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The metabolism of BW2258U89, a GRP receptor antagonist
Neuropeptides (2000) 34 (2), 108–115 © 2000 Harcourt Publishers Ltd DOI: 10.1054/npep.2000.0798, available online at http://www.idealibrary.com on
The metabolism of BW2258U89, a GRP receptor antagonist C. Marquez, A. Treston, E. Moody, S. Jakowlew, T. W. Moody Cell and Cancer Biology Department, Medicine Branch, National Cancer Institute, Rockville, USA
Summary BW2258U89 is a gastrin releasing peptide (GRP) receptor antagonist which inhibits the proliferation of the neuroendocrine tumor small cell lung cancer (SCLC). Here the biological activity of BW2258U89 and its metabolite were investigated. Using mass spectroscopy (LC-ESI/MS) techniques, three major peaks for BW2258U89 were observed with mass/charge (m/z) ratios of 1081.6, 541.4 and 361.4. After metabolism by mouse plasma enzymes, the major product had a m/z ratio of 1082.5, 541.9 and 361.8 suggesting that BW2258U89 was deamidated. Deamidated (Da) BW2258U89 was synthesized and it inhibited (125I-Tyr4) BB binding to NCI-H345 SCLC cells with an IC50 value of 450 nM; BW2258U89 had an IC50 value of 17 nM. BW2258U89 (1 µM) antagonized the ability of 50 nM BB to elevate cytosolic Ca2+ in NCI-H345 cells, whereas 1 µM (Da) BW2258U89 did not. One micromolar BW2258U89 antagonized the increase in NCI-H345 c-fos mRNA caused by 10 nM BB, whereas 1 µM (Da) BW2258U89 had little effect. One µM BW2258U89 inhibited NCI-H345 clonal growth significantly whereas 1 µM (Da) BW2258U89 did not. These data suggest that an amidated C-terminal is important for antagonism of SCLC GRP receptors by BW2258U89. © 2000 Harcourt Publishers Ltd
INTRODUCTION Bombesin (BB)-like peptides are autocrine growth factors for some small cell lung cancer (SCLC) cells (Cuttitta et al., 1985). Members of the BB family of peptides include gastrin releasing peptide (GRP) and neuromedin B (NMB), each of which have structural homology at the Cterminal (McDonald et al., 1979; Anastasi et al., 1973). GRP is detected in high concentrations in SCLC cells (Moody et al., 1981; Wood et al., 1981). GRP is secreted from NCI-H345 or NCI-H209 cells into the conditioned medium and the secretion rate is increased 3-fold by vasoactive intestinal peptide, which elevates the intracellular cAMP (Korman et al., 1986). GRP binds with high affinity, stimulates phosphatidylinositol (PI) turnover and elevates cytosolic Ca2+ using SCLC cells (Moody et al., 1985; Trepel et al., 1988). Also, GRP elevates c-fos mRNA and stimulates the growth of SCLC cells (Cuttitta et al., 1985; Draoui et al., 1995). The growth of SCLC is inhibited by monoclonal antibody 2A11, which neutralizes endogenous GRP-like peptides (Kelly et al., 1997). Received 20 September 1999 Accepted 18 February 2000
Correspondence to: Dr Terry W. Moody, Medicine Branch, Building KWC, Room 300, 9610 Medical Center Drive, Rockville, MD 20876, USA. Tel.: +1 301 402 3128; Fax: +1 301 402 4422; E-mail: [email protected]
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SCLC growth is inhibited by GRP receptor antagonists. (Psi13,14, Leu14)BB (Psi-13, 14) BB inhibited the clonal growth of NCI-H345 as well as NCI-N592 cells (Mahmoud et al., 1991). Also, (Psi-13,14) BB slowed the growth of NCI-N592 xenografts in nude mice. Similar results were obtained using the (Psi-13,14) BB fragment (3-phenylpropanoyl-His20, D-Ala24, Pro26, Psi26,27, Phe27) GRP20–27 (BW2258U89). BW2258U89 and (Psi-13, 14) BB inhibited (125I-Tyr4) BB binding to NCI-H345 cells with high affinity (IC50 values of 10 and 30 nM respectively). Also, 100 nM BW2258U89 and 1000 nM (Psi-13, 14) BB inhibited NCI-H345 proliferation using an in vitro clonogenic assay. In vivo, 0.4 mg/kg of BW2258U89 and (Psi13, 14) BB inhibited SCLC xenograft proliferation in nude mice. Nanomolar concentrations of BW2258U89 were detected in the plasma of nude mice injected subcutaneously with BW2258U89 (Moody et al., 1995; Moody and Jensen, 1996). Also, a BW2258U89 analog localized to SCLC tumors after injection into the nude mice (Moody et al., 1996). Here the half-life of BW2258U89 and metabolism of BW2258U89 was investigated. BW2258U89 was deamidated by mouse plasma enzymes and the potency of deamidated (Da) BW2258U89 was determined. The results indicate that deamidation of BW2258U89 impairs its ability to function as a GRP receptor antagonist.
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MATERIALS AND METHODS BW2258U89 and (Da) BW2258U89 were purchased from Phoenix Pharmaceuticals (Mountain View, CA, USA). Trifluoroacetic acid, formic acid and acetonitrile were purchased from Sigma Chemical Company (St Louis, MO, USA). Mass spectroscopy Stability studies were performed by incubation of 100 µg/ml BW2258U89 in mouse plasma diluted 1:4 in PBS at 37°C for 0–6 h. The reaction was stopped by addition of 10 µl of 10% trifluoroacetic acid (TFA) and the amount of BW2258U89 remaining determined by liquid chromatography-electrospray ionization/mass spectrometry (LCESI/MS). Chromatographic separations were performed on a Shimadzu LC 7A solvent delivery system in conjunction with a Vydac C18 column (2 mm × 15 cm) with a 20 mm guard column and a flow rate of 200 µl/min. The mobile phase utilized was (A) 0.1% formic acid and (B) acetonitrile/0.1% formic acid. The gradient was 0–60% (B) over an 80 min period. Electrospray ionization mass spectra were acquired on a Sciex API III plus mass spectrometer fitted with an ionspray interface. The MS conditions were: Q1 scans from 300–1250 amu, orifice potential at 60 volts.
The cells were incubated with (125I-Tyr4) BB (0.1 nM) in the presence or absence of BW2258U89 or (Da) BW2258U89; the receptor binding buffer was SIT medium (RPMI–1640 containing 3 × 10–8 M sodium selenite, 5 µg/ml bovine insulin and 10 µg/ml transferrin) with 0.25% bovine serum albumin and 250 µg/ml bacitracin. After incubation at 25°C for 20 min, bound (125ITyr4) BB was separated from free using the centrifugation techniques described previously (Moody et al., 1985). The cells which contained bound peptide were dissolved in 0.2 N NaOH and counted in a gamma counter. Cytosolic calcium Previously, we found that BB elevated cytosolic Ca2+ in NCI-H345 cells (Moody et al., 1987). Here the ability of GRP receptor antagonists to block the increase caused by BB was determined. NCI-H345 cells were harvested (2.5 × 106/ml) and incubated with Fura 2 AM at 37°C for 30 min (Moody et al., 1987). The cells, which contained Fura 2, were centrifuged at 1500 × g for 10 min and resuspended at the same concentration in new SIT medium. The fluorescence intensity was continuously monitored using a Perkin-Elmer LS2 spectrofluorometer equipped with a magnetic stirring mechanism and temperature (37°C) regulated cuvette holder prior to and after the addition of peptide.
Radioimmunoassay
Nuclear oncogenes
BW2258U89 and (Da) BW2258U89 levels in the plasma were evaluated initially by LS-ESI/MS. Subsequently, BW2258U89 concentrations were determined by radioimmunoassay (RIA). BW2258U89 (50 µg) was incubated with 1 ml of 25% plasma for 0–50 h at 37°C in the presence or absence of inhibitors such as 1 mM phenylmethylsulfonylfluoride (PMSF). The reaction was stopped by the addition of 10 µl of 10% TFA and the sample frozen and lyophilized. The sample was resuspended in radioimmunoassay buffer (0.1% BSA in PBS) and incubated with antisera (BW2 1:1000) in the presence of 10 000 cpm of 125I-BW1023U90 (Moody et al., 1996).
