Advances of radiolabeled GRPR ligands for PET/CT imaging of cancers.

GRPR is a type of seven-transmembrane G-protein coupled receptor that belongs to the bombesin protein receptor family. It is highly expressed in various cancers, including prostate cancer, breast cancer, lung cancer, gastrointestinal cancer, and so on. As a result, molecular imaging studies have been conducted using radiolabeled GRPR ligands for tumor diagnosis, as well as monitoring of recurrence and metastasis. In this paper, we provided a comprehensive overview of relevant literature from the past two decades, with a specific focus on the advancements made in radiolabeled GRPR ligands for imaging prostate cancer and breast cancer.

Assessment of the bound conformation of bombesin to the BB1 and BB2 receptors.

Bombesin is an endogenous peptide involved in a wide spectrum of physiological activities ranging from satiety, control of circadian rhythm and thermoregulation in the central nervous system, to stimulation of gastrointestinal hormone release, activation of macrophages and effects on development in peripheral tissues. Actions of the peptide are mediated through the two high affinity G-protein coupled receptors BB1R and BB2R. Under pathophysiological conditions, these receptors are overexpressed in many different types of tumors, such as prostate cancer, breast cancer, small and non-small cell lung cancer and pancreatic cancer. This observation has been used for designing cell markers, but it has not been yet exploited for therapeutical purposes. Despite the enormous biological interest of the peptide, little is known about the stereochemical features that contribute to their activity. On the one hand, mutagenesis studies identified a few receptor residues important for high bombesin affinity and on the other, a few studies focused on the relevance of diverse residues of the peptide for receptor activation. Models of the peptide bound to BB1R and BB2R can be helpful to improve our understanding of the stereochemical features granting bombesin activity. Accordingly, the present study describes the computational process followed to construct such models by means of Steered Molecular Dynamics, using models of the peptide and its receptors. Present results provide new insights into the structure-activity relationships of bombesin and its receptors, as well as render an explanation for the differential binding affinity observed towards BB1R and BB2R. Finally, these models can be further exploited to help for designing novel small molecule peptidomimetics with improved pharmacokinetics profile.

Neuromedin B-Expressing Neurons in the Retrotrapezoid Nucleus Regulate Respiratory Homeostasis and Promote Stable Breathing in Adult Mice.

Respiratory chemoreceptor activity encoding arterial Pco2 and Po2 is a critical determinant of ventilation. Currently, the relative importance of several putative chemoreceptor mechanisms for maintaining eupneic breathing and respiratory homeostasis is debated. Transcriptomic and anatomic evidence suggests that bombesin-related peptide Neuromedin-B (Nmb) expression identifies chemoreceptor neurons in the retrotrapezoid nucleus (RTN) that mediate the hypercapnic ventilatory response, but functional support is missing. In this study, we generated a transgenic Nmb-Cre mouse and used Cre-dependent cell ablation and optogenetics to test the hypothesis that RTN Nmb neurons are necessary for the CO2-dependent drive to breathe in adult male and female mice. Selective ablation of ∼95% of RTN Nmb neurons causes compensated respiratory acidosis because of alveolar hypoventilation, as well as profound breathing instability and respiratory-related sleep disruption. Following RTN Nmb lesion, mice were hypoxemic at rest and were prone to severe apneas during hyperoxia, suggesting that oxygen-sensitive mechanisms, presumably the peripheral chemoreceptors, compensate for the loss of RTN Nmb neurons. Interestingly, ventilation following RTN Nmb -lesion was unresponsive to hypercapnia, but behavioral responses to CO2 (freezing and avoidance) and the hypoxia ventilatory response were preserved. Neuroanatomical mapping shows that RTN Nmb neurons are highly collateralized and innervate the respiratory-related centers in the pons and medulla with a strong ipsilateral preference. Together, this evidence suggests that RTN Nmb neurons are dedicated to the respiratory effects of arterial Pco2/pH and maintain respiratory homeostasis in intact conditions and suggest that malfunction of these neurons could underlie the etiology of certain forms of sleep-disordered breathing in humans.SIGNIFICANCE STATEMENT Respiratory chemoreceptors stimulate neural respiratory motor output to regulate arterial Pco2 and Po2, thereby maintaining optimal gas exchange. Neurons in the retrotrapezoid nucleus (RTN) that express the bombesin-related peptide Neuromedin-B are proposed to be important in this process, but functional evidence has not been established. Here, we developed a transgenic mouse model and demonstrated that RTN neurons are fundamental for respiratory homeostasis and mediate the stimulatory effects of CO2 on breathing. Our functional and anatomic data indicate that Nmb-expressing RTN neurons are an integral component of the neural mechanisms that mediate CO2-dependent drive to breathe and maintain alveolar ventilation. This work highlights the importance of the interdependent and dynamic integration of CO2- and O2-sensing mechanisms in respiratory homeostasis of mammals.

