Dissertations / Theses on the topic 'Human neutrophil elastase inhibitors'

Dendritic cells (DC) are sentinels of the immune system that display an extraordinary capacity to present antigen to naïve T cells and initiate immune responses. DCs are distributed throughout the lungs in the conducting airways of the tracheobronchial tree and in the parenchymal lung, and play a pivotal role in controlling the immune response to inhaled antigens. The respiratory surface is continually exposed to potentially injurious particulates and pathogenic organisms, to which tightly regulated innate and adaptive immunological responses are made. The airways are usually sterile in healthy individuals. However, patients with chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) have increased susceptibility to microbial infections and increased neutrophil elastase (NE) in lung secretions. This thesis was designed to test the hypotheses that; (i) excess NE may result in a dysregulation of lung DCs function in pulmonary chronic diseases, and (ii) the natural NE inhibitors in the respiratory system are able to rescue the NE-mediated dysregulation of DCs and potentially enhance their antigen presenting activity. The data in this thesis demonstrate that purified human NE down-regulated murine bone marrow (BM)-derived DC co-stimulatory molecules (CSM; CD40, CD80 and CD86), which was due to its proteolytic activity. NE-treated LPS-matured DCs were less efficient at presenting ovalbumin (OVA) peptide to naïve OVAspecific transgenic (D011.10) T cells. In addition, immature DCs (iDC) simultaneously treated with LPS and NE failed to mature fully and produced significantly less IL-12 and TNF-α than DCs matured in the presence of LPS alone. Similarly, treatment of mature DC (mDC) with pooled and individual COPD and CF sputum samples caused a reduction in CD80 and CD86 levels (but not CD40) which positively correlated with the NE concentration present in the samples. The demonstration that NE could adversely affect DC phenotype and function suggested that augmentation of NE inhibitors could reverse this process and preserve DC function in inflammatory microenvironments. Over-expression of an NE specific inhibitor (elafin) in the lungs of mice (using either replication-deficient adenovirus [Ad] or elafin transgenic [eTg] mice) increased the number (immunofluorescence) and activation status (flow cytometric measurement) of CD11c+/MHCII+ lung DCs in in vivo models. Replication-deficient Ad vectors encoding NE inhibitors, namely elafin, secretory leukocyte protease inhibitor (SLPI) and α1-protease inhibitor (α1-PI), were also used to infect DCs in vitro, to further study the effect of these NE-inhibitors on DCs in isolation. These findings suggest that purified NE and NE-containing lung inflammatory secretions are powerful down-regulators of DC maturation, resulting in reduced capacity of these potent APCs to efficiently present antigens; whereas, NE inhibitors could boost immunity by increasing the activation state and/or number of DCs.

This thesis was an exploration of how Human Neutrophil Elastase (HNE) activity can be modulated by oxidation of methionine residues located on substrates and inhibitors. Research focused on producing a molecular toolbox of innovative HNE substrates and inhibitors specifically engineered to include methionine, then assessing the mechanism by which oxidation leads to targeted interaction with HNE. This may be an important biochemical process in chronic obstructive pulmonary disease, which is linked to HNE destruction of elastic lung tissue together with oxidative damage by cigarette smoke and neutrophil-mediated inflammation.

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 neutrophils are the most abundant type of white blood cell and provide the body with a line of defense against foreign, infectious microorganisms. Contained within the azurophilic granules in the cytoplasm of neutrophils are three serine proteases, Human Neutrophil Elastase, Cathepsin G, and Protease 3. Once a foreign bacterium is engulfed by white blood cells, these enzymes attack and degrade the invading body, thus killing it (Reeves et al., 2002). The focus of this research is centered on the production of one of the serine proteases, human neutrophil elastase (HNE), and while the importance of HNE can be seen, genetic mutations or improper regulation can compromise a person’s immunity. Neutropenia (a low neutrophil count) is one such disease caused by a genetic mutation of HNE that results in susceptibility to infection (Li and Horwitz, 2001). Additionally, HNE is a powerful enzyme that can attack the elastin of the lung if not properly controlled. Consequently, genetic deficiencies of alpha-1 proteinase inhibitor protein in the blood can result in emphysema because active HNE released from neutrophils is free to degrade lung tissue (Laurell and Eriksson, 1965). Recombinant HNE is not currently available, and the enzyme must be isolated from human blood cells, which has inherent hazards. Additionally, the lack of recombinant HNE has prevented studies involving site–directed mutagenesis to study the intracellular processing of HNE near its C-terminal end where mutations have been found to result in neutropenia. Kinetic studies of the full-length HNE might shed some light on why its C-terminal region is removed before storage in cytoplasmic granules. The HNE DNA sequence was first codon optimized for yeast and commercially synthesized. It was then fused with DNA for eGFP (enhanced green fluorescent protein) via an enterokinase cleavage site (D4K). This DNA construct (eGFP-D4K-HNE) was then inserted into the Kluyveromyces lactis (K. lactis) pKLAC1 vector, downstream of the alpha mating factor which directs proteins for secretion. Then, chemically competent GG799 cells (a strain of K. lactis) were transformed with the linearized pKLAC1-eGFP-D4K-HNE insert through a protocol from New England Biolabs. Theoretically, the gene integrates into the yeast genome upon transformation via sequences within the pKLAC1 vector that are homologous with the LAC4 gene promoter that allows for galactose utilization (Colussi 2005). Acetamide was used as a selectable marker because wild type K. lactis cells are not able to use acetamide as a nitrogen source. The pKLAC1 vector, however, contains the Aspergillus nidulans gene acetamidase (amdS) that allows only transformants to grow on plates with acetamide as the sole nitrogen source (Read 2007). Selected colonies were transferred to both liquid and agar-based synthetic media with galactose to induce transcription and translation of the HNE gene to produce the eGFP-D4K-HNE fusion, and screened via fluorescence microscopy for production of eGFP. None of the screened colonies tested positive for the presence of the fusion protein.