For the c-fos experiments, SCLC cells were cultured with SIT medium containing 0.5% fetal bovine serum (Draoui et al., 1995). After 4 h, the cells were treated with 10 nM BB in the presence or absence of GRP receptor antagonists for 60 min. Total RNA was isolated using guanidinium isothiocyanate. Ten micrograms of denatured RNA was separated in a 0.66 M formaldehyde 1% agarose gel. The gel was treated with ethidium bromide to assess RNA integrity. The RNA was blotted onto a nytran membrane overnight and the membrane hybridized with DNA probes labeled with 32p-dCTP using a Bethesda Research Laboratories random priming kit (Draoui et al., 1995). The membrane was apposed to Kodak XAR-2 film at –80°C for 1 day and the autoradiogram developed. The autoradiograms were analysed using a Molecular Dynamics densitometer.
Radioreceptor assay The receptor binding affinity for BW2258U89 and (Da) BWE2258U89 was determined. NCI-H345 cells were cultured in RPMI-1640 containing 10% heat-inactivated fetal bovine serum (Carney et al., 1985). The cells were split weekly by dilution 1/1 in new medium. The cells were mycoplasma free and were used in exponential growth phase after incubation at 37°C in 5% CO2/95% air. Receptor binding assays were conducted using (125ITyr4) BB (1100 Ci/mmol) and NCI-H345 cells (2 × 106). © 2000 Harcourt Publishers Ltd
Proliferation Growth studies were performed using cell lines NCIH345 or H720 cells and the agarose cloning system (Mahmoud et al., 1991). The base layer consisted of 3 ml of 0.5% agarose and 5% fetal bovine serum in SIT medium in 6 well plates. The top layer consisted of 3 ml Neuropeptides (2000) 34(2), 108–115
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of SIT medium containing 0.3% agarose, the peptides and 5 × 104 single viable cells. For each cell line and peptide concentration, triplicate wells were plated. After 2 weeks, 1 ml of 0.1% p-iodonitrotetrazolium violet was added and after 16 h at 37°C, the plates were screened for colony formation; the number of colonies larger than 50 µm in diameter was counted using an Omnicon image analysis system. RESULTS Mass spectroscopy BW22258U89 was metabolized by mouse plasma. BW2258U89 eluted at 53.9 min after injection into the HPLC. The mouse plasma had major peaks of absorbance at 14.1, 44.6 and 49.7 min contributing to the background. Figure 1 shows that after BW2258U89 was incubated with 25% mouse plasma for 6 h, a BW2258U89 metabolite appeared at 55.9 min. Table 1 shows that the disappearance of BW2258U89 upon incubation with mouse plasma was time dependent. In particular, after 1 and 2 h, 78 and 63% of the BW2258U89 remained respectively, and the calculated half-life was approximately 170 min. In contrast, the agonist GRP was rapidly metabolized and had a half-life of 5 min (data not shown). The mass spectra of BW2258U89 and its metabolite were determined. Figure 2 shows that singly, doubly and triply charged species were observed for BW2258U89 with m/z ratios of 1081.6, 541.3 and 361.5. For the BW2258U89 metabolite the m/z ratios were 1082.5, 541.9 and 361.8. The most likely explanation for the 1 unit difference in the m/z ratio between BW2258U89 and its metabolite is that BW2258U89 is deamidated by enzymes in mouse plasma.
Fig. 1 LC-ESI/MS profile of BW2258U89 and mouse plasma. The relative intensity of mouse plasma containing BW2258U89 and its metabolite is indicated. This experiment is representative of six others.
Table 1 Metabolism of BW2258U89 by mouse plasma Time (h)
% of BW2258U89 remaining
0 0.25 0.5 1 2
100 ± 6 90 ± 9 85 ± 7 78 ± 8 63 ± 5
BW2258U89 (100 µg) was added to 1 ml of 25% mouse plasma diluted in PBS. After incubation at 37°C, 10 µl of 10% formic acid was added and the sample injected into the LC-EIS/MS. The mean value ± S.D. of 3 determinations is indicated. The structure of BW2258U89 is (3-phenylpropanoyl-His-Trp-Ala-Val-DAla-His-Pro = Phe-NH2, which has a O = C-NH2 C-terminal. The structure of deamidated BW2258U89, which has a O = C-OH C-terminal, is (3phenylpropanoyl-His-Trp-Ala-Val-DAla-His-Pro = Phe; – is an amide bond (O = C–NH) whereas = is a reduced peptide bond CH2-NH.
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Fig. 2 Full scan mass spectrum of BW2258U89 and its metabolite. The relative intensity is indicated as a function of m/z.
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Stability of BW2258U89
Biological activity
(Da) BW2258U89 was chemically synthesized and evaluated. (Da) BW2258U89 eluted 55.9 min after injection into the HPLC and had the same m/z ratios (1082.5, 541.9 and 361.8) as the BW2258U89 metabolite. The immunoreactivity of (Da) BW2258U89 was evaluated and compared to BW2258U89. Figure 3 shows that in the RIA, (Da) BW2258U89 was less potent than was BW2258U89. BW2258U89 inhibited 125I-BW1023U90 binding to the antibody in a dose dependent manner with little inhibition occuring at 0.01 pmol and extensive inhibition at 1 pmol. The IC50 for BW2258U89 was 0.5 pmol, whereas (Da) BW2258U89 did not inhibit 125I-BW1023U90 binding to BW2 antisera even at 10 pmol. (Da) BW2258U89 was <1% as potent as BW2258U89 at binding to antibody BW2. These results suggest that the immunoreactivity detected in the plasma samples is primarily due to BW2258U89 and not its metabolite (Da) BW2258U89. Additional metabolism studies were conducted. Figure 4 shows that BW2258U89 was metabolized by mouse plasma. Initially, 3 pmol of immunoreactivity was present in the samples. In the PBS control this amount slightly declined over a 50 h period. In contrast, BW2258U89 exposed to 25% mouse plasma, was undetectable after 15 h. The half-life for BW2258U89 immunoreactivity was approximately 3 h. If the BW2258U89 was incubated with 25% mouse plasma containing 1 mM phenylmethlysulfonyl fluoride (PMSF), its dissappearance was markedly slowed. The half-life for BW2258U89 in mouse plasma containing PMSF was approximately 15 h. These results suggest that BW2258U89 is metabolized by a mouse plasma enzyme which is inhibited by PMSF.
The receptor binding activity of BW2258U89 and (Da) BW2258U89 was investigated. Figure 5 shows that BW2258U89 inhibited specific (125I-Tyr4) BB binding to NCI-H345 cells in a dose dependent manner. The IC50 for BW2258U89 was 0.017 µM. In contrast, (Da) BW2258U89 inhibited (125I-Tyr4) BB binding with an IC50 of 0.45 µM. These data indicate that (Da) BW2258U89 binds with over an order of magnitude lower affinity to SCLC GRP receptors than does BW2258U89.
Fig. 3 Radioimmunoassay. The ability of BW2258U89 (● ●) and (Da) BW2258U89 (•) to inhibit 125I-BW1023U90 binding to antibody BW2 was determined. The mean value ± SD of four determinations each repeated in duplicate is indicated.
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Fig. 4 Metabolism of BW2258U89. BW2258U89, 3 pmol, was added to 1 ml of PBS (▲), 25% plasma in 1 ml of PBS (•), and 25% mouse plasma containing 1 mM PMSF (● ●). The mean value ± SD of four determinations is indicated. This experiment is representative of two others.
Fig. 5 Receptor binding activity. The specific binding of (125I-Tyr4) BB to NCI-H345 cells is indicated as a function of BW2258U89 (●) and (Da) BW2258U89 (● ●) concentration. The mean value of three determinations each repeated in quadruplicate is indicated.
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Table 2 Effect of GRP receptor antagonists on colony formation Addition None (Da) BW2258U89, 1 µM BW2258U89, 0.1 µM BW2258U89, 1 µM
Colony number 1543 ± 110 1278 ± 156 1378 ± 191 890 ± 75*
The mean value ± SD of 3 determinations is shown; P < 0.05, * using Student’s t-test. This experiment is representative of two others.
