Submitted by PPG Biologia Celular e Molecular (bcm@pucrs.br) on 2017-07-24T18:23:29Z No. of bitstreams: 1 RAFAEL_SANGUINETTI_CZEPIELEWSKI_TES.pdf: 5487109 bytes, checksum: 88dbb99b9597e08253e2832cc01ccfce (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-07-25T17:50:46Z (GMT) No. of bitstreams: 1 RAFAEL_SANGUINETTI_CZEPIELEWSKI_TES.pdf: 5487109 bytes, checksum: 88dbb99b9597e08253e2832cc01ccfce (MD5) / Made available in DSpace on 2017-07-25T18:00:09Z (GMT). No. of bitstreams: 1 RAFAEL_SANGUINETTI_CZEPIELEWSKI_TES.pdf: 5487109 bytes, checksum: 88dbb99b9597e08253e2832cc01ccfce (MD5) Previous issue date: 2016-03-18 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Tumor microenvironment and inflammatory diseases promote alterations in our immune system along with their development. Several molecules are implicated in this modulation and are therefore considered therapeutic targets. Gastrin-releasing peptide (GRP) is produced in tumors where it promotes cellular proliferation. It is also correlated with chronic diseases, as in rheumatoid arthritis and asthma, and in the acute condition of sepsis. Recently, our group found a direct GRP action over neutrophils, promoting migration. This work aimed to study the interface between GRP-producing tumors and the recruitment of immune cells, as well as extend the cellular studies about neutrophil activation and migration processes promoted by the peptide. In tumors, we observed that a lung adenocarcinoma cell line does not proliferate in response to GRP. Yet, it is induced to migrate when exposed to the peptide, indicating a potential role for GRP in metastasis of this type of cancer. In our tumor immunology studies, we established a novel in vivo model by overexpressing GRP in a melanoma cell line (B16F10). We observed the augment of infiltrating inflammatory monocytes in the tumor microenvironment of these tumors. In parallel, we verified that reactive oxygen species production and migration in response to GRP is dependent of the NADPH oxidase complex. GRP stimulation promotes an intense activation, which culminates in neutrophil extracellular traps (NETs) release. In addition, the GRP receptor (GRPR) antagonist RC-3095 presented anti-inflammatory potential, inhibiting neutrophil migration towards IL-8 and reducing the extent of acetaminophen-induced liver damage. This effect was due to motility alterations in infiltrating neutrophils within the tissue and reduction of cell adhesion molecules. The results presented herein demonstrate the wide panorama of GRP?s interactions in tumor and immune biology. / O microambiente tumoral e as doen?as inflamat?rias promovem altera??es nas c?lulas do nosso sistema imune ? medida que progridem. Diversas mol?culas est?o envolvidas nessa modula??o, e por isso s?o alvos terap?uticos. O pept?deo liberador de gastrina (GRP) ? produzido por tumores, onde promove prolifera??o celular. Este tamb?m est? correlacionado com doen?as cr?nicas como a artrite reumatoide e asma, e em doen?as agudas, como a sepse. Recentemente, nosso grupo descobriu a??o direta do GRP em neutr?filos, promovendo indu??o de migra??o. O presente trabalho se prop?s a estudar a interface entre tumores produtores de GRP e o recrutamento celular, assim como aprofundar os estudos celulares sobre os processos de ativa??o e migra??o de neutr?filos promovidos pelo pept?deo. Em tumores, observamos que uma linhagem de adenocarcinoma pulmonar n?o prolifera quando exposto ao GRP, por?m ? induzida a migrar quando exposta ao pept?deo, estabelecendo um potencial papel deste na promo??o de met?stases para esse tipo tumoral. Na interface da imunologia tumoral, atrav?s do desenvolvimento de um modelo in vivo de superexpress?o de GRP em melanoma murino (B16F10), observamos que esse aumento do GRP induz a infiltra??o de mon?citos inflamat?rios no microambiente tumoral. Em paralelo, verificamos que a produ??o de esp?cies reativas de oxig?nio e a migra??o em dire??o ao GRP s?o dependentes do complexo NADPH oxidase. Esse est?mulo promove ativa??o intensa, culminando na produ??o de redes extracelulares de neutr?filos (NETs). J? o antagonista do seu receptor, GRPR, apresentou potencial antiinflamat?rio, sendo capaz de inibir a migra??o neutrof?lica via modula??o de IL-8 e reduzindo a extens?o da les?o hep?tica induzida por paracetamol (acetaminofeno), alterando a motilidade dos neutr?filos no tecido e a express?o de mol?culas de ades?o. Assim, os resultados aqui apresentados demonstram um panorama amplo da fun??o do GRP na biologia tumoral e no sistema imune.

