Targeting the Gastrin-Releasing Peptide Receptor (GRP-R) in Cancer Therapy: Development of Bombesin-Based Peptide–Drug Conjugates (original) (raw)
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Bombesin analogues for gastrin-releasing peptide receptor imaging
Objectives: The present study describes the design and development of a series of new bombesin (BBN) antagonist peptide ligands of the form [ 64 Cu-(NO2A-X-D-Phe 6 -BBN(6-13)NHEt)], where Cu-64=a positron emitting radiometal; NO2A=1,4,7-triazacyclononane-1,4diacetic acid; X=6-amino hexanoic acid, 8-amino octanoic acid or 9-Aminononanoic acid; and BBN(6-13)NHEt=Gln-Trp-Ala-Val-Gly-His-Leu-NHEt, an antagonist analogue of bombesin peptide for specific targeting of the gastrin-releasing peptide receptor (GRPR). Methods: [NO2A-X-D-Phe 6 -BBN(6-13)NHEt] conjugates were manually conjugated with NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), and the resulting conjugates were labeled with 64 Cu to yield [ 64 Cu-(NO2A-X-D-Phe 6 -BBN(6-13)NHEt)]. The metallated and nonmetallated conjugates were purified via reversed-phase high-performance liquid chromatography and characterized by electrospray ionization-mass spectrometry. Results: Competitive displacement binding assays displayed nanomolar binding affinities toward human GRPR for all of the newly formed peptide analogues. Biodistribution studies showed very high uptake and retention of tumor-associated radioactivity in PC-3 (a prostate tumor model known to express the GRPR) tumor-bearing rodent models. The radiolabeled conjugates also exhibited rapid urinary excretion and very high tumor to background ratios. Micro-positron emission tomography (PET) molecular imaging investigations showed clear visualization of tumors in female PC-3 tumor-bearing mice 15 h postinjection. Conclusion: The biodistribution and molecular imaging study suggests that these conjugates can be considered as potential PET tracer candidates for the diagnosis of GRPR-positive tumors in human patients. Published by Elsevier Inc.
Targeting gastrin-releasing peptide receptors for cancer treatment
Anti-Cancer Drugs, 2004
Growth factor receptors play critical roles in cancer cell proliferation and progression. A number of such receptors have been targeted for cancer treatment by either a monoclonal antibody or a specifically designed small molecule to inhibit the receptor function. Bombesin/gastrin-releasing peptide receptors (BN/GRP-Rs) are expressed in a variety of cancer cells and have limited distribution in normal human tissue. Inhibition of BN/GRP-Rs has been shown to block small cell lung cancer growth in vitro. Early phase clinical trials targeting human GRP-R showed anti-cancer activity. This review will focus on the study of the distribution of BN/GRP-Rs in normal and malignant tissues, and various approaches to targeting BN-GRP-Rs for cancer diagnosis and treatment.