Fig. 6 Cytosolic calcium. (Top) The relative excitation ratio was determined for Fura-2 loaded NCI-H345 cells after addition of 50 nM BB, 50 nM BB + 1 µM BW2258U89 and 50 nM BB + 10 µM BW2258U89. (Bottom) The relative excitation ratio was determined after the addition of 50 nM BB + 1 µM (Da) BW2258U89 and 50 nM BB + 1 µM (Da) BW2258U89. This experiment is representative of three others.
The biological activity of BW2258U89 and (Da) BW2258U89 was investigated. BW2258U89 had little effect on basal cytosolic Ca2+ of Fura-2 loaded NCI-H345 cells but antagonized the ability of 50 nM BB to elevate cytosolic Ca2+ (Fig. 6). BW2258U89 (0.1 µM and 1 µM) slightly and strongly respectively, inhibited the ability of 50 nM BB to elevate cytosolic Ca2+. In contrast, (Da) BW2258U89 (1 µM) had little effect on the ability of 10 nM BB to elevate cytosolic Ca2+. Similar results were obtained if GRP was used as the agonist instead of BB (data not shown). The ability of GRP receptor antagonists to alter nuclear oncogene expression was investigated. BB (10 nM) elevated c-fos mRNA after 45 min (Fig. 7). BW2258U89 Neuropeptides (2000) 34(2), 108–115
Fig. 7 c-fos mRNA. NCI-H345 cells were treated for 45 min with no additions, 10 nM BB + 1 µM BW2258U89, 10 nM BB + 1 µM (Da) BW2258U89 and 10 nM BB. The blot was probed for c-fos mRNA (left) or the gel was ethidium bromide stained (right). This experiment is representative of two others.
weekly (0.1 µM) and strongly (1 µM) antagonized the increase in c-fos mRNA caused by 10 nM BB. In contrast, 1 µM (Da) BW2258U89 had little effect on the ability of 10 nM BB to increase c-fos mRNA. The effects of BW2258U89 and its metabolite on proliferation were determined. Table 2 shows that 1 µM but not 0.1 µM BW2258U89 significantly decreased colony formation of NCI-H345 cells by approximately 45%. In contrast, 1 µM (Da) BW2258U89 had little effect on colony formation. © 2000 Harcourt Publishers Ltd
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DISCUSSION The present study indicates that BW2258U89 is metabolized by mouse plasma enzymes which are inhibited by PMSF. The primary product is deamidated BW2258U89 and (Da) BW2258U89 has approximately 1-order of magnitude less biological activity than BW2258U89. Previous studies showed that GRP receptors were blocked with high affinity by (des-Met14) BB antagonists such as (D-pentafluora-Phe6, D-Ala11) BB6–13 methylester ((FA) BB6–13ME) and with low affinity by substance P (SP) antagonists such as antagonist D, (D-Arg1, D-Phe5, DTrp7,9, Leu11) SP19,20. Micromolar concentrations of (FA) BB6–13ME, BW2258U89 or (D-Arg1, D-Pro2, D-Trp7,9, Leu11) substance P inhibited SCLC colony formation in soft agar (Mahmoud et al., 1991). In vivo, BW2258U89 (0.4 mg/kg) inhibits SCLC xenograft proliferation in nude mice, whereas substantially higher concentrations of (FA) BB6–13ME (Moody et al., unpublished) or antagonist D are required (Sethi et al., 1992). Because BW2258U89 may be a therapeutic agent for SCLC, additional preclinical data on its metabolism were obtained. When BW2258U89 was exposed to mouse plasma (25%), its half-life was 170 min at 37°C. The BW2258U89 metabolite was more hydrophobic than was BW2258U89 and the eluted from the LC-ESI/MS at a later time. The m/z ratio of the singly charged species for BW2258U89 and its metabolite was 1081.6 and 1082.5 respectively. Because the BW2258U89 metabolite had a mass increase of approximately 1, BW2258U89 may be deamidated by mouse plasma enzymes. Similarly, it was found that SP antagonists, which have a plasma half-life of several hours, were deamidated by mouse enzymes (Cummings et al., 1994). In contrast, GRP had a very short half-life of 5 min (Marquez, unpublished). GRP is metabolized by endopeptidase 24.11. to GRP1–25 and GRP26–27 and GRP metabolism is slowed by inclusion of thiorphan into the incubation buffer (Davis et al., 1992). To minimize degradation by endopeptidase 24.11, BW2258U89 has a D-Ala in place of a Gly. This substitution has little effect on receptor binding activity but decreases degradation by endopeptidases. BW2258U89 may be deamidated by a serine protease such as serine carboxypeptidase. In this regard, BW2258U89 metabolism by the mouse plasma was inhibited by PMSF which acetylates serines on carboxypeptidase enzymes which have deamidase activity (Jones et al., 1996). The metabolism of SP antagonist D was also inhibited by PMSF. Antagonist D was metabolized by blood, plasma or liver homogenate, suggesting that these enzymes have a broad distribution in the body. Similarly, PMSF blocks degradation of BW2258U89 by SCLC cell homogenates (Moody, unpublished). These © 2000 Harcourt Publishers Ltd
data support the hypothesis that BW2258U89 may be slowly metabolized by serine carboxypeptidases. Similarly, the structurally related antagonist, (Tpi6, Psi13,14, Tpi14) BN6–14 would likely be deamidated by serine proteases (Cai et al., 1992). It remains to be determined if BW2258U89 is metabolized by enzymes other than serine proteases. Antagonist D was metabolized by carboxypeptidase B-like enzymes after deamidation, resulting in the removal of the C-terminal leucine amino acid. It remains to be determined if BW2258U89, which has a reduced peptide bond at the penultimate position, can be metabolized by carboxypeptidases. The biological activity of the BW2258U89 metabolite was investigated. (Da) BW2258U89 had an IC50 value 15fold greater than did BW2258U89 for inhibition of (125ITyr4) BB binding to NCI-H345 cells. Similarly, 1 µM BW2258U89 antagonized the ability of BB to elevate cytosolic Ca2+ and c-fos mRNA using NCI-H345 cells whereas 1 µM (Da) BW2258U89 had little effect. Preliminary data (Moody, unpublished) indicate that higher doses of (Da) BW2258U89, e.g. 10 µM antagonized the ability of BB to elevate cytosolic Ca2+ or increase c-fos mRNA. BW2258U89 (1 µM) inhibited SCLC proliferation in vitro whereas 1 µM (Da) BW2258U89 did not. These results suggest that after BW2258U89 is metabolized by serine carboxypeptidases it is approxiately 1-order of magnitude less potent. Similarly, antagonist D metabolites were less potent than was antagonist D (Jones et al., 1996). Previously, we found that SCLC xenograft formation was strongly inhibited if BW2258U89 was placed in microspheres which slowly release BW2258U89 into the mouse over a 3 week period (Moody et al., 1996). Nanomolar concentrations of BW2258U89 were present in the mouse blood, weeks after subcutaneous injection of the microspheres. In contrast, if a single bolus dose of BW2258U89 is injected daily, its plasma concentration decreases by over 2-orders of magnitude during the course of a day (Moody, unpublished). These results suggest that if BW2258U89 is to be used as a therapeutic agent, its formulation is important. Antagonists such as BW2258U89 bind with high affinity to the GRP receptor, a 384 amino acid protein which contains 7 transmembrane domains (Battey et al., 1991; Spindel et al., 1990). In contrast, BW2258U89 binds with low affinity to the neuromedin B (NMB) receptor, which is a 390 amino acid protein (Wada et al., 1991) and bombesin receptor subtype (BRS)-3, a 399 amino acid protein (Fathi et al., 1993). Each of these receptors have approximately 50% amino acid sequence homology, but the GRP receptor prefers GRP relative to NMB and the NMB receptor prefers NMB relative to GRP; the BRS-3 receptor binds (D-Phe6, β-Ala11, Phe13, Nle14) BB6–14 but Neuropeptides (2000) 34(2), 108–115
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not GRP or NMB with high affinity (Moody and Jensen, 1996; Mantey et al., 1997). In contrast, micromolar concentrations of SP antagonists block GRP and NMB receptors as well as BRS-3 (Ryan et al., 1998). Also, somatostatin analogs inhibit NMB receptors with IC50 values of 1000 nM (Orbuch et al., 1993). Recently, nonpeptide NMB antagonists have been described which bind to NMB receptors with a Kd of 1 nM (Eden et al., 1996). Nonpeptide antagonists for GRP receptors remain unknown. In summary, BW2258U89 is a potent GRP receptor antagonist which is deamidated by mouse plasma enzymes. The resulting product is impaired in its ability to function as a GRP receptor antagonist.
