No / Whereas protease inhibitors have been developed successfully against hypertension and viral infections, they have failed thus far as cancer drugs. With advances in cancer profiling we now better understand that the tumor “degradome” (i.e. the repertoire of proteases and their natural inhibitors and interaction partners) forms a complex network in which specific nodes determine the global outcome of manipulation of the protease web. However, knowing which proteases are active in the tumor micro-environment, we may tackle cancers with the use of Protease-Activated Prodrugs (PAPs). Here we exemplify this concept for metallo-, cysteine and serine proteases. PAPs not only exist as small molecular adducts, containing a cleavable substrate sequence and a latent prodrug, they are presently also manufactured as various types of nanoparticles. Although the emphasis of this review is on PAPs for treatment, it is clear that protease activatable probes and nanoparticles are also powerful tools for imaging purposes, including tumor diagnosis and staging, as well as visualization of tumor imaging during microsurgical resections.

No description available.

Doctor of Philosophy / Department of Chemistry / Stefan H. Bossmann / This paper describes two methods for utilizing cancer associated proteases for targeting cancer therapy to the tumor. The first method is designing a drug delivery system based on liposomes that are sensitive to cancer associated proteases. Upon contact with the protease, the liposome releases its contents. The second method is designing a prodrug that is based on a porin isolated from Mycobacterium smegmatis. The porin is modified with protease consensus sequences, inhibiting its toxicity. Upon contact with the protease, the drug is activated. Protease sensitive liposomes were synthesized that were sensitive to urokinase plasminogen activator. This was done by synthesizing a cholesterol-anchored, uPA consensus – sequence-containing, acrylic acid block copolymer and using it to form a covalently bound polymer cage around the outside of a hypertonic liposome. Liposomes were synthesized that had a diameter of 136 nm. Upon addition of the polymer the diameter increased by 2.69 nm, indicating it had successfully embedded into the liposome membrane. After crosslinking with either a short peptide containing a lysine (so that it is a diamine) or ethylenediamine, the diameter increased between 5.33 nm and 14.1 nm (depending on the type and amount of the crosslinked). Fluorescence release assays showed that the polymer cage could add in excess of thirty atmospheres of osmotic pressure resistance, and, under isobaric conditions, would prevent release of much of the liposomal contents. Upon treatment with uPA, the polymer caged liposomes released a significantly larger amount of their contents making the liposomes protease sensitive. MspA was shown to be a very stable protein able to be imaged by AFM. AFM imaging demonstrated that MspA is able to form native pore structures in membranes making it a good imitator of the membrane attack complex. MspA was demonstrated to be highly cytotoxic, but poor at distinguishing between cells. Pro-MspA was synthesized by adding a hydrophilic peptide to MspA that prevents insertion. A uPA cleavage sequence embedded causes the MspA to become activated at the cancer site. This was demonstrated in tests against uPA and non-uPA producing cell lines.

The transmembrane isoform of mucin 1 (MUC1/TM) is a well recognized tumor antigen, contributing to tumorigenesis and immune evasion. While MUC1/TM has been correlated with malignancy, it appears that a secreted splice variant of MUC1 (MUC1/sec) has antitumor properties and prevents tumor development. It was discovered that MUC1/sec expressing tumor cells (DA-3/sec) have a significant reduction in expression of urokinase plasminogen activator (uPA) relative to the parental tumor line, and tumor cells expressing MUC1/TM (DA-3/TM). The serine protease uPA, has been found to be involved in growth promoting signaling, angiogenesis, and induction of matrix remodeling leading to metastasis. Furthermore, the tumor suppressive and interferon responsive Stat1 transcription factor is dramatically upregulated in DA-3/sec cells. In addition, treatment of various murine and human cell lines with conditioned media containing MUC1/sec results in up-regulation of Stat1. DA-3/sec tumor cells are also sensitized to the anti-proliferative effects of IFN-g. Furthermore, transfection of the Stat1 gene into DA-3 tumor cells leads to a downregulation of uPA, and delays tumor progression. Since myeloid-derived suppressor cells (MDSC) play a critical role in tumor-induced immunosuppression, we investigated their recruitment by DA-3/sec and DA-3/TM cells. DA-3/sec tumor cells recruit dramatically lower levels of MDSC, relative to DA-3/TM cells. Since MUC1/sec down-regulates tumor expression of uPA, its potential role in MDSC recruitment was investigated. Tumor-derived uPA is capable of recruiting MDSC, and correlates with tumor development. In addition to diminishing recruitment of MDSC, the effect of MUC1/sec on MDSC suppressive mechanisms was investigated. MUC1/sec, or its unique immunoenhancing peptide (IEP), is capable of blocking expression of arginase 1 and production of reactive oxygen species (ROS) in MDSC, implicated in the suppression of T cells. These findings demonstrate a new mechanism of MDSC recruitment, and provide evidence that MUC1/sec has antitumor properties affecting both tumor cells and MDSC. Furthermore, it was discovered that MDSC home to the liver in addition to the tumor, bone marrow, blood, and spleen of tumor bearers, as previously described. The liver is thus an organ where MDSC accumulate and can contribute to immunosuppression directly and indirectly, via interactions with a variety of immune cells.