Dans le but d'obtenir des composés originaux ayant une activité inhibitrice envers la NE, des triazoles et des 2-aminofuranes diversement substitués ont été synthétisés. Une réaction de "click chemistry" catalysée par le cuivre entre un azide et un alcyne a été appliquée pour la synthèse des triazoles. La synthèse des composés de type 2-aminofuranes est basée sur l'attaque nucléophile d'un isonitrile sur un γ-oxo butynoate d'alkyle en présence d'un aldéhyde aromatique.Les tests biologiques ont été réalisés tout au long de la thèse. Les composés présentant les meilleures IC50 envers la NE ont été sélectionnées pour une évaluation en terme de sélectivité vs les autres sérines protéases du PN (la cathepsine G et la protéinase 3). Enfin, les meilleurs inhibiteurs ont subi une évaluation de leur activité anti-oxydante selon deux méthodes: méthode au DPPH (1,1-diphényl-2-picryl-hydrazyl) et méthode au thiocyanate ferrique
To prepare new compounds with an inhibitory activity towards NE, diversely substituted triazoles and 2-aminofuranes were synthesized. Triazoles were obtained using the copper catalyzed "click chemistry" between an azide and an alkyne. The synthesis of 2-aminofuranes is based on the nucleophilic attack of an isonitrile on an alkyl γ-oxo-butynoate in the presence of an aromatic aldehyde.Biological assays were carried out throughout the thesis. Compounds owning the best IC50 towards NE were selected for evaluation in terms of selectivity vs the two other serine-proteases of the PN (cathepsin G and proteinase 3). Finally, evaluation of antioxidant activity of the best compounds was achieved using by two approachs: DPPH (1,1-diphenyl -2-picrylhydrazyl) method and ferric thiocyanate method

Behçet’s disease (BD) is a vasculitis of unknown aetiology typified by recurrent oral and genital ulcers, skin and ocular lesions. Debilitating manifestations can also affect vascular, gastrointestinal and neurological systems. Previous BD investigations showed increased circulating neutrophils and neutrophil elastase (NE), a serine protease. NE can digest connective tissues compromising their integrity if not regulated. In this study, NE and its two main inhibitors, secretory leukocyte protease inhibitor (SLPI) and alpha1- antitrypsin (α1AT), were investigated to determine if NE dysregulation is triggering oral mucosal damage. Findings were compared to healthy controls (HC) and recurrent aphthous stomatitis (RAS) patients, a disorder of episodic oral ulceration. FlowCytoMixTMmultiplex-assays compared saliva and serum inflammatory cytokines measurements where salivary levels reflected disease activity and correlated with published serum levels. Salivary NE, SLPI, and α1AT were measured by ELISA. Patients with oral ulcers had increased NE. Unexpectedly, BDq (quiescent, without ulceration) had increased NE, but SLPI was significantly lower than RASq and HC. RASq NE levels were similar to HC. Overall, NE correlated with α1AT levels, but showed an inverse relationship with SLPI. Quantitative PCR revealed significantly increased SLPI mRNA expression in both BDq and RASq buccal epithelium. High mRNA/low SLPI protein expression during ulceration could be explained by deficient translation, blocked ELISA antibody binding, or SLPI depletion. Despite high α1AT, all study groups had enzymatically active salivary NE which was successfully inhibited by recombinant SLPI. Confocal microscopy revealed BD patients’ blood neutrophils readily release neutrophil extracellular traps (NETs) in vitro compared to HCs. Antimicrobial NETs have mixed granule contents coating decondensed chromatin fibres and are associated with autoimmunity. During NET production, our novel observation that intracellular SLPI but not α1AT co-localised with NE suggests a regulatory role. This study supports the theory that a protease-antiprotease imbalance may play a role in BD oral and systemic pathology.

