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While peptide antagonists for the gastrin-releasing peptide receptor (BB2R), neuromedin B receptor (BB1R), and bombesin (BB) receptor subtype-3 (BRS-3) exist, there is a need to develop non-peptide small molecule inhibitors for all three BBR. The BB agonist (BA)1 binds with high affinity to the BB1R, BB2R, and BRS-3. In this communication, small molecule BBR antagonists were evaluated using human lung cancer cells. AM-37 and ST-36 inhibited binding to human BB1R, BB2R, and BRS-3 with similar affinity (Ki=1.4–10.8µM). AM-13 and AM-14 were approximately an order of magnitude less potent than AM-37 and ST-36. The ability of BA1 to elevate cytosolic Ca2+ in human lung cancer cells transfected with BB1R, BB2R, and BRS-3 was antagonized by AM-37 and ST-36. BA1 increased tyrosine phosphorylation of the EGFR and ERK in lung cancer cells, which was blocked by AM-37 and ST-36. AM-37 and ST-36 reduced the growth of lung cancer cells that have BBR. The results indicate that AM-37 and ST-36 function as small molecule BB receptor antagonists.
The bombesin (BB) family of peptides is biologically active in the central nervous system (CNS) and periphery. BB, a 14 amino acid peptide isolated from frog skin, has 9 of the 10 same C-terminal amino acids as does human gastrin-releasing peptide (GRP), a 27 amino acid peptide (1). GRP binds with high affinity to the BB2R, which regulates pruritus, lung development, and gastrin secretion. Neuromedin B (NMB) is a 10 amino acid peptide with 70% sequence homology to the C-terminal of BB. NMB binds with high affinity to the BB1R and causes satiety, hypothermia, and thyrotropin (TSH) secretion from the pituitary (2). BB receptor subtype-3 (BRS-3) is an orphan receptor with homology to the BB1R and BB2R, and binds the universal agonist, BB agonist (BA)1, with high affinity as does the BB1R and BB2R (3). Because BRS-3 knockout mice have impaired energy balance, glucose homeostasis, and increased body weight, BRS-3 agonists may function as satiety agents (4). In the CNS, GRP and NMB may act in a paracrine manner being released from brain neurons in the hypothalamus and dentate gyrus, respectively, activating BB2R and BB1R in adjacent cells (5).
In numerous cancers, including lung cancer, GRP and NMB function in an autocrine manner to stimulate cellular proliferation. Small cell lung cancer (SCLC), a neuroendocrine tumor, has high levels of GRP (6, 7). GRP is secreted from SCLC and binds to cell surface BB2R resulting in increased cellular proliferation (8). NMB is present in both SCLC and non-small cell lung cancer (NSCLC) cells, and after secretion it binds to cell surface BB1R stimulating proliferation (9). Because many lung cancer cells have BB1R, BB2R, and/or BRS-3 there is a need to develop antagonists that block all three receptors of the BB family.
The human BB1R, BB2R, and BRS-3 contain 390, 384, and 399 amino acids and have approximately 50% sequence homology. The BB1R, BB2R, and BRS-3 are members of the rhodopsin β group G protein-coupled receptors (GPCR) family, and they interact with Gq causing phosphatidylinositol (PI) turnover (10). PI-4,5-bisphosphate (PIP2) is metabolized to diacylglycerol, which activates protein kinase C and inositol-trisphosphate (IP3) which causes elevated cytosolic Ca2+. Neuropeptide receptors regulate the transactivation of the epidermal growth factor (EGF) receptor leading to NSCLC proliferation (11). The proliferation of NSCLC cells caused by BA1 can be inhibited by the tyrosine kinase inhibitor (TKI) gefitinib or BBR antagonists. The actions of BA1 on BB1R, BB2R, and BRS-3 are antagonized selectively by PD168368, PD176252, and Bantag-1, respectively (12).
In the present study, small molecules were synthesized and their ability to antagonize BB1R, BB2R, and BRS-3 in lung cancer cells evaluated. The results indicate that AM-37 and ST-36 are useful agents to inhibit the growth of NSCLC cells which have BB1R, BB2R, or BRS-3.