Molecular Imaging provides new aspects in cancer diagnosis and treatment. With the ap-plication of imaging and biological techniques, molecular imaging can monitor molecular and cellular changes of different diseases. To interpret the mechanism of disease, more and more at-tention is focused on the development of new probes for molecular imaging. Magnetic resonance imaging (MRI) is a powerful, non-invasive clinical diagnostic tool with high spatial resolution without the limitation of the depth of tissues. Applications of MRI contrast agents can amply the MRI signal during imaging. Many studies have been devoted to developing targeted MR contrast agents. Proteins and peptides have been widely used for target-ing cancer cells in cancer diagnosis and treatments. GRP, gastrin-releasing peptide, is one of a well-characterized group of mammalian bombesin-like peptides. GRP acts through its cell surface receptors, GRP receptor (GRPR). It has been reported that there is a high density of GRP receptors in the majority of prostate carci-noma. In contrast, the GRPRs are not highly expressed in normal cells of most tissues. Thus, this tumor specific expression pattern provides an advantage for cancer targeting. A novel class of MRI contrast agent was designed by adding the Gd3+ binding sites into a stable host protein, the domain 1 of rat CD2. Due to the unique features of the designed metal binding properties, the protein contrast agent (ProCA1) exhibits more than 10-fold enhanced MRI relaxivity compared to that of the more commonly used Gd-DTPA. The high relaxivity of the designed protein contrast agent largely overcomes the major barrier of low sensitivity of MRI techniques. A peptide of ten amino acids from the C-terminal of GRP was grafted onto ProCA1. GRP-grafted protein contrast agents (ProCA1.GRPs) showed the targeting capability to the GRPRs which are over-expressed on prostate cancer cells. Cell MRI Imaging demonstrated that ProCA1.GRP(52) grafted between Lys51 and Ser52 had better targeting capability than C-terminal one. Administration of ProCA1.GRP into xenograft tumor model enhances the contrast in the GRPR+ prostate tumor under MRI and optical imaging. Study demonstrated a potential application for disease marker targeted MR imaging by using our developed protein contrast agent.

This thesis is focused on the development, preclinical evaluation, and optimization of radiotracers for the detection of gastrin-releasing peptide receptor (GRPR)-expressing tumors. The work is divided into three distinct parts: (1) the development of bombesin (BN) antagonist (RM26)-based imaging radiotracers for the detection of GRPR-expressing tumors using different positron emission tomography (PET) and single photon emission computed tomography (SPECT) radionuclides (68Ga, 18F and 111In), (2) the establishment of a method to monitor the ligand-G protein-coupled receptor (GPCR) interaction in real time without requiring purification and stabilization of the receptors, and (3) the evaluation of radiopeptide structure-related factors (length of mini-PEG linker and composition of chelator for metal labeling) affecting the in vitro and in vivo characteristics of RM26-based tracers. We demonstrated the possibility of high-contrast in vivo imaging of GRPR-expressing xenografts despite the physiological expression of GRPR in abdominal organs. Fast radioactivity clearance from the blood and healthy organs, including receptor-positive organs, and long retention in the tumors resulted in high tumor-to-background ratios. A novel real-time assay for measuring the kinetics of the radiotracers targeting GPCR was evaluated. Living cells were used instead of purified receptors in this technology, bringing the developmental work one step closer to the true target environment (imaging in living systems). The comparative study of 68Ga-labeled NOTA-PEGn-RM26 with di-, tri-, tetra- and hexaethylene glycol chains demonstrated that the addition of only a few units of ethylene glycol to the spacer is insufficient to appreciably affect the biodistribution of the radiopeptide. Finally, a comparative study of 68Ga-labeled PEG2-RM26 analogs N-terminally conjugated to NOTA, NODAGA, DOTA or DOTAGA highlighted the influence of the chelator on the targeting properties of the radiopeptide. The main conclusion that can be drawn from this thesis is that 68Ga-NOTA-PEG2-RM26 has favorable biodistribution properties, such as rapid clearance from blood and tissues with physiological GRPR expression levels and long retention in GRPR-expressing tumors, and that this radiopeptide is potentially suitable for initial clinical investigation.