Neutrophile Granulocyten wirken als einer der ersten Abwehrmechanismen gegen invasive Mikroorganismen im angeborenen Immunsystem von Mammalia. Aktiviert durch inflammatorische Signale verlassen diese Granulocyten das vaskuläre System und migrieren durch das Gewebe zum Infektionsherd. Dort binden sie die Mikroorganismen, phagozytieren und eliminieren diese schließlich mit hoher Effizienz. Humane Neutrophile Elastase (NE) ist Bestandteil der neutrophilen Granula und spielt eine entscheidende Rolle im Abbau von Virulenzfaktoren enteroinvasiver Bakterien, einschließlich der Shigella Virulenzfaktoren IpaB (invasion antigen plasmid B) und IcsA (intracellular spread A). NE gehört zu der Familie der Chymotrypsin-ähnlichen Serinproteasen, die sich durch Sequenz- und Strukurähnlichkeit auszeichnen, jedoch sehr unterschiedliche biologische Funktionen aufweisen. Cathepsin G (CG) ist wie NE eine Chymotrypsin-ähnliche Serinprotease und ebenfalls in neutrophilen Granula lokalisiert. Allerdings zeigt CG keine Aktivität gegenüber Virulenzfaktoren von Shigella. Obwohl die Kristallstrukturen von CG und NE fast identisch sind, konnten einzelne oder mehrere Aminosäuren in der Substratbindungsspalte identifiziert werden, die zwischen den beiden Enzymen differieren. Dies legte die Vermutung nahe, dass die Spezifität von NE gegenüber Virulenzfaktoren in diesen Unterschieden codiert sein könnte. Daher wurden diese Aminosäuren durch die analogen CG Aminosäuren oder durch Alanin ersetzt. Der Vergleich der funktionellen Eigenschaften der NE Mutanten mit wildtyp NE zeigte, dass die Aminosäuren an den Positionen 98 und 216-224 entscheidend für die Substratspezifität von NE sind. Die NE Mutanten N98A, 216-218 und 216-224 waren nicht mehr in der Lage, die Virulenzfaktoren IcsA und IpaB sowie das NE Peptidsubstrat abzubauen. Stattdessen haben diese Mutanten die Fähigkeit erlangt, das CG Peptidsubstrat abzubauen. Zusammenfassend konnten wir Aminosäuren in NE identifizieren, die sowohl die Spezifität von NE für das Peptidsubstrat als auch für die Virulenzfaktoren von Shigella flexneri determinieren.
Neutrophil granulocytes are one of the first lines of defense of the mammalian innate immune system against invading microorganisms. In response to inflammatory stimuli, neutrophils migrate from the blood stream to infected tissues where they bind, engulf and inactivate microorganisms efficiently. Human neutrophil elastase (NE), a neutrophil granule component, is a key host defense protein that rapidly destroys virulence factors of enteroinvasive pathogens including IpaB (invasion plasmid antigen B) and IcsA (intracellular spread A) from Shigella. NE belongs to the family of chymotrypsin-like serine proteases with sequence and structural similarity but with very different biological functions. Cathepsin G (CG) is another abundant chymotrypsin-like serine protease in neutrophil granules. However, in contrast to NE, CG does not cleave virulence factors of Shigella. The crystallographic structures of NE and CG are very similar but we identified single or multiple residues in the substrate-binding cleft to differ in these two enzymes. We hypothesized that NE specificity for bacterial virulence factors resides within these structural differences. Therefore these specific residues in NE were replaced with the analogous amino acids of CG or with alanine. By comparing the functional properties of these NE mutants to wildtype NE we were able to show that the amino acids at position 98 and 216-224 are crucial for the substrate specificity of NE. The NE mutants N98A, 216-218 and 216-224 did not cleave the virulence factors IcsA and IpaB as well as the NE peptide substrate but cleaved the CG peptide substrate. In summary, we identified residues in NE that determine the specificity of NE for the peptide substrate and for the Shigella flexneri virulence factors.

Emphysema is a degenerative lung disease which, it is suggested, results probably from repeated periods of proteinase:antiproteinase imbalance during which excess of enzyme attacks the extracellular matrix. Of the many enzymes produced by inflammatory cells human neutrophil elastase (HNE) is thought to be the major offending enzyme. It attacks elastin, which is responsible for the elastic recoil of the lung. If emphysema was simply a result of the destruction of elastin by HNE then degradation products of elastin would inevitably be present, at least transiently, in the serum of patients. The aim of this project was to separate and characterise the soluble peptides resulting from the digestion of elastin with HNE and/or human neutrophil cathepsin G (HNCG), another neutrophilic enzyme, which is primarily bactericidal and, to determine if any of the peptides were characteristic of digestion by one enzyme or combination of enzymes. HNE and HNCG were isolated from purulent sputum, and elastin was isolated by two methods from post-mortem lungs. The digestion of the elastin by the enzymes was followed by measuring the amino groups liberated during the course of the digestion. A method was developed for the measurement of the insoluble as well as the soluble products of digestion. Initially, the amounts of soluble and insoluble products were similar, but the amount of soluble products soon exceeded the amount of insoluble products. The soluble products of digestion were separated by reverse-phase chromatography. The peptides separated into two groups (A and B), regardless of which enzyme was involved in the initial digestion. Both groups were heterogeneous mixtures of peptides. Filtration experiments and amino acid analysis showed that the groups of peptides differed in size and composition.