ACKNOWLEDGMENTS The authors thank Amy Guzzone for technical assistance and Drs J. Leban and J. McDermed for the BW2258U89.
REFERENCES Anastasi A, Erspamer V, Bucci M (1973) Isolation and amino acid sequences of alytesin and bombesin: Two analogous active tetradecapeptides from the skin of European discoglossid frogs. Archives of Biochemistry and Biophysics 148: 443–446. Battey JF, Way J, Corjay MH et al. (1991) Molecular cloning of the bombesin/GRP receptor from Swiss 3T3 cells. Proceedings of the National Academy of Science USA 88: 395–399. Cai RZ, Radulovic S, Pinski J, Nagy A, Redding TW, Olsen DB, Schally AV (1992) Pseudononapeptide bombesin antagonists containing C-terminal Trp or Tpi. Peptides 13: 267–271. Carney DN, Gazdar AF, Bepler G et al. (1985) Establishment and identification of small cell lung cancer cell lines having classic and variant features. Cancer Research 45: 2913–2923. Cummings J, Maclellan A, Langdon SP, Smythe JF (1994) Stability and in vitro metabolism of the mitogenic neuropeptide antagonists (D-Arg1, D-Phe5, D-Trp7,9, Leu11) substance P and (Arg6, D-Trp7,9, NmePhe8) substance P (6–11) characterized by high performance liquid chromatography. Journal of Pharmacentical and Biomedical Analysis 12: 811–819. Cuttitta F, Carney DN, Mulshine J, Moody TW, Fedorko J, Fischler A, Minna JD (1985) Bombesin-like peptides can function as autocrine growth factors in human small cell lung cancer. Nature 316: 823–825. Davis TP, Crowell S, Taylor J, Clark DL, Coy D, Staley J, Moody TW (1992) Metabolic stability and tumor inhibition of bombesin/GRP receptor antagonists. Peptides 13: 401–407. Draoui M, Chung P, Park M, Birrer M, Jakowlew S, Moody TW (1995) Bombesin Stimulates c-fos and c-jun mRNAs in small cell lung cancer cells. Peptides 16: 289–292. Eden JM, Hall MD, Higginbottom M et al. (1996) PD165929-The first high affinity non-peptide neuromedin B (NMB) receptor selective antagonist. Bioorganic & Medicinal Chemistry Letters 6: 2617–2623. Fathi Z, Corjay MH, Shapira H et al. (1993) BRS-3: A novel bombesin receptor subtype selectively expressed in testis and lung carcinoma cells. Journal of Biological Chemistry 268: 5979–5984. Jones DA, Cummings J, Langdon SP, Maclellan AJ, Higgins T, Rozengurt E, Smyth JF (1996) Metabolism of the broad
Neuropeptides (2000) 34(2), 108–115
spectrum neuropeptide growth factor antagonist (D-Arg1, DPhe5, D-Trp7,9, Leu11) substance P. British Journal of Cancer 73: 715–720. Kelly MJ, Linnoila RI, Avis I, Mulshine J, Johnson B (1997) Antitumor activity of a monoclonal antibody directed against gastrin releasing peptide in patients with small cell lung cancer. Chest 112: 256–261. Korman LY, Carney DN, Citron ML, Moody TW (1986) Secretin/VIP stimulated secretion of bombesin-like peptides from human small cell lung cancer. Cancer Research 46: 1214–1218. Mahmoud S, Staley J, Taylor J. et al. (1991) (Psi13,14) Bombesin analogues inhibit the growth of small cell lung cancer in vitro and in vivo. Cancer Research 51: 1798–1802. Mantey SA, Weber HC, Sainz E et al. (1997) Discovery of a high affinity radioligand for the human orphan receptor, bombesin receptor subtype 3, which demonstrates that it has a unique pharmacology compared with other mammalian bombesin receptors. Journal of Biological Chemistry 272: 26062–26071. McDonald TJ, Jornvall J, Nilsson G, Vagne M, Ghatei M, Bloom SR, Mutt V (1979) Characterization of a gastrin releasing peptide from porcine non-antral gastric tissue. Biochemical and Biophysical Research Communications 90: 227–233. Minamino N, Kangawa K, Matsuo H (1983) Neuromedin B: A novel bombesin-like peptide identified in porcine spinal cord. Biochemical and Biophysical Research Communications 114: 541–548. Moody TW, Pert CB, Gazdar AF, Carney DN, Minna JD (1981) High levels of intracellular bombesin characterize human small cell lung carcinoma. Science 214: 1246–1248. Moody TW, Carney DN, Cuttitta F, Quattrocchi K, Minna JD (1985) High affinity receptors for bombesin/GRP-like peptides on human small cell lung cancer. Life Science 37: 105–113. Moody TW, Murphy A, Mahmoud S, Fiskum G (1987) Bombesin-like peptides elevate cytosolic calcium in small cell lung cancer cells. Biochemical and Biophysical Research Communications 147: 189–195. Moody TW, Jensen RT (1996) Bombesin/GRP and vasoactive intestinal peptide/PACAP as growth factors. In: Le Roith D, Bondy C (eds) Growth Factors and Cytokines in Health and Disease JAI Press, Greenwich, 491–535. Moody TW, Venugopal R, Gozes Y, Hu V, McDermed J, Leban JJ (1996) BW1023U90: A new GRP receptor probe for small cell lung cancer cells. Peptides 17: 1337–1343. Moody TW, Venugopal R, Zia F, Patierno S, Leban JJ, McDermed J (1995) BW2258U89: A GRP receptor antagonist which inhibits small cell lung cancer growth. Life Science 5: 521–529. Orbuch M, Taylor JE, Coy DH et al. (1993) Discovery of a novel class of neuromedin B receptor antagonists, substituted somatostatin analogues. Molecular Pharmacology 44: 841–850. Ryan RR, Weber HC, Hou W et al. (1998) Ability of various bombesin receptor agonists and antagonists to alter intracellular signaling of the human orphan receptor BRS-3. Journal of Biological Chemistry 273: 13613–13624. Sausville EA, Moyer JD, Heikkila R, Neckers LM, Trepel JB (1988) A correlation of bombesin-responsiveness with myc-family gene expression in small cell lung carcinoma cell lines. Annals of the NY Academy of Science 547: 310–321. Sethi T, Langdon S, Smyth J (1992) Growth of small cell lung cancer cells: Stimulation by multiple neuropeptides and inhibition by broad spectrum antagonists in vitro and in vivo. Cancer Research 52: 2737s–42s. Spindel ER, Giladi E, Brehm TP, Goodman RH, Segerson TP (1990) Cloning and functional characterization of a cDNA encoding the murine fibroblast bombesin/GRP receptor. Molecular Endocrinology 4: 1956–1963.
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Staley J, Coy DH, Taylor JE, Kim S, Moody TW (1991) (Des-Met14) bombesin analogues function as small cell lung cancer bombesin receptor antagonists. Peptides 12: 145–149. Trepel JB, Moyer JD, Cuttitta F. et al. (1988) A novel bombesin receptor antagonist inhibits autocrine signals in a small cell lung carcinoma cell line. Biochemical and Biophysical Research Communications 156: 1383–1389. Wada E, Way J, Shapira H, Kusano K et al. (1991) cDNA Cloning, characterization and brain region-specific expression of a neuromedin-B preferring bombesin receptor. Neuron 6: 421–430.
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Woll PJ, Rozengurt E (1988) (D-Arg1, D-Phe5, D-Trp7,9, Leu11) substance P, a potent bombesin antagonist in murine Swiss 3T3 cells, inhibits the growth of human small cell lung cancer. Proceedings of the National Academy of Science USA 85: 1859–1863. Wood S, Wood J, Ghatei M, Lee U, O’Shaghnessy D, Bloom SR (1981) Bombesin, somatostatin and neurotensin-like immunoreactivity in bronchial carcinoma. Journal of Clinical Endocrinology 53: 1310–1312.