Non-small cell lung cancer cell line NCI-H1299 (ATCC, Manassas, VA, USA) was stably transfected with BB1R, BB2R, and BRS-3. The transfected cells were grown in RPMI-1640 containing 10% fetal bovine serum (FBS) with 0.3mg/ml geneticin (Invitrogen, Grand Island, NY, USA). The transfected cells, which contained approximately 100,000receptors/cell, were weekly split using trypsin/EDTA (13). In addition, lung cancer cell lines NCI-H727, H1299, and H1975 were purchased from ATCC and cultured in RPMI-1640, which contained 10% FBS. The cell types were derived from different human biopsy specimens. These studies were approved by the NIDDK biospecimens and biosafety committees.
The small molecules were synthesized as described previously (14). Figure Figure1D1D shows the structural formula of AM-37, (R)-3-(1H-indol-3-yl)-2-[3-(4-methoxyphenyl)ureido]-N-[[1-(3-pyridinyl)cyclohexyl]methyl]propanamide, and of its S-enantiomer ST-36. Figure Figure1E1E shows the structural formula of AM-13, (R)-N-[[1-(4-fluorophenyl)cyclohexyl]methyl]-3-(1H-indol-3-yl)-2-[3-(4-methoxyphenyl)ureido]propanamide, and its S-enantiomer AM-14. The molecular weight of AM-37 and ST-36 is 525.6Da, whereas the molecular weight of AM-13 and AM-14 is 542.2Da.
The ability of AM-37, ST-36, AM-13, and AM-14 to inhibit specific 125I-BA1 binding to NSCLC cells transfected stably with BB1R, BB2R, and BRS-3 was investigated. NSCLC cells were placed in 24 well plates. When confluent, the cells were washed three times with PBS. The cells were incubated with binding buffer (PBS containing 0.25% bovine serum albumin and 0.025% bacitracin, Sigma-Aldrich, St. Louis, MO, USA). Various concentrations of AM-37, ST-36, AM-13, or AM-14 were added to the cells for 10min, followed by 100,000cpm of 125I-BA1 (0.16nM) and incubated at 37°C for 30min when equilibrium of binding was reached. The cells were rinsed three times with binding buffer for 2min at 4°C. The cells that contained bound peptide dissolved in 0.2N NaOH and counted in a Wallac 1470 γ-counter. The Ki was calculated as described (15).
The ability of AM-37, ST-36, AM-13, and AM-14 to function as BBR antagonists was investigated. NSCLC cells transfected with BB1R, BB2R, and BRS-3 were harvested and loaded with Fura-2AM (Calbiochem, La Jolla, CA, USA) as described previously (16). The excitation ratio was determined at 340 and 380nm with an emission wavelength of 510nm. The lung cancer cellular calcium response was determined after the addition of AM-37, ST-36, AM-13, or AM-14 followed by 10nM BA1.
The tyrosine phosphorylation of the EGFR and ERK was investigated by western blot. NSCLC cells transfected with BB1R, BB2R, and BRS-3 were placed in 10cm dishes. When the cells were confluent, they were placed in SIT medium (RPMI-1640 containing 3×10−8M sodium selenite, 5µg/ml bovine insulin, and 10µg/ml apo-transferrin; Sigma-Aldrich, St. Louis, MO, USA) for 3h. AM-37, ST-36, AM-13, or AM-14 were added for 30min followed by 100nM BA1 for 2min. Cell extracts were made as described previously (16), and 600µg of protein extract was immunoprecipitated with 4µg anti-phosphotyrosine antibody (Becton Dickenson, USA). The immunoprecipitates were fractionated using a 4–20% polyacrylamide gel (Novex, San Diego, CA, USA). Proteins were transferred to a nitrocellulose membrane and incubated with 2µg anti-EGFR or anti-ERK antibody (Cell Signaling Technologies, Danvers, MA, USA). After washing the blot, it was incubated with enhanced chemiluminescence detection reagent (Thermo Scientific) for 5min and exposed to Biomax XAR film (Carestream, Rochester, NY, USA). The band intensity was determined using a Kodak image station 440 densitometer. Alternatively, 20µg of protein extract was loaded onto polyacrylamide gels and after transfer to nitrocellulose, the blot was probed with anti-PY1,068-EGFR, anti-EGFR, anti-PY204ERK, or anti-ERK (Cell Signaling Technologies, Danvers, MA, USA).
The proliferation of NSCLC cells was investigated using the 3-(4,5-demethylthiazol-2-yl)-2,3-diphenyl-2H-tetrazolium bromide (MTT) assay as described previously (16). NCI-H727, H1299, and H1975 cells were placed in SIT medium and varying concentration of AM-37, ST-36, AM-13, or AM-14 added. After 2days, 0.1% MTT solution (15µl) was added. After 4h, DMSO (150µl) was added and the absorbance at 570nm was determined.