The yeasts Pichia pastoris and Kluyveromyces lactis were used to express several recombinant human proteins for further biochemical characterization. Two substitution variants of recombinant human enteropeptidase light chain (rhEPL) were engineered to modify the extended substrate specificity of this serine protease. Both were secreted as active enzymes in excess of 1.7 mg/L in P. pastoris fermentation broth. The substitution variant rhEPL R96Q showed significantly reduced specificities for the preferred substrate sequences DDDDK and DDDDR; however, the rhEPL Y174R variant displayed improved specificities for these substrate sequences relative to all other reported variants of this enzyme. The neutrophil serine proteases human cathepsin G (hCatG) and human neutrophil elastase (HNE) were expressed in P. pastoris and HNE was also expressed in K. lactis. The recombinant variants rhCatG and rHNE, with intact C-terminal extensions, were expressed as fusion proteins with the soluble heme-binding domain of cytochrome B5 (CytB5) and an N-terminal hexahistidine (6xHis) tag for purification. The CytB5 domain was linked to the native N-termini of active rhCatG and rHNE by the EPLcleavable substrate sequence DDDDK~I, where ~ is the sessile bond. These fusion proteins were directed for secretion. The yeast P. pastoris expressed up to 3.5 mg/L of EPL-activable rHNE in fermentation broth; however, only 200 μg/L of rhCatG could be produced by this method. Recombinant expression in K. lactis never surpassed 100 μg/L of activable rHNE. The CytB5 fusion domain was present in the heme-bound form, conferring a red color and 410 nm absorbance peak to solutions containing the fusion proteins. This absorbance pattern was most readily visible during the purification of CytB5-rHNE from P. pastoris. Human C-reactive protein (hCRP) and the substitution variant CRP E42Q were expressed in recombinant form and secreted by P. pastoris. Both products were found to bind phosphocholine (PCh) in the same manner as native hCRP. Difficulties encountered during purification revealed that wild type recombinant CRP (rCRP) was produced at 2 different molecular masses. The P. pastoris recombinant expression system yielded better results than K. lactis. Bioreactor-scale fermentation in a 5 L vessel facilitated expression and characterization of these recombinant proteins.

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

Perseguendo la ricerca di una nuova classe di peptidi antimicrobici strutturalmente semplici, abbiamo ottimizzato una sintesi breve, economica e ad alto rendimento di derivati dell'acido α-idrazido anfifilico monocarico, con azione membranolitica. Tali composti, hanno esibito un'attività in vitro ad ampio spettro contro una varietà di batteri Gram-positivi e Gram-negativi, tra cui due ceppi multi-farmaco resistenti. Inoltre, hanno mostrato effetti sinergici con la tetraciclina nei confronti dei batteri sensibili, mentre sono stati osservati effetti sinergici o indifferenza per combinazioni con diversi antibiotici di prima linea verso ceppi multiresistenti. Nonostante la minima carica cationica, i migliori composti hanno dimostrato di essere selettivi verso le membrane cellulari batteriche rispetto alle membrane cellulari dei mammiferi. È stata anche dimostrata l'importanza di un'anfifilicità senza interruzioni. Con l'obiettivo di somministrare dosi più basse di colistina riducendo gli effetti collaterali tossici, il Dipartimento di biologia chimica dell'HZI (Braunschweig, Germania) ha sviluppato un nuovo costrutto peptide-colistina. Consiste in una miscela di cinque regioisomeri in cui ciascuno dei gruppi amminici liberi della colistina è legato al terminale C con un peptide sintetico. La scissione del gruppo ammide introdotto da parte dei neutrofili dell'elastasi umana, rilascia la colistina direttamente sulla superficie dei batteri per ucciderli. Pertanto, sfruttando la metodologia della fase solida, abbiamo preparato in modo regioselettivo gli isomeri del costrutto della colistina al fine di indagare se tutti i regioisomeri subiscono la scissione selettiva da parte dell’ elastasi con la stessa efficacia e fedeltà. Quindi, abbiamo eseguito i test antimicrobici contro un ceppo di E. coli K12 e i risultati sono stati confrontati con l'attività della miscela regioisomerica.
Pursuing the search for a new class of structurally simple mimics of antimicrobial peptides, we have optimized a short, cheap and high-yielding synthesis of mono-charged amphiphilic α-hydrazido acid derivatives, having a membranolytic action. They exhibited a broad-spectrum in vitro activity against a variety of Gram-positive and Gram-negative bacteria, including two multidrug-resistant strains. In addition, they showed synergistic effects with tetracycline toward sensitive bacteria, whereas either synergistic effects or indifference were observed for combinations with different first-line antibiotics toward multidrug-resistant strains. Despite the minimal cationic charge, the best compounds demonstrated to be selective toward bacterial cell membranes over mammalian cell membranes. The importance of a non-disrupted amphiphilicity was also demonstrated. With the aim to administer lower doses of colistin reducing the toxic side effects, the Department of Chemical Biology at HZI (Braunschweig, Germany) developed a novel peptide-colistin construct. It consists of a mixture of five regioisomers where each of its free amino groups of colistin was coupled to the C-terminal of a synthetic peptide. The cleavage of the newly introduced amide group by human elastase neutrophils, releases colistin directly on the surface of bacteria in order to kill them. Thus, exploiting solid phase methodology, we regioselectively prepared the isomers of the colistin construct in order to investigate if all the regioisomers undergo selective cleavage by elastase with the same efficacy and fidelity. Then, we carried out the antimicrobial assays against a strain of E. coli K12, and the results were compared with the activity of regioisomeric mixture.