… journal of cancer, 1994
This study examined the expression of receptors of the bombesin (BBS) family in human gastric-cancer cell lines. Of 5 cell lines screened, only one, St42. demonstrated specific binding sites for '251-Ty&BBS, which have been further characterized. This binding was saturable, and temperature-and time-dependent. Scatchard analysis of displacement data performed at 37°C revealed 2 binding sites: a high-affinity, lowcapacity site (& = 0.13 nM, B , , = 1500 sites/cell) and a lower-affinity, higher-capacity site (& = I I nM, B , , = 35,000 sites/cell); the latter was lost when internalization of peptide was prevented, suggesting that it may be an artefact. Displacement assays with gastrin-releasing peptide (GRP) and neuromedin B (NMB) revealed that the receptor was of the GRPpreferring sub-type (GRP ICSO = 0.35 nM; NMB ICs0 = I I 2 nM). Co-valent cross-linking of 1251-Tyl.A-BBS to the receptor demonstrated the presence of a single band corresponding to a molecular weight of 37 to 44 kDa on SDS-PAGE, similar to that of the cloned GRP receptor protein core. G-protein linkage of this receptor was demonstrated by selective inhibition of 12SI-Ty&BBS binding by guanosine nucleotides. The binding of BBS to the receptor resulted in a rise in intracellular calcium. Three of four structurally distinct BBS antagonists bound to the receptor with high affinity, but [DPheI2, Le~'~]-bombesin did not cause any displacement of '251-TyP-BBS even at I 0 mM. The
Bioconjugate Chemistry, 2010
The gastrin-releasing peptide receptor (GRPR) is overexpressed on a number of human tumors and has been targeted with radiolabeled bombesin analogues for the diagnosis and therapy of these cancers. Seven bombesin analogues containing various linkers and peptide sequences were designed, synthesized, radiolabeled with 18 F, and characterized in Vitro and in ViVo as potential PET imaging agents. Binding studies displayed nanomolar binding affinities toward human GRPR for all synthesized bombesin analogues. Two high-affinity peptide candidates 6b (K i ) 0.7 nM) and 7b (K i ) 0.1 nM) were chosen for further in ViVo evaluation. Both tracers revealed specific uptake in GRPR-expressing PC-3 tumors and the pancreas. Compared to [ 18 F]6b, compound [ 18 F]7b was characterized by superior tumor uptake, higher specificity of tracer uptake, and more favorable tumor-to-nontarget ratios. In ViVo PET imaging allowed for the visualization of PC-3 tumor in nude mice suggesting that [ 18 F]7b is a promising PET tracer candidate for the diagnosis of GRPR-positive tumors in humans.
Total solid-phase synthesis of porcine gut gastrin releasing peptide (GRP), a mammalian bombesin
Journal of the American Chemical Society, 1981
Recently, gastrin releasing peptide (GRP), Ala-Pro-Val-Ser-Val-Gly-Gly-Gly-Thr-Val-Leu-Ala-Lys-Met-Tyr-Pro-Arg-Gly-Asn-His-TrpAla-Val-Gly-His-Leu-Met-NH2, a mammalian bombesin, was isolated from porcine gastric mucosa and sequenced by McDonald et aL9 This polypeptide was manually synthesized by solid-phase methodology, using a benzhydrylamine-styrene-1% divinylbenzene copolymer. Deprotection and cleavage from the resin were accomplished by HF.
Discovery of potent and selective peptide agonists at the GRP-preferring bombesin receptor (BB2)
Journal of Peptide Science, 2001
Analogues of the nonselective bombesin receptor synthetic agonist H-D-Phe-Gln-Trp-Ala-Val-βAla-His-Phe-Nle-NH 2 were prepared and their biological activity assessed at the NMB-preferring/bombesin receptor (NMB-R; BB 1 ), the GRP-preferring/bombesin receptor (GRP-R; BB 2 ) and the orphan receptor bombesin receptor subtype-3 (BRS-3; BB 3 ). Progressive N -terminal deletions identified the minimum C-terminal sequences required for maintaining a significant agonist effect, whilst an alanine scan, targeted changes in stereochemistry and other pertinent substitutions identified key side-chain and stereochemical requirements for activation. Key structural elements required for functional potency at BB 1 BB 2 and BB 3 , and for selectivity between these receptor subtypes were established. Synthetic peptides were discovered, which were highly potent agonists at BB 2 and extremely selective over both BB 1 and BB 3 . tract and central nervous system (CNS), whilst BB 3 is found in the CNS as well as testis and lung cancer cells. A fourth member of the bombesin receptor family (BB 4 ) was isolated from frog brain and, to date, any mammalian equivalent has not been identified. All four receptors are members of the G-protein coupled receptor superfamily. The mammalian bombesin-like peptides elicit a wide range of biological responses in the CNS and in peripheral tissues. Their actions include stimulation of GI hormone release and activity as growth factors in human small-cell lung cancer . In the CNS, it is proposed these peptides have a role in feeding, grooming behaviour, the regulation of homeostasis, thermoregulation and metabolism.
