Preclinical Evaluation of Novel 64Cu-Labeled Gastrin-Releasing Peptide Receptor Bioconjugates for PET Imaging of Prostate Cancer.

We report herein the preclinical evaluation of new [64Cu]Cu-gastrin-releasing peptide receptor (GRPR)-targeting tracers, employing the potent peptide antagonist DPhe-Gln-Trp-Ala-VaI-Gly-His-Sta-Leu-NH2 conjugated to NOTA (in 1) or NODAGA (in 2) chelators via a 6-aminohexanoic acid linker. The Cu-1/2 metalated peptides were synthesized by reacting 1/2 with CuCl2 and were characterized by LC-ESI-MS and HR-ESI-MS. Cu-1/2 exhibited high GRPR-binding affinities with IC50 values <3 nM, as measured in a competition assay using the GRPR-expressing human PC-3 prostate cancer cell line and [125I]I-Tyr4-BBN as the competing ligand. Tracers [64Cu]Cu-1/2 were prepared in quantitative radiochemical yield (by radio-HPLC), and their identities were confirmed by coelution with their Cu-1/2 standards via comparative HPLC studies. Lipophilicity was measured in 1-octanol/PBS (pH 7.4), and the negative log D7.4 values (≤-1) confirmed the anticipated hydrophilic character for [64Cu]Cu-1/2. Both tracers demonstrated excellent in vitro stability, with ≥98% remaining intact through 24 h at physiological conditions (PBS, pH 7.4, 37 °C). Biodistribution in PC-3 tumor-bearing mice demonstrated good tumor uptake (%ID/g at 4 h: 4.34 ± 0.71 for [64Cu]Cu-1, 3.92 ± 1.03 for [64Cu]Cu-2) and rapid renal clearance (≥87% ID at 4 h). Tumor uptake was receptor-mediated, as verified by parallel GRPR-blocking studies. Small-animal PET/CT imaging studies validated the biodistribution data. These preclinical data support that the [64Cu]Cu-1/2 tracers show promise for further development as diagnostic PET imaging agents of GRPR-expressing tumors.

A New, Second Generation Trithiol Bifunctional Chelate for 72,77As: Trithiol(b)-(Ser)2-RM2.

Trithiol chelates are suitable for labeling radioarsenic (72As: 2.49 MeV ß+, 26 h; 77As: 0.683 MeV ß-, 38.8 h) to form potential theranostic radiopharmaceuticals for positron emission tomography (PET) imaging and therapy. A trithiol(b)-(Ser)2-RM2 bioconjugate and its arsenic complex were synthesized and characterized. The trithiol(b)-(Ser)2-RM2 bioconjugate was radiolabeled with no-carrier-added 77As in over 95% radiochemical yield and was stable for over 48 h, and in vitro IC50 cell binding studies of [77As]As-trithiol(b)-(Ser)2-RM2 in PC-3 cells demonstrated high affinity for the gastrin-releasing peptide (GRP) receptor (low nanomolar range). Limited biodistribution studies in normal mice were performed with HPLC purified 77As-trithiol(b)-(Ser)2-RM2 demonstrating both pancreatic uptake and hepatobiliary clearance.

Are heterobivalent GRPR- and VPAC1R-bispecific radiopeptides suitable for efficient in vivo tumor imaging of prostate carcinomas?

Receptor-specific peptides labeled with positron emitters play an important role in the clinical imaging of several malignancies by positron emission tomography (PET). Radiolabeled heterobivalent bispecific peptidic ligands (HBPLs) can target more than one receptor type and by this - besides exhibiting other advantages - increase tumor imaging sensitivity. In the present study, we show the initial in vivo evaluation of the most potent heterobivalent gastrin-releasing peptide receptor (GRPR)- and vasoactive intestinal peptide receptor subtype 1 (VPAC1R)-bispecific radiotracer and determined its tumor visualization potential via PET/CT imaging. For this purpose, the most potent described HBPL was synthesized together with its partly scrambled heterobivalent monospecific homologs and its monovalent counterparts. The agents were efficiently labeled with 68Ga3+ and evaluated in an initial PET/CT tumor imaging study in a human prostate carcinoma (PCa) xenograft rat tumor model established for this purpose. None of the three 68Ga-HBPLs enabled a clear tumor visualization and a considerably higher involvement in receptor-mediated uptake was found for the GRPR-binding part of the molecule than for the VPAC1R-binding one. Of the monovalent radiotracers, only [68Ga]Ga-NODA-GA-PESIN could efficiently delineate the tumor, confirming the results. Thus, this work sets the direction for future developments in the field of GRPR- and VPAC1R-bispecific radioligands, which should be based on other VPAC1R-specific peptides than PACAP-27.

