AM-37 and ST-36 Are Small Molecule Bombesin Receptor Antagonists

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 = 1.4–10.8 µM). 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.


inTrODUcTiOn
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.
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.

MaTerials anD MeThODs cell culture
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 0.3 mg/ml geneticin (Invitrogen, Grand Island, NY, USA). The transfected cells, which contained approximately 100,000 receptors/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.

ligand synthesis
The small molecules were synthesized as described previously (14). Figure 1D shows the structural formula of AM-37,

receptor Binding
The ability of AM-37, ST-36, AM-13, and AM-14 to inhibit specific 125 I-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 10 min, followed by 100,000 cpm of 125 I-BA1 (0.16 nM) and incubated at 37°C for 30 min when equilibrium of binding was reached. The cells were rinsed three times with binding buffer for 2 min at 4°C. The cells that contained bound peptide dissolved in 0.2 N NaOH and counted in a Wallac 1470 γ-counter. The Ki was calculated as described (15).

cytosolic ca 2+
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 380 nm with an emission wavelength of 510 nm. The lung cancer cellular calcium response was determined after the addition of AM-37, ST-36, AM-13, or AM-14 followed by 10 nM BA1.

Tyrosine Phosphorylation
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 10 cm dishes. When the cells were confluent, they were placed in SIT medium (RPMI-1640 containing 3 × 10 −8 M sodium selenite, 5 µg/ml bovine insulin, and 10 µg/ml apo-transferrin; Sigma-Aldrich, St. Louis, MO, USA) for 3 h. AM-37, ST-36, AM-13, or AM-14 were added for 30 min followed by 100 nM BA1 for 2 min. Cell extracts were made as described previously (16), and 600 µg of protein extract was immunoprecipitated with 4 µg 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 2 µg 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 5 min and exposed to Biomax XAR film (Carestream, Rochester, NY, USA). The band intensity was determined using a Kodak image station 440 densitometer. Alternatively, 20 µg of protein extract was loaded onto polyacrylamide gels and after transfer to nitrocellulose, the blot was probed with anti-PY 1,068 -EGFR, anti-EGFR, anti-PY 204 ERK, or anti-ERK (Cell Signaling Technologies, Danvers, MA, USA).

statistical analysis
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 results indicate that AM-37and ST-36 bind to BB1R, BB2R, and BRS-3 with greater affinity than does AM-13 and AM-14.

cytosolic ca 2+
The ability of the small molecules to function as BB1R, BB2R, and BRS-3 antagonists was investigated. Addition of 10 nM BA1 to NCI-H1299 cells transfected with BB1R increased the cytosolic Ca 2+ from 160 to 178 nM within seconds (Figure 2A). The response was transient and returned to baseline after 1 min. Addition of 30 µM AM-37 to NCI-H1299 cells transfected with BB1R had no effect on the basal cytosolic Ca 2+ but blocked the increase in cytosolic Ca 2+ caused by BA1 ( Figure 2B). Addition of 30 µM AM-14 had no effect of basal cytosolic Ca 2+ but partially blocked the increase caused by 10 nM BA1 ( Figure 2C). Table 2 shows that AM-37 and AM-14 significantly inhibited the ability of BA1 to increase cytosolic Ca 2+ after addition to NCI-H1299 cells transfected with BB1R. Addition of 10 nM BA1 to NCI-H1299 cells transfected with BB2R increased the cytosolic Ca 2+ from 160 to 186 nM ( Figure 2D). Addition of 30 µM ST-36 to NCI-H1299 cells transfected with BB2R had no effect on the basal cytosolic Ca 2+ but blocked the increase in cytosolic Ca 2+ caused by BA1 (Figure 2E). Addition of 30 µM AM-14 had no effect of basal cytosolic Ca 2+ but partially blocked the increase caused by 10 nM BA1 (Figure 2F). Table 2 shows that ST-36 and AM-14 significantly decreased the ability of 10 nM BA1 to elevate cytosolic Ca 2+ in NCI-H1299 cells transfected with BB2R. Addition of 10 nM BA1 to NCI-H1299 cells transfected with BRS-3 increased the cytosolic Ca 2+ from 170 to 194 nM ( Figure 2G). Addition of 30 µM ST-36 to NCI-H1299 cells transfected with BRS-3 had no effect on the basal cytosolic Ca 2+ but blocked the increase in cytosolic Ca 2+ caused by BA1 (Figure 2H). Addition of 30 µM AM-13 had no effect of basal cytosolic Ca 2+ but partially blocked the increase caused by 10 nM BA1 (Figure 2I). Table 2 shows that ST-36 and AM-13 significantly decreased the ability of 10 nM BA1 to elevate cytosolic Ca 2+ 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. 10 nM neurotensin (NT) or 5 µg/ml ionomycin (ION) strongly increased the cytosolic Ca 2+ in NSCLC cells. AM-37 or ST-36 had no effect on the ability of NT to increase cytosolic Ca 2+ in NSCLC cells. AM-13 or AM-14 had no effect on the ability of ION to increase Ca 2+ in NSCLC cells. Therefore, AM-36 and ST-37 are antagonists for the BBR but not the NTR.