Neuromedin B excites central lateral amygdala neurons and reduces cardiovascular output and fear-potentiated startle.

Neuromedin B (NMB) and gastrin-releasing peptide (GRP) are the two mammalian analogs in the bombesin peptide family that exert a variety of actions including emotional processing, appetitive behaviors, cognition, and tumor growth. The bombesin-like peptides interact with three receptors: the NMB-preferring bombesin 1 (BB1) receptors, the GRP-preferring bombesin 2 (BB2) receptors and the orphan bombesin 3 (BB3) receptors. Whereas, injection of bombesin into the central amygdala reduces satiety and modulates blood pressure, the underlying cellular and molecular mechanisms have not been determined. As administration of bombesin induces the expression of Fos in the lateral nucleus of the central amygdala (CeL) which expresses BB1 receptors, we probed the effects of NMB on CeL neurons using in vitro and in vivo approaches. We showed that activation of the BB1 receptors increased action potential firing frequency recorded from CeL neurons via inhibition of the inwardly rectifying K+ (Kir) channels. Activities of phospholipase Cß and protein kinase C were required, whereas intracellular Ca2+ release was unnecessary for BB1 receptor-elicited potentiation of neuronal excitability. Application of NMB directly into the CeA reduced blood pressure and heart rate and significantly reduced fear-potentiated startle. We may provide a cellular and molecular mechanism whereby bombesin-like peptides modulate anxiety and fear responses in the amygdala.

Intraluminal chloride regulates lung branching morphogenesis: involvement of PIEZO1/PIEZO2.

BACKGROUND: Clinical and experimental evidence shows lung fluid volume as a modulator of fetal lung growth with important value in treating fetal lung hypoplasia. Thus, understanding the mechanisms underlying these morphological dynamics has been the topic of multiple investigations with, however, limited results, partially due to the difficulty of capturing or recapitulating these movements in the lab. In this sense, this study aims to establish an ex vivo model allowing the study of lung fluid function in branching morphogenesis and identify the subsequent molecular/ cellular mechanisms. METHODS: Ex vivo lung explant culture was selected as a model to study branching morphogenesis, and intraluminal injections were performed to change the composition of lung fluid. Distinct chloride (Cl-) concentrations (5.8, 29, 143, and 715 mM) or Cl- channels inhibitors [antracene-9-carboxylic acid (A9C), cystic fibrosis transmembrane conductance regulator inhibitor172 (CFTRinh), and calcium-dependent Cl- channel inhibitorA01 (CaCCinh)] were injected into lung lumen at two timepoints, day0 (D0) and D2. At D4, morphological and molecular analyses were performed in terms of branching morphogenesis, spatial distribution (immunofluorescence), and protein quantification (western blot) of mechanoreceptors (PIEZO1 and PIEZO2), neuroendocrine (bombesin, ghrelin, and PGP9.5) and smooth muscle [alpha-smooth muscle actin (α-SMA) and myosin light chain 2 (MLC2)] markers. RESULTS: For the first time, we described effective intraluminal injections at D0 and D2 and demonstrated intraluminal movements at D4 in ex vivo lung explant cultures. Through immunofluorescence assay in in vivo and ex vivo branching morphogenesis, we show that PGP9.5 colocalizes with PIEZO1 and PIEZO2 receptors. Fetal lung growth is increased at higher [Cl-], 715 mM Cl-, through the overexpression of PIEZO1, PIEZO2, ghrelin, bombesin, MLC2, and α-SMA. In contrast, intraluminal injection of CFTRinh or CaCCinh decreases fetal lung growth and the expression of PIEZO1, PIEZO2, ghrelin, bombesin, MLC2, and α-SMA. Finally, the inhibition of PIEZO1/PIEZO2 by GsMTx4 decreases branching morphogenesis and ghrelin, bombesin, MLC2, and α-SMA expression in an intraluminal injection-independent manner. CONCLUSIONS: Our results identify PIEZO1/PIEZO2 expressed in neuroendocrine cells as a regulator of fetal lung growth induced by lung fluid.

