Glycosaminoglycans (GAGs) are linear polysaccharides whose disaccharide building blocks consist of an amino sugar and either uronic acid or galactose. They are expressed on virtually all mammalian cells, usually covalently attached to proteins, forming proteoglycans. GAGs are highly negatively charged due to an abundance of sulfate and carboxylic acid groups, and are structurally very diverse, with differences arising from chain length, the type of monomeric units, the linkages between each monomeric unit, the position of sulfate groups, and the degree of sulfation. GAGs are known to interact with a multitude of proteins, impacting diverse physiological and pathological processes. In addition, most of the biological interactions mediated by proteoglycans are believed to be primarily because of the GAG chains present on their surface. Considering the involvement of GAGs in multiple diseases, their use in the development of drugs has been of significant interest in the pharmaceutical field. Heparin, the first GAG-based drug developed in 1935, is still the most widely used anticoagulant in the world. The therapeutic potential of GAGs for the treatment of many other disease states, including cancer, inflammation, infection, wound healing, lung diseases, and Alzheimer’s disease, is being actively studied with many GAGs currently in clinical trials. However, challenges associated with the heterogeneous and complex structure of GAGs, limit their successful development. To combat such issues, our lab has focused on developing Non- Saccharide GAG Mimetics (NSGMs) as structural mimics of GAGs. NSGMs, being synthetic molecules, offer multiple advantages over GAGs. The studies mentioned here describe our efforts in the development of NSGMs as potential therapeutics for cancer, and cystic fibrosis.

Human neutrophil elastase (HNE) is a uniquely destructive serine protease with the ability to unleash a wave of proteolytic activity by destroying the inhibitors of other proteases. Although this phenomenon forms an important part of the innate immune response to invading pathogens, it is responsible for the collateral host tissue damage observed in chronic conditions such as chronic obstructive pulmonary disease (COPD), and in more acute disorders such as the lung injuries associated with COVID-19 infection. Previously, a combinatorially selected activity-based probe revealed an unexpected substrate preference for oxidised methionine, which suggests a link to oxida-tive pathogen clearance by neutrophils. Here we use oxidised model substrates and inhibitors to confirm this observation and to show that neutrophil elastase is specifically selective for the di-oxygenated methionine sulfone rather than the mono-oxygenated methionine sulfoxide. We also posit a critical role for ordered solvent in the mechanism of HNE discrimination between the two oxidised forms methionine residue. Preference for the sulfone form of oxidised methionine is especially significant. While both host and pathogens have the ability to reduce methionine sulfoxide back to methionine, a biological pathway to reduce methionine sulfone is not known. Taken to-gether, these data suggest that the oxidative activity of neutrophils may create rapidly cleaved elas-tase “super substrates” that directly damage tissue, while initiating a cycle of neutrophil oxidation that increases elastase tissue damage and further neutrophil recruitment.

Made available in DSpace on 2014-12-17T14:03:37Z (GMT). No. of bitstreams: 1 NorbertoKVM_DISSERT.pdf: 2522986 bytes, checksum: 6298d49730e0d9c9d5418ad46a9b33f5 (MD5) Previous issue date: 2011-02-22 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Studies indicate that several components were isolated from medicinal plants, which have antibacterial, antifungal, antitumor and anti-inflammatory properties. Sepsis is characterized by a systemic inflammation which leads to the production of inflammatory mediators exacerbated by excessive activation of inflammatory cells and disseminated intravascular coagulation (DIC), in which the human neutrophil elastase plays an important role in its pathogenesis. Several epidemiological studies suggest that components of plants, especially legumes, can play a beneficial role in reducing the incidence of different cancers. A chymotrypsin inhibitor of Kunitz (Varela, 2010) was purified from seeds of Erythrina velutina (Mulungu) by fractionation with ammonium sulfate, affinity chromatography on Trypsin-Sepharose, Chymotrypsin-Sepharose and ion exchange chromatography on Resource Q 1 ml (GE Healthcare) in system FPLC / AKTA. The inhibitor, called EvCI, had a molecular mass of 17 kDa determined by SDS-PAGE. The purified protein was able to inhibit human neutrophil elastase (HNE), with an IC50 of 3.12 nM. The EvCI was able to inhibit both pathways of HNE release stimulated by PAF and fMLP (75.6% and 65% respectively). The inhibitor also inhibited leukocyte migration in septic mice about 87% and prolonged the time of coagulation and inhibition factor Xa. EvCI showed neither hemolytic activity nor cytotoxicity. EvCI showed a selective antiproliferative effect to HepG2 cell lines with IC50 of 0.5 micrograms per milliliter. These results suggest EvCI as a molecule antagonist of PAF / fMLP and a potential use in fighting inflammation related disorders, disseminated intravascular coagulation (DIC) and cancer / Estudos indicam que v?rios componentes medicinais foram isolados de vegetais, os quais apresentam atividades antibacterianas, antif?ngicas, antitumorais e anti-inflamat?rias. Sepse ? caracterizada por uma inflama??o sist?mica que tem como conseq??ncia a produ??o exarcebada de mediadores inflamat?rios, pela excessiva ativa??o de c?lulas inflamat?rias e coagula??o intravascular disseminada (CIVD), na qual a elastase neutrof?lica humana exerce um papel importante na sua patog?nese. Diversos estudos epidemiol?gicos sugerem que componentes de vegetais, especialmente de leguminosas, podem desempenhar um papel ben?fico na redu??o da incid?ncia de diferentes tipos de c?ncer. Um inibidor de quimotripsina do tipo Kunitz (Varela, 2010) foi purificado de sementes de Erythrina velutina (Mulungu) por fracionamento com sulfato de am?nio, cromatografias de afinidade em Tripsina-Sepharose e Quimotripsina-Sepharose e cromatografia de troca i?nica em Resource Q 1 mL (GE Healthcare), em sistema FPLC/AKTA. O inibidor, denominado EvCI, apresentou uma massa molecular de 17 kDa, determinada por SDS-PAGE. A prote?na purificada foi capaz de inibir a elastase de neutr?filos humanos (ENH), apresentando um IC50 de 3,12 nM. O EvCI foi capaz de inibir ambas as vias de libera??o de ENH estimuladas por PAF e fMLP (75,6% e 65%, respectivamente). O inibidor tamb?m inibiu a migra??o leucocit?ria em camundongos s?pticos em cerca de 87% e prolongou o tempo de coagula??o com inibi??o do fator Xa. EvCI n?o apresentou atividade hemol?tica nem citot?xica. EvCI apresentou um efeito antiproliferativo seletivo para linhagens de c?lulas HepG2 com IC50 de 0,5 μg /mL. Estes resultados sugerem o EvCI como uma mol?cula antagonista dos receptores PAF/fMLP e um potencial emprego no combate a dist?rbios relacionados a inflama??o, coagula??o intravascular disseminada (CIVD) e cancer