Tyrosine Phosphorylation
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 3 shows that addition of 100 nM BA1 to NCI-H1299 cells transfected with BB2R increased significantly the EGFR tyrosine phosphorylation to 326%. If the cells were pretreated with 10 µM AM-37 or ST-36, addition of BA1 had little effect. In contrast, if the cells were treated with 10 µM 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).

Proliferation
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 4 shows that AM-37 had little effect at 3 µM but strongly inhibited proliferation at 30 µM. The IC50 for AM-37 was 16 µM. Similarly, ST-36 had an IC50 of 22 µM, whereas AM-14 was less potent (IC50 > 50 µM). 30 µM AM-14 followed by 10 nM BA1 and 5 µg/ml ION to increase cytosolic Ca 2+ was determined as a function of time after the addition to NCI-H1299 cells transfected with BB2R. The ability of (g) 10 nM BA1 and 10 nM NT, (h) 30 µM ST-36 followed by 10 nM BA1 and 10 nM NT, and (i) 30 µM AM-13 followed by 10 nM BA1 and 5 µg/ml ION to increase cytosolic Ca 2+ was determined as a function of time after the addition to NCI-H1299 cells transfected with bombesin receptor subtype-3. This experiment is representative of three others. The specificity of the small molecules was investigated. Table 3 shows that AM-37, ST-36, AM-13, and AM-14 (50 µM) 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.

DiscUssiOn
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, increased significantly EGFR and ERK tyrosine phosphorylation relative to the control, whereas total ERK and EGFR were unaltered; p < 0.01; ** by ANOVA. The control, BA1 + AM-37, and BA1 + ST-36 were significantly reduced relative to BA1; p < 0.01, aa by ANOVA. The experiment is representative of three others.
BRS-3 binding sites were detected in 40% of the lung cancer biopsy specimens examined (21). The EGFR is abundant on NSCLC (approximately 100,000 EGFR/cell), whereas BBR are present on most native NSCLC cells (approximately 2,000 BBR/ 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 125 I-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 Ca 2+ , increase ERK and EGFR tyrosine phosphorylation (24). Also, ML-18 inhibited NSCLC growth and increased the cytotoxicity of gefitinib. His 107 is important for BRS-3 to bind antagonists with high affinity (25). Tyr 101 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 125 I-BA1 binding to BB1R, BB2R, and BRS-3 with IC50 values of 7.9, 6.9, and 10.8 µM, 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).
AM-37 and ST-36 inhibited the proliferation of NSCLC cells such as NCI-H1299 and H727, which have BB1R, BB2R, or BRS-3.  cOnclUsiOn 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 Ca 2+ 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.

aUThOr cOnTriBUTiOns
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.