Temporal and spatial molecular imaging of disease biomarkers using non-invasive MRI with high resolution is largely limited by lack of MRI contrast agents with high sensitivity, high specificity, optimized biodistribution and pharmacokinetics. In this dissertation, I report my Ph. D. work on the development of protein-based MRI contrast agents (ProCAs) specifically targeting different cancer biomarkers, such as grastrin-releasing peptide receptor (GRPR), prostate specific membrane antigen (PSMA), and vascular endothelial growth factor receptor-2 (VEGFR-2). Similar to non-targeted ProCAs, these biomarker-targeted ProCAs exhibit 5 - 10 times higher r1 and r2 relaxivites than that of clinical MRI contrast agents. In addition, these biomarker-targeted ProCAs have high Gd3+ binding affinities and metal selectivities. The highest binding affinity of the three GRPR-targeted contrast reagents obtained by grafting a GRPR ligand binding moiety into ProCA32 for GRPR is 2.7 x 10-9 M. We further demonstrate that GRPR-targeted ProCAs were able to semi-quantitatively evaluate GRPR expression levels in xenograft mice model by MRI. In addition, we have also created a PSMA-targeted ProCA which has a binding affinity to PSMA biomarker of 5.2 x 10-7 M. Further, we developed VEGFR-targeted contrast agent which is able to image VEGFR2 in mice models using T1-weighted and T2-weighted sequences. Moreover, the relaxivities and coordination water numbers of ProCAs can be tuned by protein design of ProCA4. Since disease biomarkers are expressed in various tumors and diseases, our results may have strong preclinical and clinical implications for the diagnosis and therapeutics of cancer and other type of diseases.

臨床上止癢藥物的發展有限,故尋找具有廣泛止癢效果的藥物及探討癢覺傳遞的 神經受體機制是相當重要的。從過去研究中發現,bombesin 能在嚙齒類動物引發強 烈的搔抓行為。另外,bombesin 對 bombesin receptor family 中的 gastrin- releasing peptide receptor(GRPr)和 neuromedin B receptor(NMBr)也有高度親 和力。本研究主要目的為釐清 GRPr 和 NMBr 是否具有獨立調控癢覺搔抓行為的能 力。藉由側腦室於大白鼠中樞給藥並量化大白鼠的後肢搔抓行為,建立 GRP 與 NMB 這兩種胜肽在大白鼠上引發搔抓行為的基本藥理特性,並透過 GRPr 與 NMBr 這兩種受體的專一性拮抗劑探討受體間對於調控癢覺搔抓行為的獨立性。透過每天連 續施打 bombesin、GRP 與 NMB 這三種胜肽探討大白鼠搔抓行為的耐受性及三種胜 肽引發之搔抓行為在長時間觀測上的比較。最後,利用 GRPr 與 NMBr 這兩種受體 的專一性拮抗劑來探討 bombesin 引發搔抓行為的機制。利用側腦室給予 GRP (0.03 - 0.3 nmol)與 NMB(0.1-1 nmol)皆引發劑量相關性的搔抓行為反應。GRPr 的拮抗劑 RC-3095 (0.1-1 nmol)可以劑量相關性地阻斷 GRP 所引發的搔抓行 為,但不能阻斷 NMB 所引發的搔抓行為。相對而言,NMBr 的拮抗劑 PD168368 (0.3-3 nmol)可以劑量相關性地阻斷 NMB 引發的搔抓行為,但不能阻斷 GRP 所引 發的搔抓行為。藉由這些拮抗劑實驗,証實了 GRPr 與 NMBr 兩受體皆調控癢覺的訊 息傳遞並獨立不互相影響。Bombesin、GRP 與 NMB 都能引發搔抓行為,但程度與 持續時間上都有差異。但是每天施打這三種胜肽皆引發搔抓行為的耐受性。然而, RC-3095 與 PD168368 的有效劑量或兩個拮抗劑的組合並不能阻斷 bombesin 所引 發的搔抓行為,意指 bombesin 所引發的搔抓行為可能由其他受體調控。由本研究得 知,GRPr 與 NMBr 分別獨立調控中樞神經系統的癢覺訊息傳遞,並有程度與持續時 間上的差異。更重要的是,綜合先前文獻研究讓我們瞭解 bombesin-recognized neurons 中還有其他重要的受體機制調控於中樞神經系統的癢覺傳遞。 / Bombesin is a pruritogenic agent that causes intense itch-scratching activity in rodents. Bombesin has high affinity for the gastrin-releasing peptide receptor (GRPr) and the neuromedin B receptor (NMBr). The aim of this study was to investigate pharmacologically the ability of GRPr and NMBr to themselves elicit scratching behavior in rats. The intracerebroventricular (i.c.v.) route was selected for drug delivery because the study focused on supraspinal sites of action. The magnitude and duration of scratching produced by the naturally occurring peptides GRP and NMB were characterized. Antagonists selective for GRPr (RC-3095) and NMBr (PD168368) were used to define the role of GRPr and NMBr in the scratching response. After i.c.v. administration, GRP (0.03-0.3 nmol) and NMB (0.1-1 nmol) dose-dependently elicited marked scratching. There was a tolerance to scratching elicited by daily repeated administration of bombesin, GRP, or NMB. Presession administration of RC-3095 (0.1-1 nmol) and PD168368 (0.3-3 nmol) dose- dependently antagonized scratching elicited by GRP and NMB, respectively. More important, 1 nmol of RC-3095 failed to block NMB- elicited scratching and 3 nmol of PD168368 failed to block GRP-elicited scratching. In addition, pretreatment with effective doses of RC-3095 or PD168368 alone or in combination did not block bombesin-elicited scratching. Through the use of the selective antagonists RC-3095 and PD168368, this study demonstrates that central GRPr and NMBr may act independently to elicit scratching behavior. Although the receptor mechanisms underlying bombesin-elicited scratching elude us, this study provides a pharmacological basis for GRPr and NMBr antagonists as potential antipruritics.