München, Techn. Univ., Diss., 2004.

Urokinase-type plasminogen activator (uPA) protein is a serine protease of the trypsin family that is overexpressed by tumors cells seeking to metastasize. Molecular imaging methods using molecular imaging probe designed to target uPA could provide a method for the detection of aggressive cancers and monitoring response to treatment. Four classes of high affinity uPA inhibitors, three which were reversible and one irreversible, were used as platforms to develop radiolabeled probes for uPA. Based on structure-activity relationships, lead compounds were modified to allow for the introduction of a radiohalogen (radioiodine) at different sites in the corresponding molecules. Suitable synthetic strategies were developed to create libraries of iodinated phenyl guanidine, peptide, naphtamidine and phosphonate derivatives. For the phenylguanidines colorimetric assays showed the product had micromolar affinity while for the peptide derivatives low nanomolar affinity for the iodinated analogue was observed (1.4 nM to 2.53 nM). Unfortunately quantitative biodistribution studies showed low tumour uptake (<0.5% ID/g). More promising results were obtained for the irreversible iodinated phosphonated derivative which had an affinity of 2.1 nM. This reagent showed 1.95% ID/g tumour uptake and lower blood uptake in vivo which demonstrates advantageous properties over existing uPA probes in terms of tumour-to-blood ratios. A complementary development was also achieved in that the first example of a 125I-labelled tetrazine was prepared. This new reagent can be used in pre-targeted strategies that utilize bioorthogonal coupling between stained trans-cyclooctene (TCO) and tetrazines. The product was prepared using a concomitant oxidation iodo-destannylation reaction and the product isolated in 80% radiochemical yield. The reaction with transcycloctene proceeded rapidly to produce various isomers which were fully characterized through NMR analysis of the non-radioactive analogues. / Thesis / Doctor of Philosophy (PhD)

L'infiltration d'éosinophiles dans la muqueuse bronchique est caractéristique dans l'asthme. Pour passer du sang vers la muqueuse bronchique, l'éosinophile doit sécréter des proteases qui digéreront les protéines de la matrice extracellulaire ainsi que la membrane basale des vaisseaux sanguins. L'acide 5-oxo-6,8,11,14-éicosatétraénoïque, un lipide bioactif, provoque une transmigration des éosinophiles en activant la sécrétion de plusieurs proteases dont la matrix metalloproteinase (MMP)-9 ainsi que l'urokinase et la plasmine, deux proteases du système plasminogène/plasmine. Il active la sécrétion de la MMP-9 via la voie signalétique extracellular signal-regulated kinase (ERK)-1/2. De plus, il est décrit que l'effet de ERK-1/2 sur la migration diminue en absence du système plasminogène/plasmine. Cette étude porte sur les interactions entre les différentes protéases impliquées lors de la migration du 5-oxo-ETE, plus précisément, la capacité de l'urokinase plasminogen activator (uPA) et de la plasmine à augmenter la sécrétion de MMP-9. Elle traite également de l'implication de la voie signalétique des ERK-1/2 dans la sécrétion de la MMP-9. Elle confirme que l'uPA et de la plasmine peuvent induire la sécrétion de la MMP-9. Elle valide également l'implication de la voie signalétique des ERK-1/2 lors de ce phénomène.