Lack of bombesin receptor-activated protein homologous protein impairs hippocampal synaptic plasticity and promotes chronic unpredictable mild stress induced behavioral changes in mice.

Bombesin receptor-activated protein (BRAP) and its homologous protein in mice, which is encoded by bc004004 gene, were expressed abundantly in brain tissues with unknown functions. We treated bc004004-/- mice with chronic unpredictable mild stress (CUMS) to test whether those mice were more vulnerable to stress-related disorders. The results of forced swimming test, sucrose preference test, and open field test showed that after being treated with CUMS for 28 days or 35 days both bc004004-/- and bc004004+/+ mice exhibited behavioural changes and there was no significant difference between bc004004+/+ and bc004004-/-. However, behavioural changes were observed only in bc004004-/- mice after being exposed to CUMS for 21 days, but not in bc004004+/+ after 21-day CUMS exposure, indicating that lack of BRAP homologous protein may cause vulnerability to stress-related disorders in mice. In addition, bc004004-/- mice showed a reduction in recognition memory as revealed by novel object recognition test. Since memory changes and stress related behavioural changes are all closely related to the hippocampus function we further analyzed the changes of dendrites and synapses of hippocampal neurons as well as expression levels of some proteins closely related to synaptic function. bc004004-/- mice exhibited decreased dendritic lengths and increased amount of immature spines, as well as altered expression pattern of synaptic related proteins including GluN2A, synaptophysin and BDNF in the hippocampus. Those findings suggest that BRAP homologous protein may have a protective effect on the behavioural response to stress via regulating dendritic spine formation and synaptic plasticity in the hippocampus.

Early Prediction of Radiation-Induced Pulmonary Fibrosis Using Gastrin-Releasing Peptide Receptor-Targeted PET Imaging.

Early diagnosis of radiation-induced pulmonary fibrosis (RIPF) in lung cancer patients after radiation therapy is important. A gastrin-releasing peptide receptor (GRPR) mediates the inflammation and fibrosis after irradiation in mice lungs. Previously, our group synthesized a GRPR-targeted positron emission tomography (PET) imaging probe, [64Cu]Cu-NODAGA-galacto-bombesin (BBN), an analogue peptide of GRP. In this study, we evaluated the usefulness of [64Cu]Cu-NODAGA-galacto-BBN for the early prediction of RIPF. We prepared RIPF mice and acquired PET/CT images of [18F]F-FDG and [64Cu]Cu-NODAGA-galacto-BBN at 0, 2, 5, and 11 weeks after irradiation (n = 3-10). We confirmed that [64Cu]Cu-NODAGA-galacto-BBN targets GRPR in irradiated RAW 264.7 cells. In addition, we examined whether [64Cu]Cu-NODAGA-galacto-BBN monitors the therapeutic efficacy in RIPF mice (n = 4). As a result, the lung uptake ratio (irradiated-to-normal) of [64Cu]Cu-NODAGA-galacto-BBN was the highest at 2 weeks, followed by its decrease at 5 and 11 weeks after irradiation, which matched with the expression of GRPR and was more accurately predicted than [18F]F-FDG. These uptake results were also confirmed by the cell uptake assay. Furthermore, [64Cu]Cu-NODAGA-galacto-BBN could monitor the therapeutic efficacy of pirfenidone in RIPF mice. We conclude that [64Cu]Cu-NODAGA-galacto-BBN is a novel PET imaging probe for the early prediction of RIPF-targeting GRPR expressed during the inflammatory response.

Estimation of Human Absorbed Dose of 188Re-Hynic-Bombesin Based on Biodistribution Data in Rats.

BACKGROUND: HYNIC-Bombesin (BBN) is a potential peptide for targeted radionuclide therapy in gastrin-releasing peptide receptor (GRPr)-positive malignancies. The 188Re-HYNICBBN is a promising radiopharmaceutical for use in prostate cancer therapy. OBJECTIVE: The aim of this study was to estimate the absorbed dose due to 188Re-HYNIC-BBN radio-complex in human organs based on bio-distribution data of rats. METHODS: In this research, using bio-distribution data of 188Re-HYNIC-BBN in rats, its radiation absorbed dose of the adult human was calculated for different organs based on the MIRD dose calculation method. RESULTS: A considerable equivalent dose amount of 188Re-Hynic-BBN (0.093 mGy/MBq) was accumulated in the prostate. Moreover, all other tissues except for the kidneys and pancreas approximately received insignificant absorbed doses. CONCLUSION: Since the acceptable absorbed dose for the complex was observed in the prostate, 188Re-Hynic-Bombesin can be regarded as a new potential agent for prostate cancer therapy.