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The metabolism of BW2258U89, a GRP receptor antagonist C. Marquez, A. Treston, E. Moody, S. Jakowlew, T. W. Moody Cell and Cancer Biology Department, Medicine Branch, National Cancer Institute, Rockville, USA
Summary BW2258U89 is a gastrin releasing peptide (GRP) receptor antagonist which inhibits the proliferation of the neuroendocrine tumor small cell lung cancer (SCLC). Here the biological activity of BW2258U89 and its metabolite were investigated. Using mass spectroscopy (LC-ESI/MS) techniques, three major peaks for BW2258U89 were observed with mass/charge (m/z) ratios of 1081.6, 541.4 and 361.4. After metabolism by mouse plasma enzymes, the major product had a m/z ratio of 1082.5, 541.9 and 361.8 suggesting that BW2258U89 was deamidated. Deamidated (Da) BW2258U89 was synthesized and it inhibited (125I-Tyr4) BB binding to NCI-H345 SCLC cells with an IC50 value of 450 nM; BW2258U89 had an IC50 value of 17 nM. BW2258U89 (1 µM) antagonized the ability of 50 nM BB to elevate cytosolic Ca2+ in NCI-H345 cells, whereas 1 µM (Da) BW2258U89 did not. One micromolar BW2258U89 antagonized the increase in NCI-H345 c-fos mRNA caused by 10 nM BB, whereas 1 µM (Da) BW2258U89 had little effect. One µM BW2258U89 inhibited NCI-H345 clonal growth significantly whereas 1 µM (Da) BW2258U89 did not. These data suggest that an amidated C-terminal is important for antagonism of SCLC GRP receptors by BW2258U89. © 2000 Harcourt Publishers Ltd
INTRODUCTION Bombesin (BB)-like peptides are autocrine growth factors for some small cell lung cancer (SCLC) cells (Cuttitta et al., 1985). Members of the BB family of peptides include gastrin releasing peptide (GRP) and neuromedin B (NMB), each of which have structural homology at the Cterminal (McDonald et al., 1979; Anastasi et al., 1973). GRP is detected in high concentrations in SCLC cells (Moody et al., 1981; Wood et al., 1981). GRP is secreted from NCI-H345 or NCI-H209 cells into the conditioned medium and the secretion rate is increased 3-fold by vasoactive intestinal peptide, which elevates the intracellular cAMP (Korman et al., 1986). GRP binds with high affinity, stimulates phosphatidylinositol (PI) turnover and elevates cytosolic Ca2+ using SCLC cells (Moody et al., 1985; Trepel et al., 1988). Also, GRP elevates c-fos mRNA and stimulates the growth of SCLC cells (Cuttitta et al., 1985; Draoui et al., 1995). The growth of SCLC is inhibited by monoclonal antibody 2A11, which neutralizes endogenous GRP-like peptides (Kelly et al., 1997). Received 20 September 1999 Accepted 18 February 2000
Correspondence to: Dr Terry W. Moody, Medicine Branch, Building KWC, Room 300, 9610 Medical Center Drive, Rockville, MD 20876, USA. Tel.: +1 301 402 3128; Fax: +1 301 402 4422; E-mail: [email protected]
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SCLC growth is inhibited by GRP receptor antagonists. (Psi13,14, Leu14)BB (Psi-13, 14) BB inhibited the clonal growth of NCI-H345 as well as NCI-N592 cells (Mahmoud et al., 1991). Also, (Psi-13,14) BB slowed the growth of NCI-N592 xenografts in nude mice. Similar results were obtained using the (Psi-13,14) BB fragment (3-phenylpropanoyl-His20, D-Ala24, Pro26, Psi26,27, Phe27) GRP20–27 (BW2258U89). BW2258U89 and (Psi-13, 14) BB inhibited (125I-Tyr4) BB binding to NCI-H345 cells with high affinity (IC50 values of 10 and 30 nM respectively). Also, 100 nM BW2258U89 and 1000 nM (Psi-13, 14) BB inhibited NCI-H345 proliferation using an in vitro clonogenic assay. In vivo, 0.4 mg/kg of BW2258U89 and (Psi13, 14) BB inhibited SCLC xenograft proliferation in nude mice. Nanomolar concentrations of BW2258U89 were detected in the plasma of nude mice injected subcutaneously with BW2258U89 (Moody et al., 1995; Moody and Jensen, 1996). Also, a BW2258U89 analog localized to SCLC tumors after injection into the nude mice (Moody et al., 1996). Here the half-life of BW2258U89 and metabolism of BW2258U89 was investigated. BW2258U89 was deamidated by mouse plasma enzymes and the potency of deamidated (Da) BW2258U89 was determined. The results indicate that deamidation of BW2258U89 impairs its ability to function as a GRP receptor antagonist.
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MATERIALS AND METHODS BW2258U89 and (Da) BW2258U89 were purchased from Phoenix Pharmaceuticals (Mountain View, CA, USA). Trifluoroacetic acid, formic acid and acetonitrile were purchased from Sigma Chemical Company (St Louis, MO, USA). Mass spectroscopy Stability studies were performed by incubation of 100 µg/ml BW2258U89 in mouse plasma diluted 1:4 in PBS at 37°C for 0–6 h. The reaction was stopped by addition of 10 µl of 10% trifluoroacetic acid (TFA) and the amount of BW2258U89 remaining determined by liquid chromatography-electrospray ionization/mass spectrometry (LCESI/MS). Chromatographic separations were performed on a Shimadzu LC 7A solvent delivery system in conjunction with a Vydac C18 column (2 mm × 15 cm) with a 20 mm guard column and a flow rate of 200 µl/min. The mobile phase utilized was (A) 0.1% formic acid and (B) acetonitrile/0.1% formic acid. The gradient was 0–60% (B) over an 80 min period. Electrospray ionization mass spectra were acquired on a Sciex API III plus mass spectrometer fitted with an ionspray interface. The MS conditions were: Q1 scans from 300–1250 amu, orifice potential at 60 volts.
The cells were incubated with (125I-Tyr4) BB (0.1 nM) in the presence or absence of BW2258U89 or (Da) BW2258U89; the receptor binding buffer was SIT medium (RPMI–1640 containing 3 × 10–8 M sodium selenite, 5 µg/ml bovine insulin and 10 µg/ml transferrin) with 0.25% bovine serum albumin and 250 µg/ml bacitracin. After incubation at 25°C for 20 min, bound (125ITyr4) BB was separated from free using the centrifugation techniques described previously (Moody et al., 1985). The cells which contained bound peptide were dissolved in 0.2 N NaOH and counted in a gamma counter. Cytosolic calcium Previously, we found that BB elevated cytosolic Ca2+ in NCI-H345 cells (Moody et al., 1987). Here the ability of GRP receptor antagonists to block the increase caused by BB was determined. NCI-H345 cells were harvested (2.5 × 106/ml) and incubated with Fura 2 AM at 37°C for 30 min (Moody et al., 1987). The cells, which contained Fura 2, were centrifuged at 1500 × g for 10 min and resuspended at the same concentration in new SIT medium. The fluorescence intensity was continuously monitored using a Perkin-Elmer LS2 spectrofluorometer equipped with a magnetic stirring mechanism and temperature (37°C) regulated cuvette holder prior to and after the addition of peptide.
Radioimmunoassay
Nuclear oncogenes
BW2258U89 and (Da) BW2258U89 levels in the plasma were evaluated initially by LS-ESI/MS. Subsequently, BW2258U89 concentrations were determined by radioimmunoassay (RIA). BW2258U89 (50 µg) was incubated with 1 ml of 25% plasma for 0–50 h at 37°C in the presence or absence of inhibitors such as 1 mM phenylmethylsulfonylfluoride (PMSF). The reaction was stopped by the addition of 10 µl of 10% TFA and the sample frozen and lyophilized. The sample was resuspended in radioimmunoassay buffer (0.1% BSA in PBS) and incubated with antisera (BW2 1:1000) in the presence of 10 000 cpm of 125I-BW1023U90 (Moody et al., 1996).
