ML-18 is a non-peptide bombesin receptor subtype-3 antagonist which inhibits lung cancer growth

Abstract

Bombesin receptor subtype (BRS)-3 is a G protein coupled receptor (GPCR) for the bombesin (BB)-family of peptides. BRS-3 is an orphan GPCR and little is known of its physiological role due to the lack of specific agonists and antagonists. PD168368 is a nonpeptide antagonist for the neuromedin B (NMB) receptor (R) whereas PD176252 is a nonpeptide antagonist for the gastrin releasing peptide (GRP) R and NMBR but not BRS-3. Here nonpeptide analogs of PD176252 e.g. the S-enantiomer ML-18, and the R-enantiomer, EMY-98, were investigated as BRS-3 antagonists using lung cancer cells. ML-18 and EMY-98 inhibited specific 125I-BA1 (DTyr-Gln-Trp-Ala-Val-βAla-His-Phe-Nle-NH2)BB6-14 binding to NCI-H1299 lung cancer cells stably transfected with BRS-3 with IC50 values of 4.8 and > 100 μM, respectively. In contrast, ML-18 bound with lower affinity to the GRPR and NMBR with IC50 values of 16 and >100 μM, respectively. ML-18 (16 μM), but not its enantiomer EMY-98, inhibited the ability of 10 nM BA1 to elevate cytosolic Ca2+ in a reversible manner using lung cancer cells loaded with FURA2-AM. ML-18 (16 μM), but not EMY-98, inhibited the ability of 100 nM BA1 to cause tyrosine phosphorylation of the EGFR and ERK in lung cancer cells. ML-18 but not EMY-98 inhibited the proliferation of lung cancer cells. The results indicate that ML-18 is a nonpeptide BRS-3 antagonist that should serve as a template to improve potency and selectivity.

Bombesin receptor subtype 3 as a potential target for obesity and diabetes

“…Transactivation of the EGFR by GRPR and NMBR, has been shown to be an important signaling cascade mediating growth effects of these receptors on a number of different neoplasms including cancers of the lung (especially nonsmall cell tumors), head and neck squamous cells, neuroendocrine cells and the prostate[8,17,37,52,56–58,60–62]. Studies with BRS-3 in this area are more limited, although recently a number of studies of BRS-3 growth stimulatory effects in lung cancer cells report a similar mechanism with its growth stimulatory effects occurring primarily via EGFR transactivation[53,57]. In a number of studies the transactivation of EGFR by BRS-3 and the other mammalian BnRs has been shown to involve activation of phospholipase C, Src kinases, stimulation of matrix metalloproteinases and shedding of EGF-related ligands, as well as the stimulation of reactive oxygen species[8,9,17,52,54,57].…”

“…Two different BRS-3 antagonists have recently been described: ML-18[53] is a peptoid(S-enantiomer of PD176252, a GRP preferring antagonist[89,99]) and the other is a peptide antagonist, Bantag-1(Table 2)[31,34]. ML-18 has both a low affinity and low (<5-fold) selectivity for hBRS-3 over GRPR or NMBR (Table 2)[53].…”

“…Two different BRS-3 antagonists have recently been described: ML-18[53] is a peptoid(S-enantiomer of PD176252, a GRP preferring antagonist[89,99]) and the other is a peptide antagonist, Bantag-1(Table 2)[31,34]. ML-18 has both a low affinity and low (<5-fold) selectivity for hBRS-3 over GRPR or NMBR (Table 2)[53]. In contrast, Bantag-1 (Boc-Phe-His-4-amino-5-cyclohexyl-2,4,5-trideoxypentonyl-Leu-(3-dimethylamino)benzylamide N-methylammonium trifluoroacetate) was reported to be a potent, selective peptide antagonist for the BRS-3-receptor in human, rat and mouse[31,34].…”

“… Data for selective BRS-3 ligands is shown in shaded boxes a Data are from [14,24,25,27,28,65,66,71,72,89,133] b Data are from [24,66,71,72] c Data are from [25] d Data are from [30] e Data are from [53] f Data are from [34] g Data are from [86] h Data are from [81] i Data are from [83] j Data are from [72] k Data are from [88]: These data are not Ki’s determining by binding studies, but EC50’s determining by using a cellular FLIPR dye Calcium assay. l Data are from [85]: m Data are from [76]: These data are not Ki’s determining by binding studies, but EC50’s determining by using a IP-One HTRF assay which assesses phospholipase C activity and accumulation of IP1. Abbreviations: See Table 1 for structures of 9f, 9g, 17c, Alytesin, Bantag-1, Bn, GRP, Litorin, MK-5046, PD168368, Ranatensin. Ac , acetyl; Apa , 3-amino,propionic acid; Apa-4Cl , 4-chloro,3-amino,propionic acid; Bag-1 , (2-(4-(2-[5-(2,2-dimethylbutyl)-1H-imidazol-2-yl]ethyl)phenyl)pyridine[34]; Bag-2 (4′-(2-[5-(cyclopentylmethyl)- 1H-imidazol-2-yl]-1,2-ditritium-ethyl)biphenyl-2-carboxylic acid[34]; Cpa , chlorophenylalamine,; His(tBzl), histidine(tBenzl ); Cmpd 9, 2-(2-[4-(pyridin-2-yl)phenyl] ethyl)-5-(2,2-dimethylbutyl)- 1H-imidazole; Cmpd 16, phenyacetyl-Ala,DTrp, phenthylamide[68,69]; ML-18 , S-enantiomer of PD176252; Cpd 1t [88]: MK-7725 ,2-(3-(6-((4-(trifluoromethoxy)phenyl)sulfonyl)-2-(trifluoromethyl)-6,11-dihydro-5H-benzo[b]pyrido[2,3-e][1,4]diazepin-7-yl)-1,2,4-oxadiazol-5-yl)propan-2-ol[80,81]; Nip, piperidine-3 carboxylic acid; Nal, β-napthylalanine; Orn , ornithine; ψ ; pseudopeptide bond, ( i.e., CONH changed to CH 2 NH); Ph-Pr , phenylpropanolamine; PD176252 , (3-(1H-Indol-3-yl)-N-[1-(5-methoxy-pyridin-2-yl)-cyclohexylmethyl]-2-methyl-2-[3-(4-nitro-phenyl)-ureido]-propionamide); RY-337 [83].…”

Molecular basis for high affinity and selectivity of peptide antagonist, Bantag-1, for the orphan BB3 receptor

“…This has occurred because its native ligand is unknown, it has low affinity for all natural Bn peptides, and unlike BB 2 or BB 1 receptor, for which numerous selective agonists and antagonists are described [2,6–11], until recently no agonist or antagonist with sufficient selectivity to be useful for in vivo studies, existed for BB 3 receptor [2,3,12–17]. A high affinity BB 3 receptor agonist has been described, [D-Tyr 6 , β-Ala 11 , Phe 13 , Nle 14 ]Bn(6–14) (peptide #1), which allowed studies of BB 3 receptor’s signaling cascades, demonstrating it was coupled to phospholipase C, A2 and D activation as well as tyrosine kinase cascades [4,14,18–21]. However, peptide #1 was not useful for pharmacological/pathological studies because it was nonselective, having high affinity for BB 2 receptor / BB 1 receptor in all species [12,22–24], as well as human BB 3 receptor [25–27], but not rat/mouse BB 3 receptor [27,28].…”