Lack of bombesin receptor-activated protein attenuates bleomycin-induced pulmonary fibrosis in mice.

Bombesin receptor-activated protein (BRAP) was found to express in the interstitial cells of human fibrotic lungs with unknown function. Its homologous protein, encoded by BC004004 gene, was also present in mouse lung tissues. We used BC004004 -/- mice which lack BRAP homologous protein expression to establish a bleomycin-induced lung fibrotic model. After bleomycin treatment, BC004004 -/- mice exhibited attenuation of pulmonary injury and less pulmonary fibrosis. Fibroblasts from BC004004 -/- mice proliferated at a lower rate and produced less collagen. Autophagy-related gene 5 (ATG5) was identified as a partner interacting with human BRAP. Lacking BRAP homologous protein led to enhanced autophagy activity in mouse lung tissues as well as in isolated lung fibroblasts, indicating a negative regulatory role of this protein in autophagy via interaction with ATG5. Enhanced autophagy process in fibroblasts due to lack of BRAP homologous protein might contribute to the resistance of BC004004 -/- mice to pulmonary fibrosis.

The Nonpeptide Agonist MK-5046 Functions As an Allosteric Agonist for the Bombesin Receptor Subtype-3.

Allosteric ligands of various G-protein-coupled receptors are being increasingly described and are providing important advances in the development of ligands with novel selectivity and efficacy. These unusual properties allow expanded opportunities for pharmacologic studies and treatment. Unfortunately, no allosteric ligands are yet described for the bombesin receptor family (BnRs), which are proposed to be involved in numerous physiologic/pathophysiological processes in both the central nervous system and peripheral tissues. In this study, we investigate the possibility that the bombesin receptor subtype-3 (BRS-3) specific nonpeptide receptor agonist MK-5046 [(2S)-1,1,1-trifluoro-2-[4-(1H-pyrazol-1-yl)phenyl]-3-(4-[[1-(trifluoromethyl)cyclopropyl]methyl]-1H-imidazol-2-yl)propan-2-ol] functions as a BRS-3 allosteric receptor ligand. We find that in BRS-3 cells, MK-5046 only partially inhibits iodine-125 radionuclide (125I)-Bantag-1 [Boc-Phe-His-4-amino-5-cyclohexyl-2,4,5-trideoxypentonyl-Leu-(3-dimethylamino) benzylamide N-methylammonium trifluoroacetate] binding and that both peptide-1 (a universal BnR-agonist) and MK-5046 activate phospholipase C; however, the specific BRS-3 peptide antagonist Bantag-1 inhibits the action of peptide-1 competitively, whereas for MK-5046 the inhibition is noncompetitive and yields a curvilinear Schild plot. Furthermore, MK-5046 shows other allosteric behaviors, including slowing dissociation of the BRS-3 receptor ligand 125I-Bantag-1, dose-inhibition curves being markedly affected by increasing ligand concentration, and MK-5046 leftward shifting the peptide-1 agonist dose-response curve. Lastly, receptor chimeric studies and site-directed mutagenesis provide evidence that MK-5046 and Bantag-1 have different binding sites determining their receptor high affinity/selectivity. These results provide evidence that MK-5046 is functioning as an allosteric agonist at the BRS-3 receptor, which is the first allosteric ligand described for this family of receptors. SIGNIFICANCE STATEMENT: G-protein-coupled receptor allosteric ligands providing higher selectivity, selective efficacy, and safety that cannot be obtained using usual orthosteric receptor-based strategies are being increasingly described, resulting in enhanced usefulness in exploring receptor function and in treatment. No allosteric ligands exist for any of the mammalian bombesin receptor (BnR) family. Here we provide evidence for the first such example of a BnR allosteric ligand by showing that MK-5046, a nonpeptide agonist for bombesin receptor subtype-3, is functioning as an allosteric agonist.

BR-bombesin: a novel bombesin-related peptide from the skin secretion of the Chaco tree frog (Boana raniceps) with physiological gastric effects.

