells that have been stably transfected with BRS-3 with IC50 values of 4.8 μM. ML-18 exhibits a reduced affinity for binding the GRPR and NMBR, with IC50 values exceeding 100 μM and 16 μM, respectively. ML-18 at 16 μM reversibly prevents 10 nM BA1 from increasing cytosolic Ca²⁺ in lung cancer cells that have been loaded with FURA2-AM. ML-18 at 16 μM prevents 100 nM BA1 from causing EGFR and ERK tyrosine phosphorylation in lung cancer cells. It prevents lung cancer cells from proliferating[1].

ML-18 blocks specific ¹²⁵I-BA1 (DTyr-Gln-Trp-Ala-Val-βAla-His-Phe-Nle-NH2)BB6-14 binding to NCI-H1299 lung cancer cells that have been stably transfected with BRS-3 with IC50 values of 4.8 μM. ML-18 exhibits a reduced affinity for binding the GRPR and NMBR, with IC50 values exceeding 100 μM and 16 μM, respectively. ML-18 at 16 μM reversibly prevents 10 nM BA1 from increasing cytosolic Ca²⁺ in lung cancer cells that have been loaded with FURA2-AM. ML-18 at 16 μM prevents 100 nM BA1 from causing EGFR and ERK tyrosine phosphorylation in lung cancer cells. It prevents lung cancer cells from proliferating[1].

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ML-18 inhibited specific ¹²⁵I-BA1 binding to NCI-H1299 lung cancer cells stably transfected with BRS-3 with an IC₅₀ of 4.8 ± 0.6 μM, while its R-enantiomer EMY-98 had an IC₅₀ >100 μM. In NCI-H727 lung cancer cells, the IC₅₀ for ML-18 was 6.4 ± 1.1 μM.
ML-18 (16 μM), but not EMY-98, antagonized the ability of 10 nM BRS-3 agonist BA1 to elevate cytosolic Ca²⁺ in a reversible manner in both BRS-3-transfected NCI-H1299 and NCI-H727 lung cancer cells pre-loaded with Fura-2AM. The antagonist effect was overcome by subsequent addition of higher concentrations of BA1 (0.1 and 1 μM).
ML-18 (16 μM), but not EMY-98, also antagonized the increase in cytosolic Ca²⁺ induced by 10 nM of the selective BRS-3 agonist MK-5046.
Treatment of BRS-3-transfected NCI-H1299 cells with 0.1 μM BA1 for 2 minutes significantly increased tyrosine phosphorylation of FAK (10.9-fold), ERK (2.9-fold), and EGFR (5.9-fold). This BA1-induced phosphorylation was significantly inhibited by pre-treatment with 16 μM ML-18 for 30 minutes, but not by lower concentrations (0.16 or 1.6 μM).
In a 2-day MTT proliferation assay, ML-18 inhibited the growth of BRS-3-transfected NCI-H1299 cells in a dose-dependent manner, with moderate and strong inhibition observed at 16 μM and 48 μM, respectively. The IC₅₀ for gefitinib alone was >30 μM, but co-treatment with ML-18 (48 μM) shifted the gefitinib dose-response curve to the left, yielding an approximate IC₅₀ of 4.5 μM for gefitinib.
In a 2-week clonogenic assay using NCI-H727 cells, ML-18 (1.6 μM) alone significantly reduced colony number. Furthermore, ML-18 (1.6 μM) inhibited the increase in colony number stimulated by 0.01 μM BA1. The combination of ML-18 and gefitinib (1 μM) resulted in a stronger reduction in colony number than either agent alone. [1]

The cells are cultured in SIT buffer supplemented with varying concentrations of unlabelled competitor (ML-18), 0.25% bovine serum albumin, 250 μg/mL bacitracin, and ¹²⁵³I-BA1 (100,000 cpm). Following a 30-minute incubation period at 37°C, free ¹²⁵³I-BA1 is eliminated by three rounds of buffer washing, and the cells containing bound ¹²⁵³I-BA1 are dissolved in 0.2 N NaOH and counted using a gamma counter. For every competitor without a label, the IC50 is computed[1].

The MTT assay is used to determine cell viability. After transfecting NCI-H727 or NCI-H1299 cells with BRS-3, ML-18 (0, 4.8, 16, 48 μM) or gefitinib is added. 15 μL of 0.1% MTT solution was added after two days. 150 μL of DMSO is added after 4 hours. The optical density at 570 nm is calculated after 16 hours[1].

[1]. ML-18 is a non-peptide bombesin receptor subtype-3 antagonist which inhibits lung cancer growth. Peptides. 2015 Feb;64:55-61.

ML-18 is the (S)-enantiomer of the PD176252 analogue with a molecular weight of 569.9 Daltons. It is a stereoselective, reversible non-peptide BRS-3 antagonist. The compound was synthesized from N-BOC-S-tryptophan and purified by chiral HPLC to an enantiomer excess of >95%. In the experiment, it was dissolved in DMSO to prepare a 10 mM stock solution. The study concluded that ML-18 is the first reported non-peptide BRS-3 antagonist and can be used as a lead compound for the development of drugs with higher affinity and specificity for BRS-3. [1]

C32H35N5O5

569.66

569.263

C, 67.47; H, 6.19; N, 12.29; O, 14.04

1422269-30-4

91827363

White to yellow solid powder

1.3±0.1 g/cm3

803.5±75.0 °C at 760 mmHg

439.7±37.1 °C

0.0±3.0 mmHg at 25°C

1.652

8.03

4

5

9

42

906

1

O=C([C@]([H])(C([H])([H])C1=C([H])N([H])C2=C([H])C([H])=C([H])C([H])=C12)N([H])C(N([H])C1C([H])=C([H])C(=C([H])C=1[H])[N+](=O)[O-])=O)N([H])C([H])([H])C1(C2C([H])=C([H])C(=C([H])C=2[H])OC([H])([H])[H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C1([H])[H]

JOKVJNCYOSFDGC-LJAQVGFWSA-N

InChI=1S/C32H35N5O5/c1-42-26-15-9-23(10-16-26)32(17-5-2-6-18-32)21-34-30(38)29(19-22-20-33-28-8-4-3-7-27(22)28)36-31(39)35-24-11-13-25(14-12-24)37(40)41/h3-4,7-16,20,29,33H,2,5-6,17-19,21H2,1H3,(H,34,38)(H2,35,36,39)/t29-/m0/s1

(2S)-3-(1H-indol-3-yl)-N-[[1-(4-methoxyphenyl)cyclohexyl]methyl]-2-[(4-nitrophenyl)carbamoylamino]propanamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: 2.5 mg/mL (4.39 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (4.39 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)