DESIGN AND SYNTHESIS OF HUMAN NEUTROPHIL ELASTASE (HNE) INHIBITORS Dottorato in area del Farmaco e Trattamenti Innovativi PhD student: Antonella Iacovone Scientific tutor: Prof.ssa Maria Paola Giovannoni Theoretical tutor: Prof.ssa Elisabetta Teodori Human Neutrophil Elastase (HNE) is an enzyme belonging to the family of serine proteases. It is a small, soluble and basic glycoprotein of approximately 30 kDa containing 218 amino acid residues that are stabilized by four disulfide bridges. HNE carries out its activity through the catalytic triad, consisting of Ser195, His57 and Asp102. It is involved in the killing of pathogens, in the regulation of inflammation and tissue homeostasis due to its proteolytic action against a variety of extracellular matrix proteins, such as elastin, collagen, fibronectin, laminin, and proteoglycans. In physiological conditions, HNE proteolytic activity is regulated by endogenous inhibitors belonging to the serpins family, including α1-antitripsin (α1-AT), α2-macroglobuline, elafin and secretory leucocyte protease inhibitor (SLPI). Alteration of the balance between HNE and serpins activity can contribute to the develop or the worsening of certain pathologies, especially affecting the respiratory system. The main pulmonary diseases involving HNE are chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), acute respiratory distress syndrome (ARDS) and acute lung injury (ALI). Additionally, HNE is also involved in other inflammatory disorders including psoriasis, dermatitis, atherosclerosis, rheumatoid arthritis and various types of cancer. Many examples of peptide and non-peptide HNE inhibitors have been reported in the literature, however, only two drugs are currently available for clinical use: Prolastin (purified α1-AT), a peptide drug synthetized by recombinant DNA techniques and used for the treatment of α1-antitripsin deficiency (AATD) and Sivelestat (Elaspol®100), a low molecular weight non-peptide molecule. The use of Sivelestat for the treatment of acute lung injury associated with systemic inflammation is approved only in Japan and Korea. The research performed in this period as a PhD student consisted in the design and synthesis of new potential HNE inhibitors. In particular, on one side we focused on the synthesis of compounds with 7-azaindole structure, as isomers of the potent indazoles, previously investigated in our research group. On the other side, we moved our attention on the investigation of new small and flexible compounds with isoxazol-5(2H)-one scaffold; additionally we synthetized some products with benzo[c]isoxazol-3(1H)-one nucleus, as elaboration of the isoxazolone derivatives. All new products were tested as HNE inhibitors in the laboratory of Prof. Quinn, Montana University, and many of these compounds have proved to be very potent inhibitors, recording activity values in the nanomolar range. On selected isoxazolones derivatives molecular modeling studies were performed to clarify the interaction with the target enzyme, as well as studies on stability in aqueous buffer and on kinetic of HNE inhibition. References 1. Hoenderdos, K.; Condliffe, A. The neutrophil in chronic obstructive pulmonary diseases. Am. J. Respir. Cell. Mol. Biol. 2013, 48, 531-539. 2. Cools-Lartigue, J.; Spicer, J.; Najmeh, S.; Ferri, L. Neutrophil extracellular traps in cancer progression. Cell. Mol. Life Sci. 2014, 71, 4179-4194. 3. Pham, C. T. Neutrophil serine proteases: specific regulators of inflammation. Nat. Rev. Immunol. 2006, 6, 541-550. 4. Crocetti, L.; Giovannoni, M. P.; Schepetkin, I. A.; Quinn, M. T.; Khlebnikov, A. I.; Cilibrizzi, A.; Dal Piaz, V.; Graziano, A.; Vergelli, C. Design, synthesis and evaluation of N-benzoylindazole derivatives and analogues as inhibitors of human neutrophil elastase. Bioorg. Med. Chem. 2011, 19, 4460-4472. 5. Crocetti, L.; Schepetkin, I. A.; Cilibrizzi, A.; Graziano, A.; Vergelli, C.; Giomi, D.; Khlebnikov, A. I.; Quinn, M. T.; Giovannoni, M. P. Optimization of N-benzoylindazole derivatives as inhibitors of human neutrophil elastase. J. Med. Chem. 2013, 56, 6259-6272. 6. Giovannoni, M. P.; Schepetkin, I. A.; Crocetti, L.; Ciciani, G.; Cilibrizzi, A.; Guerrini, G.; Khelebnikov, A. I.; Quinn, M. I.; Vergelli, C. Cinnoline derivatives as human neutrophil elastase inhibitors. J. Enz. Inhib. Med. Chem. 2016, 31(4), 628-639. 7. Crocetti, L.; Schepetkin, I. A.; Ciciani, G.; Giovannoni, M. P.; Guerrini, G.; Iacovone, A.; Khlebnikov, A. I.; Kirpotina, L. N.; Quinn, M. T. Synthesis and farmacological evaluation of indole derivative as deaza analogues of potent human neutrophil elastase inhibitors. Drug Dev. Res. 2016, 77(6), 285-289. 8. Vergelli, C.; Schepetkin, I. A.; Crocetti, L.; Iacovone, A.; Giovannoni, M. P.; Guerrini, G.; Khlebnikov, A.I.; Ciattini, S.; Ciciani, G.; Quinn, M. T. Isoxazol-5(2H)-one: a new scaffold for potent human neutrophil elastase (HNE) inhibitors. J. Enz. Inhib. Med. Chem. 2017, 32(1), 821-831. SOLID PHASE PEPTIDE SYNTHESIS OF SMALL PEPTIDE-BASED METAL CHELATORS Supervisor: Professor Robert Hider During the last year of PhD, a visiting period at King’s College of London was spent. The host research group of Professor Robert Hider is involved for many years in the design and synthesis of Iron and Gallium chelators. In particular, as regard the Iron chelators they follow two main project. The first one concerns the synthesis of small molecules used for the treatment of the pathologies characterized by an iron overload including hemochromatosis