Bombesin mediates several biological activities in the gastrointestinal (GI) tract and central nervous system in mammals, including smooth muscle contraction, secretion of GI hormones and regulation of homeostatic mechanisms. Here, we report a novel bombesin-like peptide isolated from Boana raniceps. Its amino acid sequence, GGNQWAIGHFM-NH2, was identified and structurally confirmed by HPLC, MS/MS and 454-pyrosequencing; the peptide was named BR-bombesin. The effect of BR-bombesin on smooth muscle contraction was assessed in ileum and esophagus, and its anti-secretory activity was investigated in the stomach. BR-bombesin exerted significant contractile activity with a concentration-response curve similar to that of commercially available bombesin in ileum strips of Wistar rats. In esophageal strips, BR-bombesin acted as an agonist, as many other bombesin-related peptides act, although with different behavior compared to the muscarinic agonist carbachol. Moreover, BR-bombesin inhibited stomach secretion by approximately 50% compared to the untreated control group. This novel peptide has 80% and 70% similarity with the 10-residue C-terminal domain of human neuromedin B (NMB) and human gastrin releasing peptide (GRP10), respectively. Molecular docking analysis revealed that the GRP receptor had a binding energy equal to - 7.3 kcal.mol-1 and - 8.5 kcal.mol-1 when interacting with bombesin and BR-bombesin, respectively. Taken together, our data open an avenue to investigate BR-bombesin in disorders that involve gastrointestinal tract motility and acid gastric secretion.

Gastrin-releasing peptide regulates fear learning under stressed conditions via activation of the amygdalostriatal transition area.

The amygdala, a critical brain region responsible for emotional behavior, is crucially involved in the regulation of the effects of stress on emotional behavior. In the mammalian forebrain, gastrin-releasing peptide (GRP), a 27-amino-acid mammalian neuropeptide, which is a homolog of the 14-amino-acid amidated amphibian peptide bombesin, is highly expressed in the amygdala. The levels of GRP are markedly increased in the amygdala after acute stress; therefore, it is known as a stress-activated modulator. To determine the role of GRP in emotional behavior under stress, we conducted some behavioral and biochemical experiments with GRP-knockout (KO) mice. GRP-KO mice exhibited a longer freezing response than wild-type (WT) littermates in both contextual and auditory fear (also known as threat) conditioning tests only when they were subjected to acute restraint stress 20 min before the conditioning. To identify the critical neural circuits associated with the regulation of emotional memory by GRP, we conducted Arc/Arg3.1-reporter mapping in the amygdala with an Arc-Venus reporter transgenic mouse line. In the amygdalostriatal transition area (AST) and the lateral side of the basal nuclei, fear conditioning after restraint stress increased neuronal activity significantly in WT mice, and GRP KO was found to negate this potentiation only in the AST. These results indicate that the GRP-activated neurons in the AST are likely to suppress excessive fear expression through the regulation of downstream circuits related to fear learning following acute stress.

Bombesin-like peptide recruits disinhibitory cortical circuits and enhances fear memories.

Disinhibitory neurons throughout the mammalian cortex are powerful enhancers of circuit excitability and plasticity. The differential expression of neuropeptide receptors in disinhibitory, inhibitory, and excitatory neurons suggests that each circuit motif may be controlled by distinct neuropeptidergic systems. Here, we reveal that a bombesin-like neuropeptide, gastrin-releasing peptide (GRP), recruits disinhibitory cortical microcircuits through selective targeting and activation of vasoactive intestinal peptide (VIP)-expressing cells. Using a genetically encoded GRP sensor, optogenetic anterograde stimulation, and trans-synaptic tracing, we reveal that GRP regulates VIP cells most likely via extrasynaptic diffusion from several local and long-range sources. In vivo photometry and CRISPR-Cas9-mediated knockout of the GRP receptor (GRPR) in auditory cortex indicate that VIP cells are strongly recruited by novel sounds and aversive shocks, and GRP-GRPR signaling enhances auditory fear memories. Our data establish peptidergic recruitment of selective disinhibitory cortical microcircuits as a mechanism to regulate fear memories.

The gastrin-releasing peptide/bombesin system revisited by a reverse-evolutionary study considering Xenopus.