Functional Hybrid Molecules for the Visualization of Cancer: PESIN-Homodimers Combined with Multimodal Molecular Imaging Probes for Positron Emission Tomography and Optical Imaging: Suited for Tracking of GRPR-Positive Malignant Tissue*.

We describe multimodal imaging probes for gastrin-releasing peptide receptor (GRPR)-specific targeting suited for positron emission tomography and optical imaging (PET/OI), consisting of PESIN (PEG3 -BBN7-14 ) dimers connected to multimodal imaging subunits. These multimodal agents comprise a fluorescent dye for OI and the chelator ((1,4,7-triazacyclononane-4,7-diyl)diacetic acid-1-glutaric acid) (NODA-GA) for PET radiometal isotope labelling. Special focus was put on the influence of the used dyes on the properties of the whole bioconjugates. For this, several compounds with different fluorescent dyes and non-dye carrying subunits were synthesized and investigated. As fluorescent dyes, dansyl, NBD, derivatives of fluorescein, coumarin and rhodamine as well as three pyrilium-based dyes were employed. Considerable influence of the charge of the colored unit on hydrophilicity as well as in vitro target receptor binding was observed and classified. High radiochemical yields and purities were found during radiolabeling of the multimodal imaging subunits as well as their GRPR-specific bioconjugates with 68 Ga. Examinations of the photophysical properties of both molecule species displayed no loss or alteration of fluorescence characteristics.

On-cell saturation transfer difference NMR study of Bombesin binding to GRP receptor.

We report the NMR characterization of the molecular interaction between Gastrin Releasing Peptide Receptor (GRP-R) and its natural ligand bombesin (BN). GRP-R is a transmembrane G-protein coupled receptor promoting the stimulation of cancer cell proliferation; in addition, being overexpressed on the surface of different human cancer cell lines, it is ideal for the development of new strategies for the selective targeted delivery of anticancer drugs and diagnostic devices to tumor cells. However, the design of new GRP-R binders requires structural information on receptor interaction with its natural ligands. The experimental protocol presented herein, based on on-cell STD NMR techniques, is a powerful tool for the screening and the epitope mapping of GRP-R ligands aimed at the development of new anticancer and diagnostic tools. Notably, the study can be carried out in a physiological environment, at the surface of tumoral cells overespressing GRP-R. Moreover, to the best of our knowledge, this is the first example of an NMR experiment able to detect and investigate the structural determinants of BN/GRP-R interaction.

Stability Evaluation and Stabilization of a Gastrin-Releasing Peptide Receptor (GRPR) Targeting Imaging Pharmaceutical.