and transfusion-dependent thalassemia; the second one concerns the synthesis of peptide as novel biosensors for the mitochondrial Labile Iron Pool (LIP) in order to better understand the role of the LIP in mitochondrial oxidative disorders. About the gallium chelators, peptide molecules have recently been developed as radiopharmaceuticals for PET imaging in particular to detect and monitor prostate cancer. In this context, the entrusted project focused on the solid-phase synthesis of tripeptide (Lys-Lys-Lys) and tetrapeptide (Lys-Lys-Lys-Lys) derivatives bearing 3,4-dihydroxy picolinic acid and the 3-(3-hydroxy-2-methyl-4-oxopyridin-1(4H)-yl)propionic acid (as chelating groups) on the amino groups of the side chains. All final compounds will be tested in the near future as iron and gallium chelators for biological and clinical applications. References 1. Hider R. C.; Zhou T. The design of orally active iron chelators. Ann N. Y. Acad. Sci. 2005, 1054, 141-154. 2. Abbate, V.; Reelfs, O.; Hider, R. C.; Pourzand, C. Design of novel fluorescent mitochondria-targeted peptides with iron selective sensing activity. Biochem. J. 2015, 469 (3), 357-366. 3. Cusnir, R.; Imberti, C.; Hider, R. C.; Blower, P. J.; Ma, M.T. Hydroxypyridinone chelators: from iron scavenging to radiopharmaceuticals for PET imaging with Gallium-68. J. Mol. Sci. 2017, 18, 116.

Tese de mestrado, Química Farmacêutica e Terapêutica, Universidade de Lisboa, Faculdade de Farmácia, 2019
A elastase neutrófila humana (HNE) é uma protease que se caracteriza pela presença de um resíduo de serina no sitio ativo da enzima. Esta enzima está envolvida na degradação das proteínas da matriz, desempenhando um papel importante na modulação da inflamação. Assim, o excesso de HNE pode desencadear várias condições patológicas, tais como a psoríase. Substâncias ativas podem ser administradas através da pele (administração tópica) utilizando veículos adequados tais como, emulsões e microemulsões. Tendo em conta estudos anteriores elaboradores pelo nosso grupo de investigação, neste trabalho foram sintetizados compostos diferentes, que apresentam na sua estrutura uma 4-oxo-β-lactama, que favorece a atividade contra a HNE. A caracterização total dos compostos foi elaborada através de RMN, IV, ponto de fusão, espectrometria de massa e análise elementar. Para facilitar a localização e quantificação do composto durante os ensaios de libertação de fármacos, um fluoróforo foi sintetizado e acoplado nos compostos que apresentaram a maior viabilidade celular (18 e 22) originando dois novos compostos 27 e 28. Estes dois compostos também foram caracterizados por RMN, IV, ponto de fusão, espectrometria de massa, análise elementar. Através da análise da caracterização realizada para todos os compostos, é possível verificar estes compostos foram obtidos. Posteriormente, foram realizados ensaios in vitro de citotoxicidade, atividade inibitória e seletividade para a HNE, de forma a determinar a viabilidade celular, atividade e a seletividade de cada composto para esta enzima. Os compostos 27 e 28, demonstram atividades e seletividades semelhantes para a HNE, porém o composto 27 apresentou uma viabilidade celular de 91.6%, sendo o escolhido para ser incorporado nas formulações. Deste modo, os resultados obtidos in vitro, demonstraram que é fundamental que os inibidores contenham uma 4-oxo-β-lactama e que através da introdução de alguns substituintes nesta estrutura foi possível manter a atividade e melhorar a viabilidade celular. Sistemas de administração tópica (emulsões óleo-em-água (O/A) e microemulsões) foram desenvolvidos, otimizados e caracterizados. Foram estudados diferentes humectantes e conservantes bem como a influência de diferentes concentrações de álcool cetílico nas propriedades finais das emulsões O/A. Óleos contendo diferentes cadeias de carbono foram incorporados nas microemulsões para mimetizar a camada lipídica da pele. Todas as formulações foram caracterizadas (características organoléticas, pH e reologia). Distribuição do tamanho das gotículas, estudos de eficácia biológica in vivo (hidratação, TEWL e avaliação da hidratação) e análise sensorial foram realizadas nas formulações finais. Todas as formulações apresentaram propriedades físico-químicas tópicas adequadas. Em relação à avaliação biológica in vivo, não se verificaram diferenças significativas entre as formulações e a área de controlo. A partir da análise dos dados obtidos do estudo sensorial, os voluntários preferiram a emulsão O/A à microemulsão, uma vez que esta formulação apresentava fácil aplicação, baixa oleosidade e brilho. Por fim, o composto 27 foi incorporado nas duas formulações: AAN-27 E (emulsão) e AAN-27 ME (microemulsão). Foram realizados estudos de caracterização físico-química (características organoléticas, pH, reologia, distribuição do tamanho de gotículas), libertação de fármacos in vitro e atividade antipsoriática in vivo para avaliar as propriedades e a eficácia de ambas as formulações. Para concluir, os estudos de libertação de fármacos in vitro demonstraram que a quantidade de composto libertada é depende totalmente do tipo de formulação, onde a AAN-27 E apresenta uma maior quantidade de libertação de fármaco, quando comparado à AAN-27 ME. Através do estudo da atividade antipsoriática in vivo verificou-se que as microemulsões são as menos indicadas para aplicação na pele e a emulsão pode ser considerada uma formulação eficaz, porém não existem diferenças significativas entre o placebo e a formulação AAN-27 E.