For the c-fos experiments, SCLC cells were cultured with SIT medium containing 0.5% fetal bovine serum (Draoui et al., 1995). After 4 h, the cells were treated with 10 nM BB in the presence or absence of GRP receptor antagonists for 60 min. Total RNA was isolated using guanidinium isothiocyanate. Ten micrograms of denatured RNA was separated in a 0.66 M formaldehyde 1% agarose gel. The gel was treated with ethidium bromide to assess RNA integrity. The RNA was blotted onto a nytran membrane overnight and the membrane hybridized with DNA probes labeled with 32p-dCTP using a Bethesda Research Laboratories random priming kit (Draoui et al., 1995). The membrane was apposed to Kodak XAR-2 film at –80°C for 1 day and the autoradiogram developed. The autoradiograms were analysed using a Molecular Dynamics densitometer.
Radioreceptor assay The receptor binding affinity for BW2258U89 and (Da) BWE2258U89 was determined. NCI-H345 cells were cultured in RPMI-1640 containing 10% heat-inactivated fetal bovine serum (Carney et al., 1985). The cells were split weekly by dilution 1/1 in new medium. The cells were mycoplasma free and were used in exponential growth phase after incubation at 37°C in 5% CO2/95% air. Receptor binding assays were conducted using (125ITyr4) BB (1100 Ci/mmol) and NCI-H345 cells (2 × 106). © 2000 Harcourt Publishers Ltd
Proliferation Growth studies were performed using cell lines NCIH345 or H720 cells and the agarose cloning system (Mahmoud et al., 1991). The base layer consisted of 3 ml of 0.5% agarose and 5% fetal bovine serum in SIT medium in 6 well plates. The top layer consisted of 3 ml Neuropeptides (2000) 34(2), 108–115
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of SIT medium containing 0.3% agarose, the peptides and 5 × 104 single viable cells. For each cell line and peptide concentration, triplicate wells were plated. After 2 weeks, 1 ml of 0.1% p-iodonitrotetrazolium violet was added and after 16 h at 37°C, the plates were screened for colony formation; the number of colonies larger than 50 µm in diameter was counted using an Omnicon image analysis system. RESULTS Mass spectroscopy BW22258U89 was metabolized by mouse plasma. BW2258U89 eluted at 53.9 min after injection into the HPLC. The mouse plasma had major peaks of absorbance at 14.1, 44.6 and 49.7 min contributing to the background. Figure 1 shows that after BW2258U89 was incubated with 25% mouse plasma for 6 h, a BW2258U89 metabolite appeared at 55.9 min. Table 1 shows that the disappearance of BW2258U89 upon incubation with mouse plasma was time dependent. In particular, after 1 and 2 h, 78 and 63% of the BW2258U89 remained respectively, and the calculated half-life was approximately 170 min. In contrast, the agonist GRP was rapidly metabolized and had a half-life of 5 min (data not shown). The mass spectra of BW2258U89 and its metabolite were determined. Figure 2 shows that singly, doubly and triply charged species were observed for BW2258U89 with m/z ratios of 1081.6, 541.3 and 361.5. For the BW2258U89 metabolite the m/z ratios were 1082.5, 541.9 and 361.8. The most likely explanation for the 1 unit difference in the m/z ratio between BW2258U89 and its metabolite is that BW2258U89 is deamidated by enzymes in mouse plasma.
Fig. 1 LC-ESI/MS profile of BW2258U89 and mouse plasma. The relative intensity of mouse plasma containing BW2258U89 and its metabolite is indicated. This experiment is representative of six others.
Table 1 Metabolism of BW2258U89 by mouse plasma Time (h)
% of BW2258U89 remaining
0 0.25 0.5 1 2
100 ± 6 90 ± 9 85 ± 7 78 ± 8 63 ± 5
BW2258U89 (100 µg) was added to 1 ml of 25% mouse plasma diluted in PBS. After incubation at 37°C, 10 µl of 10% formic acid was added and the sample injected into the LC-EIS/MS. The mean value ± S.D. of 3 determinations is indicated. The structure of BW2258U89 is (3-phenylpropanoyl-His-Trp-Ala-Val-DAla-His-Pro = Phe-NH2, which has a O = C-NH2 C-terminal. The structure of deamidated BW2258U89, which has a O = C-OH C-terminal, is (3phenylpropanoyl-His-Trp-Ala-Val-DAla-His-Pro = Phe; – is an amide bond (O = C–NH) whereas = is a reduced peptide bond CH2-NH.
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Fig. 2 Full scan mass spectrum of BW2258U89 and its metabolite. The relative intensity is indicated as a function of m/z.
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Stability of BW2258U89
Biological activity
(Da) BW2258U89 was chemically synthesized and evaluated. (Da) BW2258U89 eluted 55.9 min after injection into the HPLC and had the same m/z ratios (1082.5, 541.9 and 361.8) as the BW2258U89 metabolite. The immunoreactivity of (Da) BW2258U89 was evaluated and compared to BW2258U89. Figure 3 shows that in the RIA, (Da) BW2258U89 was less potent than was BW2258U89. BW2258U89 inhibited 125I-BW1023U90 binding to the antibody in a dose dependent manner with little inhibition occuring at 0.01 pmol and extensive inhibition at 1 pmol. The IC50 for BW2258U89 was 0.5 pmol, whereas (Da) BW2258U89 did not inhibit 125I-BW1023U90 binding to BW2 antisera even at 10 pmol. (Da) BW2258U89 was <1% as potent as BW2258U89 at binding to antibody BW2. These results suggest that the immunoreactivity detected in the plasma samples is primarily due to BW2258U89 and not its metabolite (Da) BW2258U89. Additional metabolism studies were conducted. Figure 4 shows that BW2258U89 was metabolized by mouse plasma. Initially, 3 pmol of immunoreactivity was present in the samples. In the PBS control this amount slightly declined over a 50 h period. In contrast, BW2258U89 exposed to 25% mouse plasma, was undetectable after 15 h. The half-life for BW2258U89 immunoreactivity was approximately 3 h. If the BW2258U89 was incubated with 25% mouse plasma containing 1 mM phenylmethlysulfonyl fluoride (PMSF), its dissappearance was markedly slowed. The half-life for BW2258U89 in mouse plasma containing PMSF was approximately 15 h. These results suggest that BW2258U89 is metabolized by a mouse plasma enzyme which is inhibited by PMSF.
The receptor binding activity of BW2258U89 and (Da) BW2258U89 was investigated. Figure 5 shows that BW2258U89 inhibited specific (125I-Tyr4) BB binding to NCI-H345 cells in a dose dependent manner. The IC50 for BW2258U89 was 0.017 µM. In contrast, (Da) BW2258U89 inhibited (125I-Tyr4) BB binding with an IC50 of 0.45 µM. These data indicate that (Da) BW2258U89 binds with over an order of magnitude lower affinity to SCLC GRP receptors than does BW2258U89.
Fig. 3 Radioimmunoassay. The ability of BW2258U89 (● ●) and (Da) BW2258U89 (•) to inhibit 125I-BW1023U90 binding to antibody BW2 was determined. The mean value ± SD of four determinations each repeated in duplicate is indicated.
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Fig. 4 Metabolism of BW2258U89. BW2258U89, 3 pmol, was added to 1 ml of PBS (▲), 25% plasma in 1 ml of PBS (•), and 25% mouse plasma containing 1 mM PMSF (● ●). The mean value ± SD of four determinations is indicated. This experiment is representative of two others.
Fig. 5 Receptor binding activity. The specific binding of (125I-Tyr4) BB to NCI-H345 cells is indicated as a function of BW2258U89 (●) and (Da) BW2258U89 (● ●) concentration. The mean value of three determinations each repeated in quadruplicate is indicated.
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Table 2 Effect of GRP receptor antagonists on colony formation Addition None (Da) BW2258U89, 1 µM BW2258U89, 0.1 µM BW2258U89, 1 µM
Colony number 1543 ± 110 1278 ± 156 1378 ± 191 890 ± 75*
The mean value ± SD of 3 determinations is shown; P < 0.05, * using Student’s t-test. This experiment is representative of two others.