Bombesin is a putative antibacterial peptide isolated from the skin of the frog, Bombina bombina. Two related (bombesin-like) peptides, gastrin-releasing peptide (GRP) and neuromedin B (NMB) have been found in mammals. The history of GRP/bombesin discovery has caused little attention to be paid to the evolutionary relationship of GRP/bombesin and their receptors in vertebrates. We have classified the peptides and their receptors from the phylogenetic viewpoint using a newly established genetic database and bioinformatics. Here we show, by using a clawed frog (Xenopus tropicalis), that GRP is not a mammalian counterpart of bombesin and also that, whereas the GRP system is widely conserved among vertebrates, the NMB/bombesin system has diversified in certain lineages, in particular in frog species. To understand the derivation of GRP system in the ancestor of mammals, we have focused on the GRP system in Xenopus. Gene expression analyses combined with immunohistochemistry and Western blotting experiments demonstrated that GRP peptides and their receptors are distributed in the brain and stomach of Xenopus. We conclude that GRP peptides and their receptors have evolved from ancestral (GRP-like peptide) homologues to play multiple roles in both the gut and the brain as one of the 'gut-brain peptide' systems.

Application of Alanine Scanning to Determination of Amino Acids Essential for Peptide Adsorption at the Solid/Solution Interface and Binding to the Receptor: Surface-Enhanced Raman/Infrared Spectroscopy versus Bioactivity Assays.

The article describes the application of the alanine-scanning technique used in combination with Raman, surface-enhanced Raman, attenuated total reflection Fourier transform infrared, and surface-enhanced infrared absorption (SEIRA) spectroscopies, which allowed defining the role of individual amino acid residues in the C-terminal 6-14 fragment of the bombesin chain (BN6-14) on the path of its adsorption on the surface of Ag (AgNPs) and Au nanoparticles (AuNPs). A reliable analysis of the SEIRA spectra of these peptides was possible, thanks to a curve fitting of these spectra. By combining alanine-scanning with biological activity studies using cell lines overexpressing bombesin receptors and the intracellular inositol monophosphate assay, it was possible to determine which peptide side chains play a significant role in binding a peptide to membrane-bound G protein-coupled receptors (GPCRs). Based on the analysis of spectral profiles and bioactivity results, conclusions for the specific peptide-metal and peptide-GPCR interactions were drawn and compared.

Sorbitol as a Polar Pharmacological Modifier to Enhance the Hydrophilicity of 99mTc-Tricarbonyl-Based Radiopharmaceuticals.

The organometallic technetium-99m tricarbonyl core, [99mTc][Tc(CO)3(H2O)3]+, is a versatile precursor for the development of radiotracers for single photon emission computed tomography (SPECT). A drawback of the 99mTc-tricarbonyl core is its lipophilicity, which can influence the pharmacokinetic properties of the SPECT imaging probe. Addition of polar pharmacological modifiers to 99mTc-tricarbonyl conjugates holds the promise to counteract this effect and provide tumor-targeting radiopharmaceuticals with improved hydrophilicities, e.g., resulting in a favorable fast renal excretion in vivo. We applied the "Click-to-Chelate" strategy for the assembly of a novel 99mTc-tricarbonyl labeled conjugate made of the tumor-targeting, modified bombesin binding sequence [Nle14]BBN(7-14) and the carbohydrate sorbitol as a polar modifier. The 99mTc-radiopeptide was evaluated in vitro with PC-3 cells and in Fox-1nu mice bearing PC-3 xenografts including a direct comparison with a reference conjugate lacking the sorbitol moiety. The glycated 99mTc-tricarbonyl peptide conjugate exhibited an increased hydrophilicity as well as a retained affinity toward the Gastrin releasing peptide receptor and cell internalization properties. However, there was no significant difference in vivo in terms of pharmacokinetic properties. In particular, the rate and route of excretion was unaltered in comparison to the more lipophilic reference compound. This could be attributed to the intrinsic properties of the peptide and/or its metabolites. We report a novel glycated (sorbitol-containing) alkyne substrate for the "Click-to-Chelate" methodology, which is potentially of general applicability for the development of 99mTc-tricarbonyl based radiotracers displaying an enhanced hydrophilicity.

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.

Neuropeptide bombesin receptor activation stimulates growth of lung cancer cells through HER3 with a MAPK-dependent mechanism.