Human neutrophil elastase (HNE) is a serine protease characterized by a serine residue in the active site. This enzyme is involved in the degradation of matrix proteins playing an important role in inflammation modulation. Hereupon, the excess of HNE may trigger several pathological conditions, such as psoriasis. Active substances may be administered through the skin (topical delivery), allowing the treatment of skin diseases, using suitable vehicles such as emulsions and microemulsions. In this work different compounds containing a 4-oxo-β-lactams scaffold, that could act against HNE were synthesized, taking into account the preceding studies carried out by our group. The total characterization through NMR, FTIR, melting point, mass spectrometry and elemental analysis, was made for all. To facilitate the localization and quantification of compounds during the drug release studies, a fluorophore was synthesized and coupled to the compounds with highest cell viability (18 and 22) originating two new compounds, 27 and 28. These new compounds were also characterized through NMR, FTIR, melting point, mass spectrometry and elemental analysis. Through the analysis of all characterization performed for the compounds, it could be noted that all compounds were successfully synthesized. In vitro cytotoxicity and serine proteases assays were performed to determine the activity of each compound regarding cell viability and enzyme selectivity. Compounds 27 and 28 showed similar activities and selectivity against HNE, however compound 27 presented a cell viability of 91.6%, being chosen to be incorporated into the topical delivery systems. Therefore, the results showed that the 4-oxo-β-lactams scaffold is fundamental to the development of HNE inhibitors and some substituents in this scaffold could maintain the activity and improve the cell viability. Topical delivery systems (oil-in-water (O/W) emulsions and microemulsions) were developed, optimized and characterized. Different humectants, preservatives were studied as well as the influence of different concentration of cetyl alcohol in the final properties of the O/W emulsions. Oils containing different carbon chains were incorporated into microemulsions to mimic the skin lipid layer. All formulations were characterized through organoleptic characteristics, pH and rheology. Droplet size distribution, in vivo biological efficacy (hidratation, TEWL and moisture evaluation) and sensorial analysis were performed on final formulations. All formulations present suitable topical physicochemical properties. There were no significant differences among the formulations and with the control area concerning the in vivo biological evaluation. From the sensory data analysis, volunteers preferred the O/W emulsion to microemulsion since this formulation presented an easy application, low oiliness and low shine. Finally, compound 27 was incorporated in the two formulations: AAN-27 E (emulsion) and AAN-27 ME (microemulsion). Physicochemical characterization (organoleptic characteristics, pH, rheology, droplet size distribution), in vitro drug release and in vivo antipsoriatic activity studies were performed to evaluate their properties and efficacy. The in vitro drug release studies showed that the amount of AAN-27 released is totally dependent on the type of formulation, where the AAN-27 E releases a higher amount of drug, when compared with AAN-27 ME. The in vivo antipsoriatic activity showed that microemulsions are the less indicated for skin application and emulsion can be considered an effective formulation, but it should be noted that there are no significant differences between the placebo and AAN-27 E formulation.