The prostate-specific membrane antigen (PSMA) and gastrin-releasing peptide receptor (GRPR) are identified as important targets on prostate cancer. Receptor-targeting radiolabeled imaging pharmaceuticals with high affinity and specificity are useful in studying and monitoring biological processes and responses. Two potential imaging pharmaceuticals, AMBA agonist (where AMBA = DO3A-CH2CO-G-[4-aminobenzyl]- Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2) and RM1 antagonist (where RM1 = DO3A-CH2CO-G-[4-aminobenzyl]-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2), have demonstrated high binding affinity (IC50) to GRP receptors and high tumor uptake. Antagonists, despite the poor tumor cell internalization properties, can show clearer images and pharmacokinetic profiles by virtue of their higher tumor uptake in animal models compared to agonists. For characterization, development, and translation of a potential imaging pharmaceutical into the clinic, it must be evaluated in a series of tests, including in vitro cell binding assays, in vitro buffer and serum stability studies, the biodistribution of the radiolabeled material, and finally imaging studies in preclinical animal models. Data related to acetate buffer, mouse, canine, and human sera stability of 177Lu-labeled RM1 are presented here and compared with the acetate buffer and sera stability data of AMBA agonist. The samples of 177Lu-labeled RM1 with a high radioconcentration degrade faster than low-radioconcentration samples upon storage at 2-8 °C. Addition of stabilizers, ascorbic acid and gentisic acid, improve the stability of 177Lu-labeled RM1 significantly with gentisic acid being more efficient than ascorbic acid as a stabilizer. The degradation kinetics of 177Lu-labeled AMBA and RM1 in sera follow the order (fastest to slowest): mouse > canine > human sera. Finally, 177Lu-labeled RM1 antagonist is slower to degrade in mouse, canine, and human sera than 177Lu-labeled AMBA agonist, further suggesting that an antagonist is a more promising candidate than agonist for the positron emission tomography (PET) imaging and therapy of prostate cancer patients.

Next Step toward Optimization of GRP Receptor Avidities: Determination of the Minimal Distance between BBN(7-14) Units in Peptide Homodimers.

As the gastrin releasing peptide receptor (GRPR) is overexpressed on several tumor types, it represents a promising target for the specific in vivo imaging of these tumors using positron emission tomography (PET). We were able to show that PESIN-based peptide multimers can result in substantially higher GRPR avidities, highly advantageous in vivo pharmacokinetics and tumor imaging properties compared to the respective monomers. However, the minimal distance between the peptidic binders, resulting in the lowest possible system entropy while enabling a concomitant GRPR binding and thus optimized receptor avidities, has not been determined so far. Thus, we aimed here to identify the minimal distance between two GRPR-binding peptides in order to provide the basis for the development of highly avid GRPR-specific PET imaging agents. We therefore synthesized dimers of the GRPR-binding bombesin analogue BBN(7-14) on a dendritic scaffold, exhibiting different distances between both peptide binders. The homodimers were further modified with the chelator NODAGA, radiolabeled with (68)Ga, and evaluated in vitro regarding their GRPR avidity. We found that the most potent of the newly developed radioligands exhibits GRPR avidity twice as high as the most potent reference compound known so far, and that a minimal distance of 62 bond lengths between both peptidic binders within the homodimer can result in concomitant peptide binding and optimal GRPR avidities. These findings answer the question as to what molecular design should be chosen when aiming at the development of highly avid homobivalent peptidic ligands addressing the GRPR.

Conjugation with receptor-targeted histidine-rich peptides enhances the pharmacological effectiveness of antisense oligonucleotides.

Ineffective delivery to intracellular sites of action is one of the key limitations to the use of antisense and siRNA oligonucleotides as therapeutic agents. Here, we describe molecular scale antisense oligonucleotide conjugates that bind selectively to a cell surface receptor, are internalized, and then partially escape from nonproductive endosomal locations to reach their sites of action in the nucleus. Peptides that include bombesin sequences for receptor targeting and a run of histidine residues for endosomal disruption were covalently linked to a splice switching antisense oligonucleotide. The conjugates were tested for their ability to correct splicing and up-regulate expression of a luciferase reporter in prostate cancer cells that express the bombesin receptor. We found that trivalent conjugates that included both the targeting sequence and several histidine residues were substantially more effective than conjugates containing only the bombesin or histidine moieties. This demonstrates the potential of creating molecular scale oligonucleotide conjugates with both targeting and endosome escape capabilities.

The effect of mini-PEG-based spacer length on binding and pharmacokinetic properties of a 68Ga-labeled NOTA-conjugated antagonistic analog of bombesin.