Nuclear Medicine and Biology, 2014
Sulfonated aluminum phthalocyanines (AlPcS) are potent photosensitizers for the photodynamic therapy (PDT) of cancer. In this study we evaluate the possibility to improve the efficacy of AlPcS-PDT for prostate cancer by targeting tetrasulfonated aluminum phthalocyanines (AlPcS 4) to the gastrin-releasing peptide receptor (GRPR) through coupling to bombesin. A mono-carbohexyl derivative of AlPcS 4 is attached to 8-Aoc-bombesin(7-14)NH 2 via an amide bridge to yield a bombesin-AlPcS 4 conjugate linked by a C-14 spacer chain. The conjugate is characterized by mass spectroscopy and shown to bind to the GRPR with a relative binding affinity (RBA) of 2.3, taking bombesin (RBA = 100) as unity. The in vitro photodynamic efficacy of the conjugate against PC-3 human prostate cancer cells is improved by a factor 2.5 over the non-conjugated mono-carbohexyl derivative of AlPcS 4 .
Journal of Cancer Research and Clinical Oncology, 1994
We investigated the effect of bombesin/gastrinreleasing peptide (GRP) antagonist RC-3095 and other analogs on the growth of Hs746T human gastric cancer cells implanted in nude mice or cultured in vitro and on the binding of bombesin to its receptors. Nude mice bearing xenografts of the Hs746T cell line received s.c. injections of RC-3095 (10 gg twice daily) or the vehicle (control) for 21 days. Administration of antagonist RC-3095 inhibited the growth of Hs746T tumors. Treatment with RC-3095 produced a significant decrease in tumor volume, prolonged the tumor volume doubling time from 3.6 days to 5.1 days, and decreased the tumor growth rate by 76.9%. The tumor growth delay time in mice treated with RC-3095 was 2.8 days. Treatment with RC-3095 also decreased the final tumor weight by 88.3% and reduced DNA and protein contents in tumors by 91.5% and 89.5%, respectively, as compared to controls. The presence of specific receptors for bombesin/GRP was investigated on the crude membranes of implanted tumors of Hs746T cells. Saturation binding assays showed that the binding of [125I-Tyr4]bombesin to the membranes was saturable and reversible. Scatchard analysis indicated the presence of a single class of binding sites with a high affinity (Kd = 0.24 _+ 0.07 nM) and a low binding capacity (Bmax = 57.0 _+ 0.9 fmol/mg protein). In displacement studies, the binding of [ 12 s i_Tyr ~] bombesin was inhibited in a dose-dependent manner by unlabelled bombesin(1-14), [Tyr4]-bombesin and GRP(14-27), but not by structurally unrelated peptides. Synthetic bombesin/GRP antagonists RC-3095, RC-3110, and RC-3950-II were all able to inhibit effectively the binding of [125 i_Tyr41bombesin to the membranes of Hs746T cells. RC-3950-II showed a higher binding affinity for bombesin Abbreviations.receptors than RC-3095 or RC-3110. Addition of the non-hydrolyzable guanine-nucleotide analog GTP IS] to the binding buffer caused a significant reduction in the amount of [125I-Tyr4]bombesin bound to the cells, indicating that the bombesin receptor is coupled to a G-protein. In cell cultures, bombesin significantly stimulated the growth of Hs746T cells in vitro as shown by an increase in the uptake of [3H]thymidine. Bombesin antagonist RC-3095 could effectively inhibit the bombesinstimulated growth of Hs746T cells in cultures. These observations suggest that bombesin/GRP may act as growth factors through specific receptors present on the membranes of Hs746T cells. Bombesin/GRP antagonists appear to nullify the effects of bombesin/GRP and may be useful for the treatment of gastric cancers.