Au cours de l'invasion tumorale, la matrice extracellulaire du tissu broncho-pulmonaire, riche en élastine, subit de nombreux remaniements. La dégradation de cette élastine conduit à la production de peptides bioactifs. Ces peptides d'élastine (PE) possèdent un récepteur spécifique, le complexe récepteur de l'élastine (CRE), et peuvent également interagir avec l'intégrine alphavbeta3 et la galectine-3. Dans cette étude, nous avons étudié le rôle des PE et de leurs récepteurs dans la progression tumorale des carcinomes broncho-pulmonaires.Des cellules épithéliales bronchiques tumorales sont incubées in vitro avec un mélange de PE, la kappa-élastine (kE), ou avec des peptides synthétiques. Le traitement par les peptides entraine une augmentation de la capacité infiltrante des cellules invasives associée à un relargage précoce de MMP 2, MMP 9 et uPA mais n'a pas d'effet sur la prolifération et le phénotype cellulaire. Les niveaux d'ARNm des 3 protéases stimulées ne sont pas modifiés et ni l'actinomycine D, ni le cycloheximide ou la bréfeldine A ne sont capables d'inhiber les effets liés à la kE. Ces effets ne sont pas non plus inhibés par le lactose et les autres antagonistes des trois récepteurs. Enfin, les peptides VGVAPG et GRKRK, présentant les séquences spécifiques reconnues par les récepteurs, ne réussissent pas à reproduire les effets observés avec la kE, alors que des nonapeptides les reproduisent de façon quasi-identique.Ces résultats montrent que les PE régulent la capacité invasive des carcinomes broncho-pulmonaires, via le relargage d'enzymes protéolytiques. Cette modulation mettrait en jeu des mécanismes post-traductionnels et un récepteur lactose-insensible, différent du CRE, de l'intégrine alphavbeta3 et de la galectine-3, et reconnaissant des nonapeptides d'élastine. / Elastin-rich lung extra-cellular matrix is largely remodeled during tumor invasion. Elastin degradation produces peptides displaying a wide range of biological activities. These elastin derived peptides (EP) interact with the Elastin Receptor Complex (ERC) but also bind to alphaVbeta3 integrin and galectin-3. In this study, we explored the role of EP and their receptors in tumor progression of lung carcinomas.In vitro, lung tumor cells were incubated in presence of kappa-elastin (kE), a mix of EP or with synthetic elastin peptides. EP treatment induced an increase of invasive capacity of invasive cells with quickly increased levels of MMP-2, MMP 9 and uPA but had no effect on cell proliferation and phenotype. Interestingly, protease regulation was not observed at the mRNA level and actinomycin D, cycloheximide and brefeldin A were unable to inhibit kE effects. These effects could not be inhibited either by classical receptor antagonists including lactose or blocking antibodies. Finally, synthetic peptides VGVAPG and GRKRK, displaying receptor-specific sequences, failed to reproduce kE effects whereas nonapeptides partially mimicked them.These results demonstrate that treatment with EP up-regulates invasiveness of lung tumor cells via the release of proteolytic enzymes. This modulation involves post-translational mechanisms and a lactose-insensitive receptor, different from the ERC, alphaVbeta3 integrin and galectin-3 and recognizing nonapeptidic sequences.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Doctor of Philosophy / Department of Chemistry / Stefan H. Bossmann / Cancer is one of the leading causes of death in the world. Billions of dollars are spent to treat cancer every year. This clearly shows the need for developing improved treatment techniques that are affordable to every person. Early diagnosis and imaging of tumors is equally important for the battle against this disease. This dissertation will discuss new approaches for discovering and developing novel detection and treatment techniques for cancer using organic ligands, and Fe/Fe3O4 core/shell magnetic nanoparticles. A series of o-phenylenediamine derivatives with nitro-, methyl- and chloro- substituents were synthesized and studied their ability to act as anticancer agents by using steady-state, UV/Vis-, and fluorescence spectroscopy. In the absence of zinc(II), intercalation with DNA is the most probable mode of interaction. Upon addition of zinc(II), DNA-surface binding of the supramolecular aggregates was observed. The interaction of the supramolecular (-ligand-Zn2+-)n aggregates with MDA 231 breast cancer cells led to significant cell death in the presence of UVA at λ=313 nm displaying their potential as anticancer agents. Bimagnetic Fe/Fe3O4 core/shell nanoparticles (MNPs) were designed for cancer targeting after intratumoral or intravenous administration. Their inorganic center was protected by dopamine-oligoethylene glycol ligands. TCPP (4-tetracarboxyphenyl porphyrin), a fluorescent dye, was attached to the dopamine-oligoethylene glycol ligands. These modified nanoparticles have the ability to selectively accumulate within the cancerous cells. They are suitable candidates for local hyperthermia treatment. We have observed a temperature increase of 11 ºC in live mice when subcutaneously injecting the MNPs at the cancer site and applying an alternating magnetic field The system is also suitable for Magnetic Resonance Imaging (MRI), which is a diagnostic tool to obtain images of the tumors. Our superparamagnetic iron oxide nanoparticles have the ability to function as T1 weighted imaging agents or positive contrasting agents. We were able to image tumors in mice using MRI. Various proteases are over-expressed by numerous cancer cell lines and, therefore, of diagnostic value. Our diagnostic nanoplatforms, designed for the measurement of protease activities in various body fluids (blood, saliva, and urine), comprise Fe/Fe3O4 core/shell nanoparticles featuring consensus sequences, which are specific for the target protease. Linked to the consensus sequence is a fluorescent organic dye (e.g. TCPP). Cleavage of the sequence by the target protease can be detected as a significant increase in fluorescence occurring from TCPP. We were able to correlate our diagnostic results with cancer prognosis.