The overexpression of gastrin-releasing peptide receptor (GRPR) in cancer can be used for peptide-receptor mediated radionuclide imaging and therapy. We have previously shown that an antagonist analog of bombesin RM26 conjugated to 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA) via a diethyleneglycol (PEG2) spacer (NOTA-PEG2-RM26) and labeled with 68Ga can be used for imaging of GRPR-expressing tumors. In this study, we evaluated if a variation of mini-PEG spacer length can be used for optimization of targeting properties of the NOTA-conjugated RM26. A series of analogs with different PEG-length (n = 2, 3, 4, 6) was synthesized, radiolabeled and evaluated in vitro and in vivo. The IC50 values of natGa-NOTA-PEGn-RM26 (n = 2, 3, 4, 6) were 3.1 ± 0.2, 3.9 ± 0.3, 5.4 ± 0.4 and 5.8 ± 0.3 nM, respectively. In normal mice all conjugates demonstrated similar biodistribution pattern, however 68Ga-NOTA-PEG3-RM26 showed lower liver uptake. Biodistribution of 68Ga-NOTA-PEG3-RM26 was evaluated in nude mice bearing PC-3 (prostate cancer) and BT-474 (breast cancer) xenografts. High uptake in tumors (4.6 ± 0.6%ID/g and 2.8 ± 0.4%ID/g for PC-3 and BT-474 xenografts, respectively) and high tumor-to-background ratios (tumor/blood of 44 ± 12 and 42 ± 5 for PC-3 and BT-474 xenografts, respectively) were found already at 2 h p.i. of 68Ga-NOTA-PEG3-RM26. Results of this study suggest that variation in the length of the PEG spacer can be used for optimization of targeting properties of peptide-chelator conjugates. However, the influence of the mini-PEG length on biodistribution is minor when di-, tri-, tetra- and hexaethylene glycol are compared.

International Union of Pharmacology. LXVIII. Mammalian bombesin receptors: nomenclature, distribution, pharmacology, signaling, and functions in normal and disease states.

The mammalian bombesin receptor family comprises three G protein-coupled heptahelical receptors: the neuromedin B (NMB) receptor (BB(1)), the gastrin-releasing peptide (GRP) receptor (BB(2)), and the orphan receptor bombesin receptor subtype 3 (BRS-3) (BB(3)). Each receptor is widely distributed, especially in the gastrointestinal (GI) tract and central nervous system (CNS), and the receptors have a large range of effects in both normal physiology and pathophysiological conditions. The mammalian bombesin peptides, GRP and NMB, demonstrate a broad spectrum of pharmacological/biological responses. GRP stimulates smooth muscle contraction and GI motility, release of numerous GI hormones/neurotransmitters, and secretion and/or hormone release from the pancreas, stomach, colon, and numerous endocrine organs and has potent effects on immune cells, potent growth effects on both normal tissues and tumors, potent CNS effects, including regulation of circadian rhythm, thermoregulation; anxiety/fear responses, food intake, and numerous CNS effects on the GI tract as well as the spinal transmission of chronic pruritus. NMB causes contraction of smooth muscle, has growth effects in various tissues, has CNS effects, including effects on feeding and thermoregulation, regulates thyroid-stimulating hormone release, stimulates various CNS neurons, has behavioral effects, and has effects on spinal sensory transmission. GRP, and to a lesser extent NMB, affects growth and/or differentiation of various human tumors, including colon, prostate, lung, and some gynecologic cancers. Knockout studies show that BB(3) has important effects in energy balance, glucose homeostasis, control of body weight, lung development and response to injury, tumor growth, and perhaps GI motility. This review summarizes advances in our understanding of the biology/pharmacology of these receptors, including their classification, structure, pharmacology, physiology, and role in pathophysiological conditions.

Multifunctionalized gold nanoparticles with peptides targeted to gastrin-releasing peptide receptor of a tumor cell line.

Functionalization of gold nanoparticles (AuNPs) with both a targeting peptide (an analogue of the peptide Bombesin) and a drug peptide ligand (an analogue of the RAF peptide) with the aim of improving selectivity in the delivery of the conjugates as well as the antitumor activity is described. Studies on the internalization mechanism of peptide-AuNP conjugates and viability of cells were carried out. An enhancement of the activity and selectivity of the peptide multifunctionalized conjugates was observed.