The results are expressed as the mean±SD. Statistical significance of differences was performed by a one-way or two-way repeated measures of variance. The binding curves were drawn using PRISM.
The ability of the small molecules to bind to BB1R, BB2R, and BRS-3 was investigated. AM-37 (R-enantiomer) inhibited specific 125I-BA1 binding to BB1R, BB2R, and BRS-3 in a dose-dependent manner with Ki values 3.6, 1.4, and 5.5µM, respectively (Figure (Figure1).1). ST-36 (S-enantiomer) inhibited specific 125I-BA1 binding to BB1R, BB2R, and BRS-3 with Ki values of 7.9, 6.9, and 10.8µM, respectively (Figure (Figure1).1). In contrast, AM-13 (R-enantiomer) and AM-14 (S-enantiomer) inhibited specific 125I-BA1 binding to BB1R, BB2R, and BRS-3 with Ki>20µM. The results indicate that AM-37and ST-36 bind to BB1R, BB2R, and BRS-3 with greater affinity than does AM-13 and AM-14.
The specificity of binding was investigated. Table Table11 shows that BA1 bound with high affinity (Ki=0.002, 0.0005, and 0.004µM) to BB1R, BB2R, and BRS-3. AM-37, ST-36, AM-13, and AM-14 inhibited specific 125I-BA1 binding (Ki=1.4, 6.9, 27, and 45µM) to BB2R. ST-36 inhibited specific 125I-BA1 binding (Ki=7.9 and 10.8µM) to BB1R and BRS-3, respectively. AM-13 and AM-14 bind with low affinity to BB1R and BRS-3 (Ki>100µM and >100μM, respectively).
The ability of the small molecules to function as BB1R, BB2R, and BRS-3 antagonists was investigated. Addition of 10nM BA1 to NCI-H1299 cells transfected with BB1R increased the cytosolic Ca2+ from 160 to 178nM within seconds (Figure (Figure2A).2A). The response was transient and returned to baseline after 1min. Addition of 30µM AM-37 to NCI-H1299 cells transfected with BB1R had no effect on the basal cytosolic Ca2+ but blocked the increase in cytosolic Ca2+ caused by BA1 (Figure (Figure2B).2B). Addition of 30µM AM-14 had no effect of basal cytosolic Ca2+ but partially blocked the increase caused by 10nM BA1 (Figure (Figure2C).2C). Table Table22 shows that AM-37 and AM-14 significantly inhibited the ability of BA1 to increase cytosolic Ca2+ after addition to NCI-H1299 cells transfected with BB1R. Addition of 10nM BA1 to NCI-H1299 cells transfected with BB2R increased the cytosolic Ca2+ from 160 to 186nM (Figure (Figure2D).2D). Addition of 30µM ST-36 to NCI-H1299 cells transfected with BB2R had no effect on the basal cytosolic Ca2+ but blocked the increase in cytosolic Ca2+ caused by BA1 (Figure (Figure2E).2E). Addition of 30µM AM-14 had no effect of basal cytosolic Ca2+ but partially blocked the increase caused by 10nM BA1 (Figure (Figure2F).2F). Table Table22 shows that ST-36 and AM-14 significantly decreased the ability of 10nM BA1 to elevate cytosolic Ca2+ in NCI-H1299 cells transfected with BB2R. Addition of 10nM BA1 to NCI-H1299 cells transfected with BRS-3 increased the cytosolic Ca2+ from 170 to 194nM (Figure (Figure2G).2G). Addition of 30µM ST-36 to NCI-H1299 cells transfected with BRS-3 had no effect on the basal cytosolic Ca2+ but blocked the increase in cytosolic Ca2+ caused by BA1 (Figure (Figure2H).2H). Addition of 30µM AM-13 had no effect of basal cytosolic Ca2+ but partially blocked the increase caused by 10nM BA1 (Figure (Figure2I).2I). Table Table22 shows that ST-36 and AM-13 significantly decreased the ability of 10nM BA1 to elevate cytosolic Ca2+ in NCI-H1299 cells transfected with BRS-3. The results indicate that AM-37 and ST-36 are antagonists for BB1R, BB2R, and BRS-3. In contrast, AM-13 and AM-14 are weak antagonists for the BBR family.