We have investigated the role of the metabotropic glutamate receptor 7 (mGluR7) and the gastrin releasing peptide receptor (Grpr) population that are involved at different levels of itch transmission. We found that mGuR7 deficient mice displayed an anaphylaxis-like behavior when provoked with histamine. Analysis of blood revealed elevated plasma levels of histamine and mouse mast cell protease-1 (mMCP1), two indicators of anaphylaxis, in mGluR7 deficient mice compared with control mice. Inhibition of the neurokinin 1 receptor, by preventing binding of the corresponding ligand substance P (SP), prior to provocation with histamine prevented the development of anaphylaxis in mGluR7 deficient animals. However, blocking GRPR (gastrin releasing peptide receptor) only resulted in decreased itch levels in mGluR7 deficient mice but did not prevent the systemic anaphylaxis-like behavior. Our findings indicate that mGluR7 normally functions as a brake on histaminergic itch that is mediated through GRPR as well as anaphylaxis through Substance P. Grpr has previously been shown to mediate both histaminergic and non-histaminergic itch but little is known about the GRPR neuronal population. We used a BAC cloning strategy to construct a Grpr-Cre line, which we crossed with the reporter lines tdTomato and Viaat-egfp as well as with Vglut2-lox. We could conclude that Grpr-Cre neurons are mainly excitatory interneurons located in lamina II-IV, that convey itch using VGLUT2-mediated glutamatergic transmission to the next, currently unknown, step in the labeled line of chemical itch. To eventually deduce the function of the endogenous opioids dynorphin and enkephalin, which are hypothesized to be involved in gating pain and itch in the spinal cord, we constructed two Cre lines using BAC cloning that targeted the precursor proteins preprodynorphin and preproenkephalin, respectively. Preprodynorphin-Cre neurons were mainly located in lamina II-IV and overlapped to 47% with Vglut2 mRNA, while the co-expression with the inhibitory markers Viaat-egfp and PAX2 was 13% and 28% respectively in the spinal cord. Preproenkephalin neurons were more localized to lamina III in the dorsal horn, furthermore single cell analysis showed that they overlapped to 94% with Vglut2 mRNA while 7% and 13% expressed Viaat-egfp and PAX2 respectively.

Coordination of transition metals to peptides, either through the incorporation of unnatural chelating groups or amino acid ligating side-chains, expands the utility of peptides for biological studies. The first part of this project describes induction of α-helical secondary structure in pentapeptides upon side-chain coordination of inert transition metal ions. The second part of this project describes the syntheses of biologically active peptide species that contain a macrobicyclic hexaamine ligand that can complex radioactive metal ions for diagnostic imaging purposes. / Short peptide sequences do not form thermodynamically stable α-helices in water. The capacity of two metal clips, cis-[Ru(NH3)4(solvent)2]2+ and cis [Pd(en)(solvent)2]2+ to induce α-helicity in peptides that are five amino acids long, Ac HARAH NH2 and Ac MARAM-NH2 has been explored. In all cases at pH < 5, the metal ions bind to the side-chains of amino acid residues at positions i, i+4 of the pentapeptides resulting in formation of bidentate macrocyclic species. Circular dichroism and 1H nuclear magnetic resonance data indicate that the metal complexes of Ac-MARAM-NH2 are highly α helical in water, and in the most spectacular case, coordination of Ac-MARAM-NH2 to cis-[Ru(NH3)4(solvent)2]2+ results in up to 80% α-helicity. In contrast, metal complexes of Ac-HARAH-NH2 exhibit significantly less α-helicity in water. / 64Cu-radiolabelled peptides have been investigated for their ability to target specific tissue or cell types. These peptides require a chelating group that binds copper ions strongly. Macrobicyclic hexaamine ligands, based on the compound commonly referred to as “sarcophagine”, have demonstrated extremely high stability under biological conditions. Here we describe the synthesis of diaminosarcophagine chelators with carboxylate groups for conjugation to peptides. These new chelators have been attached to the N-terminus or lysine side-chain of biologically-active peptides, including Tyr3 octreotate, Lys3-bombesin and an integrin targeting peptide. Spectroscopic and voltammetric studies of these species suggest that the conjugated sarcophagine group retains the high metal binding affinity and structural properties of the parent species, diaminosarcophagine. These are among the first sarcophagine-peptide compounds that have been properly characterised. The new sarcophagine-peptide conjugates can be easily radiolabelled with 64Cu2+ over a wide pH range at ambient temperature.

No description available.