Gastrin releasing protein receptor specific gold nanorods: breast and prostate tumor avid nanovectors for molecular imaging.

Gastrin releasing protein receptor specific bombesin (BBN) peptide-gold nanoconjugates were successfully synthesized using gold nanorods and dithiolated peptide. The gold nanorod-bombesin (GNR-BBN) conjugates showed extraordinary in vitro stabilities against various biomolecules including NaCl, cysteine, histidine, bovine serum albumin, human serum albumin, and dithiothreitol. Quantitative measurements on the binding affinity (IC(50)) of GNR-BBN conjugates toward prostate and breast tumor cells were evaluated. The IC(50) values establish that GNR-BBN conjugates have strong affinity toward the gastrin releasing peptide receptors on both the tumors. Detailed cellular interaction studies of GNR-BBN conjugates revealed that nanorods internalize via a receptor-mediated endocytosis pathway. The receptor specific interactions of GNR-BBN conjugates provide realistic opportunities in the design and development of in vivo molecular imaging and therapy agents for cancer.

Radiolabeled GRPR Antagonists for Imaging of Disseminated Prostate Cancer - Influence of Labeling Chemistry on Targeting Properties.

BACKGROUND: Radionuclide molecular imaging of Gastrin-Releasing Peptide Receptor (GRPR) expression promises unparalleled opportunities for visualizing subtle prostate tumors, which due to small size, adjacent benign tissue, or a challenging location would otherwise remain undetected by conventional imaging. Achieving high imaging contrast is essential for this purpose and the molecular design of any probe for molecular imaging of prostate cancer should be aimed at obtaining as high tumor-to-organ ratios as possible. OBJECTIVE: This short review summarizes the key imaging modalities currently used in prostate cancer, with a special focus on radionuclide molecular imaging. Emphasis is laid mainly on the issue of radiometals labeling chemistry and its influence on the targeting properties and biodistribution of radiolabeled GRPR antagonists for imaging of disseminated prostate cancer. METHODS: A comprehensive literature search of the PubMed/MEDLINE, and Scopus library databases was conducted to find relevant articles. RESULTS: The combination of radionuclide, chelator and required labeling chemistry was shown to have a significant influence on the stability, binding affinity and internalization rate, off-target interaction with normal tissues and blood proteins, interaction with enzymes, activity uptake and retention in excretory organs and activity uptake in tumors of radiolabeled bombesin antagonistic analogues. CONCLUSION: Labeling chemistry has a very strong impact on the biodistribution profile of GRPRtargeting peptide based imaging probes and needs to be considered when designing a targeting probe for high contrast molecular imaging. Taking into account the complexity of in vivo interactions, it is not currently possible to accurately predict the optimal labeling approach. Therefore, a detailed in vivo characterization and optimization is essential for the rational design of imaging agents.

Gastrin releasing peptide receptor targeted nano-graphene oxide for near-infrared fluorescence imaging of oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is the most common malignant tumor that occurs in the oral mucosa. Pathological biopsy is still the current gold standard for OSCC diagnosis; however, some drawbacks need to be overcome. Therefore, it is urgently needed to find a non-invasive targeted technology for OSCC early diagnosis. Fluorescent optical imaging using near infrared (NIR) dyes tagged to tumor specific target will benefit such developments. Gastrin releasing peptide receptor (GRPR) is an attractive target for OSCC imaging and therapy. In this study, we synthesized nano-graphene oxide (NGO) nanoparticles with GRPR-specific peptides AF750-6Ahx-Sta-BBN via hydrogen bond and π-π bonds (NGO-BBN-AF750), and investigated their receptor binding, cell uptake and internalization in HSC-3 cells. NGO-BBN-AF750 and AF750-6Ahx-Sta-BBN showed a similar binding affinity to GRPR on HSC-3 cells. In contrast to AF750-6Ahx-Sta-BBN antagonist peptide, NGO-BBN-AF750 showed cellular internalization property. Overall, this study proposes a NGO nanoclusters-based nanoprobe for GRPR targeted near-infrared fluorescence imaging for OSCC. Nanoparticle-based delivery systems have shown highly significant potential in the delivery of a wide range of therapeutic agents.