Fig. 6 Cytosolic calcium. (Top) The relative excitation ratio was determined for Fura-2 loaded NCI-H345 cells after addition of 50 nM BB, 50 nM BB + 1 µM BW2258U89 and 50 nM BB + 10 µM BW2258U89. (Bottom) The relative excitation ratio was determined after the addition of 50 nM BB + 1 µM (Da) BW2258U89 and 50 nM BB + 1 µM (Da) BW2258U89. This experiment is representative of three others.
The biological activity of BW2258U89 and (Da) BW2258U89 was investigated. BW2258U89 had little effect on basal cytosolic Ca2+ of Fura-2 loaded NCI-H345 cells but antagonized the ability of 50 nM BB to elevate cytosolic Ca2+ (Fig. 6). BW2258U89 (0.1 µM and 1 µM) slightly and strongly respectively, inhibited the ability of 50 nM BB to elevate cytosolic Ca2+. In contrast, (Da) BW2258U89 (1 µM) had little effect on the ability of 10 nM BB to elevate cytosolic Ca2+. Similar results were obtained if GRP was used as the agonist instead of BB (data not shown). The ability of GRP receptor antagonists to alter nuclear oncogene expression was investigated. BB (10 nM) elevated c-fos mRNA after 45 min (Fig. 7). BW2258U89 Neuropeptides (2000) 34(2), 108–115
Fig. 7 c-fos mRNA. NCI-H345 cells were treated for 45 min with no additions, 10 nM BB + 1 µM BW2258U89, 10 nM BB + 1 µM (Da) BW2258U89 and 10 nM BB. The blot was probed for c-fos mRNA (left) or the gel was ethidium bromide stained (right). This experiment is representative of two others.
weekly (0.1 µM) and strongly (1 µM) antagonized the increase in c-fos mRNA caused by 10 nM BB. In contrast, 1 µM (Da) BW2258U89 had little effect on the ability of 10 nM BB to increase c-fos mRNA. The effects of BW2258U89 and its metabolite on proliferation were determined. Table 2 shows that 1 µM but not 0.1 µM BW2258U89 significantly decreased colony formation of NCI-H345 cells by approximately 45%. In contrast, 1 µM (Da) BW2258U89 had little effect on colony formation. © 2000 Harcourt Publishers Ltd
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DISCUSSION The present study indicates that BW2258U89 is metabolized by mouse plasma enzymes which are inhibited by PMSF. The primary product is deamidated BW2258U89 and (Da) BW2258U89 has approximately 1-order of magnitude less biological activity than BW2258U89. Previous studies showed that GRP receptors were blocked with high affinity by (des-Met14) BB antagonists such as (D-pentafluora-Phe6, D-Ala11) BB6–13 methylester ((FA) BB6–13ME) and with low affinity by substance P (SP) antagonists such as antagonist D, (D-Arg1, D-Phe5, DTrp7,9, Leu11) SP19,20. Micromolar concentrations of (FA) BB6–13ME, BW2258U89 or (D-Arg1, D-Pro2, D-Trp7,9, Leu11) substance P inhibited SCLC colony formation in soft agar (Mahmoud et al., 1991). In vivo, BW2258U89 (0.4 mg/kg) inhibits SCLC xenograft proliferation in nude mice, whereas substantially higher concentrations of (FA) BB6–13ME (Moody et al., unpublished) or antagonist D are required (Sethi et al., 1992). Because BW2258U89 may be a therapeutic agent for SCLC, additional preclinical data on its metabolism were obtained. When BW2258U89 was exposed to mouse plasma (25%), its half-life was 170 min at 37°C. The BW2258U89 metabolite was more hydrophobic than was BW2258U89 and the eluted from the LC-ESI/MS at a later time. The m/z ratio of the singly charged species for BW2258U89 and its metabolite was 1081.6 and 1082.5 respectively. Because the BW2258U89 metabolite had a mass increase of approximately 1, BW2258U89 may be deamidated by mouse plasma enzymes. Similarly, it was found that SP antagonists, which have a plasma half-life of several hours, were deamidated by mouse enzymes (Cummings et al., 1994). In contrast, GRP had a very short half-life of 5 min (Marquez, unpublished). GRP is metabolized by endopeptidase 24.11. to GRP1–25 and GRP26–27 and GRP metabolism is slowed by inclusion of thiorphan into the incubation buffer (Davis et al., 1992). To minimize degradation by endopeptidase 24.11, BW2258U89 has a D-Ala in place of a Gly. This substitution has little effect on receptor binding activity but decreases degradation by endopeptidases. BW2258U89 may be deamidated by a serine protease such as serine carboxypeptidase. In this regard, BW2258U89 metabolism by the mouse plasma was inhibited by PMSF which acetylates serines on carboxypeptidase enzymes which have deamidase activity (Jones et al., 1996). The metabolism of SP antagonist D was also inhibited by PMSF. Antagonist D was metabolized by blood, plasma or liver homogenate, suggesting that these enzymes have a broad distribution in the body. Similarly, PMSF blocks degradation of BW2258U89 by SCLC cell homogenates (Moody, unpublished). These © 2000 Harcourt Publishers Ltd
data support the hypothesis that BW2258U89 may be slowly metabolized by serine carboxypeptidases. Similarly, the structurally related antagonist, (Tpi6, Psi13,14, Tpi14) BN6–14 would likely be deamidated by serine proteases (Cai et al., 1992). It remains to be determined if BW2258U89 is metabolized by enzymes other than serine proteases. Antagonist D was metabolized by carboxypeptidase B-like enzymes after deamidation, resulting in the removal of the C-terminal leucine amino acid. It remains to be determined if BW2258U89, which has a reduced peptide bond at the penultimate position, can be metabolized by carboxypeptidases. The biological activity of the BW2258U89 metabolite was investigated. (Da) BW2258U89 had an IC50 value 15fold greater than did BW2258U89 for inhibition of (125ITyr4) BB binding to NCI-H345 cells. Similarly, 1 µM BW2258U89 antagonized the ability of BB to elevate cytosolic Ca2+ and c-fos mRNA using NCI-H345 cells whereas 1 µM (Da) BW2258U89 had little effect. Preliminary data (Moody, unpublished) indicate that higher doses of (Da) BW2258U89, e.g. 10 µM antagonized the ability of BB to elevate cytosolic Ca2+ or increase c-fos mRNA. BW2258U89 (1 µM) inhibited SCLC proliferation in vitro whereas 1 µM (Da) BW2258U89 did not. These results suggest that after BW2258U89 is metabolized by serine carboxypeptidases it is approxiately 1-order of magnitude less potent. Similarly, antagonist D metabolites were less potent than was antagonist D (Jones et al., 1996). Previously, we found that SCLC xenograft formation was strongly inhibited if BW2258U89 was placed in microspheres which slowly release BW2258U89 into the mouse over a 3 week period (Moody et al., 1996). Nanomolar concentrations of BW2258U89 were present in the mouse blood, weeks after subcutaneous injection of the microspheres. In contrast, if a single bolus dose of BW2258U89 is injected daily, its plasma concentration decreases by over 2-orders of magnitude during the course of a day (Moody, unpublished). These results suggest that if BW2258U89 is to be used as a therapeutic agent, its formulation is important. Antagonists such as BW2258U89 bind with high affinity to the GRP receptor, a 384 amino acid protein which contains 7 transmembrane domains (Battey et al., 1991; Spindel et al., 1990). In contrast, BW2258U89 binds with low affinity to the neuromedin B (NMB) receptor, which is a 390 amino acid protein (Wada et al., 1991) and bombesin receptor subtype (BRS)-3, a 399 amino acid protein (Fathi et al., 1993). Each of these receptors have approximately 50% amino acid sequence homology, but the GRP receptor prefers GRP relative to NMB and the NMB receptor prefers NMB relative to GRP; the BRS-3 receptor binds (D-Phe6, β-Ala11, Phe13, Nle14) BB6–14 but Neuropeptides (2000) 34(2), 108–115
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not GRP or NMB with high affinity (Moody and Jensen, 1996; Mantey et al., 1997). In contrast, micromolar concentrations of SP antagonists block GRP and NMB receptors as well as BRS-3 (Ryan et al., 1998). Also, somatostatin analogs inhibit NMB receptors with IC50 values of 1000 nM (Orbuch et al., 1993). Recently, nonpeptide NMB antagonists have been described which bind to NMB receptors with a Kd of 1 nM (Eden et al., 1996). Nonpeptide antagonists for GRP receptors remain unknown. In summary, BW2258U89 is a potent GRP receptor antagonist which is deamidated by mouse plasma enzymes. The resulting product is impaired in its ability to function as a GRP receptor antagonist.