Despite recent advances in treatment of non-small cell lung cancer (NSCLC), prognosis still remains poor and new therapeutic approaches are needed. Studies demonstrate the importance of the EGFR/HER-receptor family in NSCLC growth, as well as that of other tumors. Recently, HER3 is receiving increased attention because of its role in drug resistance and aggressive growth. Activation of overexpressed G-protein-coupled receptors (GPCR) can also initiate growth by transactivating EGFR/HER-family members. GPCR transactivation of EGFR has been extensively studied, but little is known of its ability to transactivate other EGFR/HER-members, especially HER3. To address this, we studied the ability of bombesin receptor (BnR) activation to transactivate all EGFR/HER-family members and their principal downstream signaling cascades, the PI3K/Akt- and MAPK/ERK-pathways, in human NSCLC cell-lines. In all three cell-lines studied, which possessed EGFR, HER2 and HER3, Bn rapidly transactivated EGFR, HER2 and HER3, as well as Akt and ERK. Immunoprecipitation studies revealed Bn-induced formation of both HER3/EGFR- and HER3/HER2-heterodimers. Specific EGFR/HER3 antibodies or siRNA-knockdown of EGFR and HER3, demonstrated Bn-stimulated activation of EGFR/HER members is initially through HER3, not EGFR. In addition, specific inhibition of HER3, HER2 or MAPK, abolished Bn-stimulated cell-growth, while neither EGFR nor Akt inhibition had an effect. These results show HER3 transactivation mediates all growth effects of BnR activation through MAPK. These results raise the possibility that targeting HER3 alone or with GPCR activation and its signal cascades, may be a novel therapeutic approach in NSCLC. This is especially relevant with the recent development of HER3-blocking antibodies.

Comparison of biological properties of [177 Lu]Lu-ProBOMB1 and [177 Lu]Lu-NeoBOMB1 for GRPR targeting.

The gastrin-releasing peptide receptor (GRPR) is overexpressed in prostate cancer and other solid malignancies. Following up on our work on [68 Ga]Ga-ProBOMB1 that had better imaging characteristics than [68 Ga]Ga-NeoBOMB1, we investigated the effects of substituting 68 Ga for 177 Lu to determine if the resulting radiopharmaceuticals could be used with a therapeutic aim. We radiolabeled the bombesin antagonist ProBOMB1 (DOTA-pABzA-DIG-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-ψ-Pro-NH2 ) with lutetium-177 and compared it with [177 Lu]Lu-NeoBOMB1 (obtained in 54.2 ± 16.5% isolated radiochemical yield with >96% radiochemical purity and 440.8 ± 165.1 GBq/µmol molar activity) for GRPR targeting. Lu-NeoBOMB1 had better binding affinity for GRPR than Lu-ProBOMB1 (Ki values: 2.26 ± 0.24 and 30.2 ± 3.23nM). [177 Lu]Lu-ProBOMB1 was obtained in 53.7 ± 5.4% decay-corrected radiochemical yield with 444.2 ± 193.2 GBq/µmol molar activity and >95% radiochemical purity. In PC-3 prostate cancer xenograft mice, tumor uptake of [177 Lu]Lu-ProBOMB1 was 3.38 ± 1.00, 1.32 ± 0.24, and 0.31 ± 0.04%ID/g at 1, 4, and 24 hours pi. However, the uptake in tumor was lower than [177 Lu]Lu-NeoBOMB1 at all time points. [177 Lu]Lu-ProBOMB1 was inferior to [177 Lu]Lu-NeoBOMB1, which had better therapeutic index for the organs receiving the highest doses.

Bombesin conjugated solid lipid nanoparticles for improved delivery of epigallocatechin gallate for breast cancer treatment.

Epigallocatechin-gallate (EGCG) is a potent anti-cancer therapeutic which effectively controls the growth of cancerous cells through a variety of different pathways. However, its molecular structure is susceptible to modifications due to cellular enzymes affecting its stability, bioavailability and hence, overall efficiency. In this study, we have initially encapsulated EGCG in the matrix of solid lipid nanoparticles to provide a stable drug carrier. To confer additional specificity towards gastrin releasing peptide receptors (GRPR) overexpressed in breast cancer, EGCG loaded nanoparticles were conjugated with a GRPR-specific peptide. In-vitro cytotoxicity studies showed that the peptide-conjugated formulations possessed greater cytotoxicity to cancer cell lines compared to the non-conjugated formulations. Further, in-vivo studies performed on C57/BL6 mice showed greater survivability and reduction in tumour volume in mice treated with peptide-conjugated formulation as compared to the mice treated with non-conjugated formulation or with plain EGCG. These results warrant the potential of the system designed in this study as a novel and effective drug delivery system in breast cancer therapy.