Beta-arrestin-based Bret2 screening assay for the "non"-beta-arrestin binding CB1 receptor.

CB1 receptor (CB1R) antagonists have been demonstrated to be effective in treating obesity and related disorders. This study has been focused on establishing a beta-arrestin 2-based screening assay for the CB1R using BRET2 technology. When the existing BRET2 screening platform was applied to the CB1R, the authors discovered that the receptor interacted weakly with beta-arrestin 2, resulting in unsatisfactory assay performance. To enhance the beta-arrestin binding capacity, they replaced the C-terminal tail of the CB1R with tails from either the V2 or BRS3 receptors, both of which interact strongly with beta-arrestin 2. Using this chimeric approach, the authors screened a small compound library and identified 21 antagonist and inverse agonist hits with IC50 and EC50 values ranging from 0.3 nM to 7.5 microM. Both primary and secondary screening were performed with Z'>0.5, suggesting that the assay is a robust and cost-effective alternative to existing cell-based assays.

Molecular basis for agonist selectivity and activation of the orphan bombesin receptor subtype 3 receptor.

Bombesin receptor subtype (BRS)-3, a G-protein-coupled orphan receptor, shares 51% identity with the mammalian bombesin (Bn) receptor for gastrin-releasing peptide. There is increasing interest in BRS-3 because it is important in energy metabolism, glucose control, motility, and tumor growth. BRS-3 has low affinity for all Bn-related peptides; however, recently synthetic high-affinity agonists, [d-Tyr(6)/d-Phe(6),betaAla(11),Phe(13),Nle(14)]Bn-(6-14), were described, but they are nonselective for BRS-3 over other Bn receptors. Based on these peptides, three BRS-3-selective ligands were developed: peptide 2, [d-Tyr(6)(R)-3-amino-propionic acid(11),Phe(13),Nle(14)]Bn(6-14); peptide 3, [d-Tyr(6),(R)-Apa(11),4Cl-Phe(13),Nle(14)]Bn(6-14); and peptide 4, acetyl-Phe-Trp-Ala-His-(tBzl)-piperidine-3 carboxylic acid-Gly-Arg-NH(2). Their molecular determinants of selectivity/high affinity for BRS-3 are unknown. To address this, we used a chimeric/site mutagenesis approach. Substitution of extracellular domain 2 (EC2) of BRS-3 by the comparable gastrin-releasing peptide receptor (GRPR) domain decreased 26-, 4-, and 0-fold affinity for peptides 4, 3, and 2. Substitution of EC3 decreased affinity 4-, 11-, and 0-fold affinity for peptides 2 to 4. Ten-point mutations in the EC2 and adjacent transmembrane regions (TM2) 2 and 3 of BRS-3 were made. His107 (EC2-BRS-3) for lysine (H107K) (EC2-GRPR) decreased affinity (25- and 0-fold) for peptides 4 and 1; however, it could not be activated by either peptide. Its combination with Val101 (TM2), Gly112 (EC2), and Arg127 (TM3) resulted in complete loss-of-affinity of peptide 4. Receptor-modeling showed that each of these residues face inward and are within 4 A of the binding pocket. These results demonstrate that Val101, His107, Gly112, and Arg127 in the EC2/adjacent upper TMs of BRS-3 are critical for the high BRS3 selectivity of peptide 4. His107 in EC2 is essential for BRS-3 activation, suggesting amino-aromatic ligand/receptor interactions with peptide 4 are critical for both binding and activation. Furthermore, these result demonstrate that even though these three BRS-3-selective agonists were developed from the same template peptide, [d-Phe(6),betaAla(11),Phe(13),Nle(14)]Bn-(6-14), their molecular determinants of selectivity/high affinity varied considerably.

99mTc(CO)3-DTMA bombesin conjugates having high affinity for the GRP receptor.