The specificity of AM-37, ST-36, AM-13, and AM-14 was investigated. 10nM neurotensin (NT) or 5µg/ml ionomycin (ION) strongly increased the cytosolic Ca2+ in NSCLC cells. AM-37 or ST-36 had no effect on the ability of NT to increase cytosolic Ca2+ in NSCLC cells. AM-13 or AM-14 had no effect on the ability of ION to increase Ca2+ in NSCLC cells. Therefore, AM-36 and ST-37 are antagonists for the BBR but not the NTR.
The ability of the small molecules to impair EGFR transactivation was investigated. Previously, we found that the BB1R and BRS-3 regulate EGFR tyrosine phosphorylation (13, 16). Figure Figure33 shows that addition of 100nM BA1 to NCI-H1299 cells transfected with BB2R increased significantly the EGFR tyrosine phosphorylation to 326%. If the cells were pretreated with 10µM AM-37 or ST-36, addition of BA1 had little effect. In contrast, if the cells were treated with 10µM AM-13, BA1 increased strongly EGFR tyrosine phosphorylation. Similarly, BA1 addition to NCI-H1299 cells transfected with BB2R increased ERK tyrosine phosphorylation to 277%. This increase in ERK tyrosine phosphorylation was decreased significantly in the cells pretreated with AM-37 or ST-36 but not AM-13. Similarly, AM-14 had little effect on EGFR or ERK tyrosine phosphorylation (data not shown). The results indicate that AM-37 and ST-36 antagonize the ability of the BB2R to regulate tyrosine phosphorylation of the EGFR and ERK. Similar transactivation results were obtained for NSCLC cells transfected with BB1R or BRS-3 (data not shown).
The ability of the small molecules to inhibit lung cancer proliferation was investigated. AM-37 inhibited NCI-H1299 proliferation in a dose-dependent manner. Figure Figure44 shows that AM-37 had little effect at 3µM but strongly inhibited proliferation at 30µM. The IC50 for AM-37 was 16µM. Similarly, ST-36 had an IC50 of 22µM, whereas AM-14 was less potent (IC50>50µM).
The specificity of the small molecules was investigated. Table Table33 shows that AM-37, ST-36, AM-13, and AM-14 (50µM) inhibited significantly the proliferation of NCI-H727 cells, which have mRNA for BB1R, BB2R, and BRS-3. In contrast, AM-37, ST-36, AM-13, and AM-14 had little effect on NCI-H1975 cells, which lack BB1R, BB2R, and BRS-3. These results indicate that the BBR is essential for AM-37, ST-36, AM-13, or AM-14 to inhibit cancer cellular proliferation.
While NSCLC patients are traditionally treated with combination chemotherapy, the 5-year survival rate is only 16% (17). Some NSCLC patients (13%) have L858R EGFR mutations, and these patients respond to TKI such as gefitinib or erlotinib; however, secondary EGFR mutations can occur such as T790M resulting in TKI resistance (18). Numerous GPCR are expressed in lung cancer cell lines and biopsy specimens. BB2R mRNA is expressed in 46–67% of the lung cancer cell lines examined (19). BB1R mRNA is present in 81% of the NSCLC cell lines examined (20). Using autoradiographic techniques, BRS-3 binding sites were detected in 40% of the lung cancer biopsy specimens examined (21). The EGFR is abundant on NSCLC (approximately 100,000EGFR/cell), whereas BBR are present on most native NSCLC cells (approximately 2,000BBR/cell) (22).
Addition of GRP to NSCLC cells causes transactivation of the EGFR (23). The effects of GRP on NSCLC tyrosine phosphorylation of the EGFR are impaired by gefitinib, a TKI, and PD176252, a peptoid BB2R antagonist. Because the ERK and EGFR tyrosine phosphorylation caused by GRP was impaired by marimastat, GM6001 and antibodies to TGFα, matrix metalloproteases may regulate the cellular shedding of TGFα from NSCLC cells. The TGFα may then bind to the EGFR causing its tyrosine phosphorylation. The results indicate that the BB2R regulates EGFR transactivation in NSCLC cells.