ACKNOWLEDGMENTS The authors thank Amy Guzzone for technical assistance and Drs J. Leban and J. McDermed for the BW2258U89.
REFERENCES Anastasi A, Erspamer V, Bucci M (1973) Isolation and amino acid sequences of alytesin and bombesin: Two analogous active tetradecapeptides from the skin of European discoglossid frogs. Archives of Biochemistry and Biophysics 148: 443–446. Battey JF, Way J, Corjay MH et al. (1991) Molecular cloning of the bombesin/GRP receptor from Swiss 3T3 cells. Proceedings of the National Academy of Science USA 88: 395–399. Cai RZ, Radulovic S, Pinski J, Nagy A, Redding TW, Olsen DB, Schally AV (1992) Pseudononapeptide bombesin antagonists containing C-terminal Trp or Tpi. Peptides 13: 267–271. Carney DN, Gazdar AF, Bepler G et al. (1985) Establishment and identification of small cell lung cancer cell lines having classic and variant features. Cancer Research 45: 2913–2923. Cummings J, Maclellan A, Langdon SP, Smythe JF (1994) Stability and in vitro metabolism of the mitogenic neuropeptide antagonists (D-Arg1, D-Phe5, D-Trp7,9, Leu11) substance P and (Arg6, D-Trp7,9, NmePhe8) substance P (6–11) characterized by high performance liquid chromatography. Journal of Pharmacentical and Biomedical Analysis 12: 811–819. Cuttitta F, Carney DN, Mulshine J, Moody TW, Fedorko J, Fischler A, Minna JD (1985) Bombesin-like peptides can function as autocrine growth factors in human small cell lung cancer. Nature 316: 823–825. Davis TP, Crowell S, Taylor J, Clark DL, Coy D, Staley J, Moody TW (1992) Metabolic stability and tumor inhibition of bombesin/GRP receptor antagonists. Peptides 13: 401–407. Draoui M, Chung P, Park M, Birrer M, Jakowlew S, Moody TW (1995) Bombesin Stimulates c-fos and c-jun mRNAs in small cell lung cancer cells. Peptides 16: 289–292. Eden JM, Hall MD, Higginbottom M et al. (1996) PD165929-The first high affinity non-peptide neuromedin B (NMB) receptor selective antagonist. Bioorganic & Medicinal Chemistry Letters 6: 2617–2623. Fathi Z, Corjay MH, Shapira H et al. (1993) BRS-3: A novel bombesin receptor subtype selectively expressed in testis and lung carcinoma cells. Journal of Biological Chemistry 268: 5979–5984. Jones DA, Cummings J, Langdon SP, Maclellan AJ, Higgins T, Rozengurt E, Smyth JF (1996) Metabolism of the broad
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spectrum neuropeptide growth factor antagonist (D-Arg1, DPhe5, D-Trp7,9, Leu11) substance P. British Journal of Cancer 73: 715–720. Kelly MJ, Linnoila RI, Avis I, Mulshine J, Johnson B (1997) Antitumor activity of a monoclonal antibody directed against gastrin releasing peptide in patients with small cell lung cancer. Chest 112: 256–261. Korman LY, Carney DN, Citron ML, Moody TW (1986) Secretin/VIP stimulated secretion of bombesin-like peptides from human small cell lung cancer. Cancer Research 46: 1214–1218. Mahmoud S, Staley J, Taylor J. et al. (1991) (Psi13,14) Bombesin analogues inhibit the growth of small cell lung cancer in vitro and in vivo. Cancer Research 51: 1798–1802. Mantey SA, Weber HC, Sainz E et al. (1997) Discovery of a high affinity radioligand for the human orphan receptor, bombesin receptor subtype 3, which demonstrates that it has a unique pharmacology compared with other mammalian bombesin receptors. Journal of Biological Chemistry 272: 26062–26071. McDonald TJ, Jornvall J, Nilsson G, Vagne M, Ghatei M, Bloom SR, Mutt V (1979) Characterization of a gastrin releasing peptide from porcine non-antral gastric tissue. Biochemical and Biophysical Research Communications 90: 227–233. Minamino N, Kangawa K, Matsuo H (1983) Neuromedin B: A novel bombesin-like peptide identified in porcine spinal cord. Biochemical and Biophysical Research Communications 114: 541–548. Moody TW, Pert CB, Gazdar AF, Carney DN, Minna JD (1981) High levels of intracellular bombesin characterize human small cell lung carcinoma. Science 214: 1246–1248. Moody TW, Carney DN, Cuttitta F, Quattrocchi K, Minna JD (1985) High affinity receptors for bombesin/GRP-like peptides on human small cell lung cancer. Life Science 37: 105–113. Moody TW, Murphy A, Mahmoud S, Fiskum G (1987) Bombesin-like peptides elevate cytosolic calcium in small cell lung cancer cells. Biochemical and Biophysical Research Communications 147: 189–195. Moody TW, Jensen RT (1996) Bombesin/GRP and vasoactive intestinal peptide/PACAP as growth factors. In: Le Roith D, Bondy C (eds) Growth Factors and Cytokines in Health and Disease JAI Press, Greenwich, 491–535. Moody TW, Venugopal R, Gozes Y, Hu V, McDermed J, Leban JJ (1996) BW1023U90: A new GRP receptor probe for small cell lung cancer cells. Peptides 17: 1337–1343. Moody TW, Venugopal R, Zia F, Patierno S, Leban JJ, McDermed J (1995) BW2258U89: A GRP receptor antagonist which inhibits small cell lung cancer growth. Life Science 5: 521–529. Orbuch M, Taylor JE, Coy DH et al. (1993) Discovery of a novel class of neuromedin B receptor antagonists, substituted somatostatin analogues. Molecular Pharmacology 44: 841–850. Ryan RR, Weber HC, Hou W et al. (1998) Ability of various bombesin receptor agonists and antagonists to alter intracellular signaling of the human orphan receptor BRS-3. Journal of Biological Chemistry 273: 13613–13624. Sausville EA, Moyer JD, Heikkila R, Neckers LM, Trepel JB (1988) A correlation of bombesin-responsiveness with myc-family gene expression in small cell lung carcinoma cell lines. Annals of the NY Academy of Science 547: 310–321. Sethi T, Langdon S, Smyth J (1992) Growth of small cell lung cancer cells: Stimulation by multiple neuropeptides and inhibition by broad spectrum antagonists in vitro and in vivo. Cancer Research 52: 2737s–42s. Spindel ER, Giladi E, Brehm TP, Goodman RH, Segerson TP (1990) Cloning and functional characterization of a cDNA encoding the murine fibroblast bombesin/GRP receptor. Molecular Endocrinology 4: 1956–1963.
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Staley J, Coy DH, Taylor JE, Kim S, Moody TW (1991) (Des-Met14) bombesin analogues function as small cell lung cancer bombesin receptor antagonists. Peptides 12: 145–149. Trepel JB, Moyer JD, Cuttitta F. et al. (1988) A novel bombesin receptor antagonist inhibits autocrine signals in a small cell lung carcinoma cell line. Biochemical and Biophysical Research Communications 156: 1383–1389. Wada E, Way J, Shapira H, Kusano K et al. (1991) cDNA Cloning, characterization and brain region-specific expression of a neuromedin-B preferring bombesin receptor. Neuron 6: 421–430.
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Woll PJ, Rozengurt E (1988) (D-Arg1, D-Phe5, D-Trp7,9, Leu11) substance P, a potent bombesin antagonist in murine Swiss 3T3 cells, inhibits the growth of human small cell lung cancer. Proceedings of the National Academy of Science USA 85: 1859–1863. Wood S, Wood J, Ghatei M, Lee U, O’Shaghnessy D, Bloom SR (1981) Bombesin, somatostatin and neurotensin-like immunoreactivity in bronchial carcinoma. Journal of Clinical Endocrinology 53: 1310–1312.
Neuropeptides (2000) 34(2), 108–115