INTRODUCTION: Targeted diagnosis of specific human cancer types continues to be of significant interest in nuclear medicine. 99mTc is ideally suited as a diagnostic radiometal for in vivo tumor targeting due to its ideal physical characteristics and diverse labeling chemistries in numerous oxidation states. METHODS: In this study, we report a synthetic approach toward design of a new tridentate amine ligand for the organometallic aqua-ion [99mTc(H2O)3(CO)3]+. The new chelating ligand framework, 2-(N,N'-Bis(tert-butoxycarbonyl)diethylenetriamine) acetic acid (DTMA), was synthesized from a diethylenetriamine precursor and fully characterized by mass spectrometry and nuclear magnetic resonance spectroscopy (1H and 13C). DTMA was conjugated to H2N-(X)-BBN(7-14)NH2, where X=an amino acid or aliphatic pharmacokinetic modifier and BBN=bombesin peptide, by means of solid phase peptide synthesis. DTMA-(X)-BBN(7-14)NH2 conjugates were purified by reversed-phase high-performance chromatography and characterized by electrospray-ionization mass spectrometry. RESULTS: The new conjugates were radiolabeled with [99mTc(H2O)3(CO)3]+ produced via Isolink radiolabeling kits to produce [99mTc(CO)3-DTMA-(X)-BBN(7-14)NH2]. Radiolabeled conjugates were purified by reversed-phase high-performance chromatography. Effective receptor binding behavior was evaluated in vitro and in vivo. CONCLUSIONS: [99mTc(CO)3-DTMA-(X)-BBN(7-14)NH2] conjugates displayed very high affinity for the gastrin releasing peptide receptor in vitro and in vivo. Therefore, these conjugates hold some propensity to be investigated as molecular imaging agents that specifically target human cancers uniquely expressing the gastrin releasing peptide receptor subtypes.

Discovery of small molecule agonists for the bombesin receptor subtype 3 (BRS-3) based on an omeprazole lead.

Starting from a weak omeprazole screening hit, replacement of the pyridine with a 1,3-benzodioxole moiety, modification of the thioether linkage, and substitution of the benzimidazole pharmacophore led to the discovery of nanomolar BRS-3 agonists.

A coupling of homology modeling with multiple molecular dynamics simulation for identifying representative conformation of GPCR structures: a case study on human bombesin receptor subtype-3.

In this study, a computational pipeline was therefore devised to overcome homology modeling (HM) bottlenecks. The coupling of HM with molecular dynamics (MD) simulation is useful in that it tackles the sampling deficiency of dynamics simulations by providing good-quality initial guesses for the native structure. Indeed, HM also relaxes the severe requirement of force fields to explore the huge conformational space of protein structures. In this study, the interaction between the human bombesin receptor subtype-3 and MK-5046 was investigated integrating HM, molecular docking, and MD simulations. To improve conformational sampling in typical MD simulations of GPCRs, as in other biomolecules, multiple trajectories with different initial conditions can be employed rather than a single long trajectory. Multiple MD simulations of human bombesin receptor subtype-3 with different initial atomic velocities are applied to sample conformations in the vicinity of the structure generated by HM. The backbone atom conformational space distribution of replicates is analyzed employing principal components analysis. As a result, the averages of structural and dynamic properties over the twenty-one trajectories differ significantly from those obtained from individual trajectories.

Structural Determinants in the Binding of BB2 Receptor Ligands: In Silico, X-Ray and NMR Studies in PD176252 Analogues.

BACKGROUND: The mammalian bombesin receptor family comprises three G proteincoupled receptors: the neuromedin B receptor, the gastrin-releasing peptide receptor (BB2), and the bombesin receptor subtype 3. BB2 receptor plays a role in gastrointestinal functions; however, at present the role of this subtype in physiological and pathological conditions is unknown due to the lack of specific binders for all subclasses of bombesin receptors. RESULTS: Here, we present a study focused on the properties of the peptoid bombesin antagonist called PD176252, and other structural analogues with the aim to elucidate causes of their different affinity towards the BB2 receptor. CONCLUSION: By means of computational techniques, based on QSAR, docking and homology building, supported by experimental data (X-ray diffraction and NMR spectroscopy) fresh insights on binding modes of this class of biological targets were achieved.