The BB1R regulates EGFR transactivation (16). The increase in EGFR and ERK tyrosine phosphorylation caused by NMB addition to NSCLC cells was impaired by PD168368, a BB1R peptoid antagonist, as well as gefitinib. The increase in EGFR tyrosine phosphorylation caused by NMB was impaired by N-acetyl cysteine (NAC), an antioxidant, or tiron, a superoxide scavenger. NMB increased reactive oxygen species (ROS) in NSCLC cells, and the increase was inhibited by Tiron. It remains to be determined if the ROS impair protein tyrosine phosphatases in NSCLC cells, which remove phosphate from the P-EGFR. Activation of BRS-3 with BA1 increased EGFR and ERK tyrosine phosphorylation (13). The increase in EGFR tyrosine phosphorylation caused by BA1 is impaired by NAC, tiron, and diphenyleneiodonium, an inhibitor of NADPH oxidase enzymes.
ML-18 is a small molecule that prefers BRS-3 relative to BB1R or BB2R (24). ML-18, an S-enantiomer, inhibits 125I-BA1 binding to BRS-3, BB2R, and BB1R with IC50 values of 4.8, 16, and >100 μM, respectively, whereas the R-enantiomer EMY-98 is inactive. ML-18 is a BRS-3 antagonist, which inhibits the ability of BA1 to increase cytosolic Ca2+, increase ERK and EGFR tyrosine phosphorylation (24). Also, ML-18 inhibited NSCLC growth and increased the cytotoxicity of gefitinib. His107 is important for BRS-3 to bind antagonists with high affinity (25). Tyr101 of the BB2R is important for binding of non-peptide antagonists (26). Similarly, this Tyr is conserved in the BB1R and BRS-3. It remains to be determined if this Tyr is essential for binding of AM-37 to the BB1R, BB2R, or BRS-3. ST-36, which is an S-enantiomer, inhibited specific 125I-BA1 binding to BB1R, BB2R, and BRS-3 with IC50 values of 7.9, 6.9, and 10.8µM, respectively. It is surprising that AM-37, which is the R-enantiomer, binds with slightly higher affinity to BBR than does ST-36. Previously, the BB1R was found to prefer PD168,368, which is an S-isomer, relative to the R-isomer (27).
(D-Arg1, D-Trp5,7,9, Leu11)substance P (SP) is an inhibitor of signal transduction and growth of SCLC cells (28). (D-Arg1, D-Trp5,7,9, Leu11)SP impaired the ability of BB, vasopressin, or bradykinin to increase cytosolic Ca2+ and ERK activity. (D-Arg1, D-Trp5,7,9, Leu11)SP decreased SCLC growth in vitro, and (D-Arg1, D-Trp5,7,9, Leu11)SP has a unique tertiary structure in with two type IV non-standard turns, which juxtapose the N- and C-terminal adjacent to one another (29). Due to this unique structure (D-Arg1, D-Trp5,7,9, Leu11)SP may be able to interact with multiple GPCR. In contrast, AM-37 and ST-36 are small molecules that have a different structure from that of (D-Arg1, D-Trp5,7,9, Leu11)SP.
AM-37 and ST-36 inhibited the proliferation of NSCLC cells such as NCI-H1299 and H727, which have BB1R, BB2R, or BRS-3. In contrast, AM-37 and ST-36 have little effect on NSCLC cell line NCI-H1975, which lacks BB1R, BB2R, and BRS-3. It remains to be determined if AM-37 or ST-36 are synergistic with gefitinib at inhibiting the growth of NSCLC. A goal is to identify GPCR antagonists, which potentiate the action of TKI in NSCLC patients.
AM-37 and ST-36 are small molecules, which bind to the BB1R, BB2R, and BRS-3. Because AM-37 and ST-36 inhibit the ability of BA1 to increase cytosolic Ca2+ as well as increase EGFR and ERK tyrosine phosphorylation, they function as BB1R, BB2R, and BRS-3 antagonists. A particular advantage of AM-37 and ST-36 is that they will inhibit the growth of NSCLC cells if they have BB1R, BB2R, or BRS-3.
TM and SM were responsible for the receptor binding studies. TM and NT were responsible for the cell culture and calcium experiments. TM, PM, and IR-A were responsible for the transactivation and growth experiments. ML was responsible for the synthesis of the small molecules. TM and RJ were responsible for the writing of the manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.
The authors thank Drs. M. Nicklaus, E. Lacivita, D. Venzon, and M. Peach for helpful discussions.
Funding. This research was supported by the intramural programs of the NCI and NIDDK of the NIH.