Human Neutrophil Elastase (HNE) is a serine protease responsible for cleavage of peptide bonds conferring elasticity to the connecting tissues. For this reason, this enzyme is mainly found in the lungs, arteries and ligaments [1-2]. In case of over-expression, HNE enables the appearance of some diseases, such as Chronic Obstructive Pulmonary Disease (COPD), Rheumatoid Arthritis, Psoriasis and Arteriosclerosis [3-4]. Currently, diseases affecting the respiratory tract are one of the major causes of death in the world, so HNE is a potential drug target of considerable interest [4]. Porcine Pancreatic Elastase (PPE) is commonly used as a model for HNE, sharing 37% of amino acid sequence identity [5]. According to previous studies, the catalytic serine performs a nucleophilic attack on a carbonyl group present in the inhibitors [6]. The focus of this work was the three-dimensional structure determination of elastases (PPE and HNE) in complex with inhibitors by X-ray crystallography to characterize their interactions at atomic level. The rational is to correlate structure and function and contribute to the design of more potent and specific inhibitors. These newly synthetic compounds were provided by the group of Prof. Rui Moreira, Instituto de Investigação do Medicamento, Faculdade de Farmácia, Universidade de Lisboa. X-ray diffraction data of PPE crystals were collected at a synchrotron source and three 3D-structures of PPE in complex with inhibitors were determined at resolutions around 1.4 Ǻ. Analysis of the electron density maps revealed that the nucleophilic attack occurred at the sulfonyl group of the inhibitors, contrary to what was initially expected (which would be in the carbonyl group). In silico energy minimization studies of the docked ligand structure into the active site of HNE, show no relevant structural modifications of the protein structure upon ligand binding. Finally, crystals of HNE have already been obtained and experiments are ongoing to grow complexes of HNE with various inhibitors. References: [1] A. Thomson and S. B. Kapadia, “The specificity of the S1 and S2 subsites of elastase,” Eur. J. Biochem., vol. 102, pp. 111–116, 1979. [2] Z. Werb, M. J. Banda, J. H. McKerrow, and R. A. Sandhaus, “Elastases and elastin degradation.,” J. Invest. Dermatol., vol. 79 Suppl 1, p. 154s–159s, Jul. 1982. [3] E. F. P. Ruivo, L. M. Gonåalves, L. A. R. Carvalho, R. C. Guedes, S. Hofbauer, J. A. Brito, M. Archer, R. Moreira, and S. D. Lucas, “Clickable 4-Oxo- b -lactam-Based Selective Probing for Human Neutrophil Elastase Related Proteomes,” pp. 1–7, 2016. [4] L. R. P. Areias, E. F. P. Ruivo, M. T. Duarte, R. Moreira, S. D. Lucas, and R. C. Guedes, “RSC Advances PAPER A uni fi ed approach toward the rational design of selective low nanomolar human neutrophil elastase,” pp. 51717–51721, 2015. [5] “Uniprot.” [Online]. Available: http://www.uniprot.org/. [Accessed: 16-Aug-2017]. [6] W. Huang, Y. Yamamoto, Y. Li, D. Dou, K. R. Alliston, R. P. Hanzlik, T. D. Williams, and W. C. Groutas, “X-ray Snapshot of the Mechanism of Inactivation of Human Neutrophil Elastase by 1,2,5-Thiadiazolidin-3-one 1,1-Dioxide Derivatives.,” Society, pp. 2003–2008, 2008.
Elastase Neutrófila Humana (HNE) é uma protease de serina responsável pela clivagem das ligações peptídicas que conferem elasticidade aos tecidos de conexão. Por esta razão, esta enzima é encontrada principalmente nos pulmões, artérias e ligamentos [1-2]. Em casos de sobre-expressão, esta permite o aparecimento de algumas doenças, como Doença Pulmonar Obstrutiva Crónica (DPOC), Artrite Reumatóide, Psoríase e Arteriosclerose [3-4]. Atualmente, as doenças que afetam o trato respiratório são uma das principais causas de morte no mundo, sendo então a HNE um potencial alvo terapêutico de considerável interesse [4]. A Elastase Pancreática Suína (PPE) é normalmente usada como modelo para HNE, compartilhando 37% de identidade de sequência primária [5]. De acordo com estudos anteriores, a serina catalítica realiza um ataque nucleofílico ao grupo carbonilo presente nos inibidores [6]. O foco deste trabalho foi a determinação por cristalografia de raios-X da estrutura tridimensional de elastases (HNE e PPE) complexadas com inibidores, de modo a caracterizar as respetivas interações a nível atómico. O racional é correlacionar a estrutura com a função e contribuir para o desenho de inibidores mais fortes e mais específicos. Estes novos compostos sintéticos foram fornecidos pelo grupo do Prof. Rui Moreira, Instituto de Investigação do Medicamento, Faculdade de Farmácia, Universidade de Lisboa. Os dados de difração de raios-X dos cristais de PPE foram recolhidos numa fonte de sincrotrão e três estruturas 3D de três complexos da PPE com inibidores foram determinadas com resoluções em torno dos 1,4 Å. A análise dos mapas de densidade eletrónica revelaram que o ataque nucleofílico ocorreu no grupo sulfonilo dos inibidores ao contrário do que era inicialmente esperado (que seria no grupo carbonilo). A minimização de energia in silico da estrutura do ligando acoplado no centro ativo da HNE não mostra modificações relevantes na estrutura da proteína após a ligação do ligando. Finalmente, já foram obtidos cristais de HNE, estando já em curso experiencias para o crescimento de cristais de complexos de HNE com vários inibidores. Referências: [1] A. Thomson and S. B. Kapadia, “The specificity of the S1 and S2 subsites of elastase,” Eur. J. Biochem., vol. 102, pp. 111–116, 1979. [2] Z. Werb, M. J. Banda, J. H. McKerrow, and R. A. Sandhaus, “Elastases and elastin degradation.,” J. Invest. Dermatol., vol. 79 Suppl 1, p. 154s–159s, Jul. 1982. [3] E. F. P. Ruivo, L. M. Gonåalves, L. A. R. Carvalho, R. C. Guedes, S. Hofbauer, J. A. Brito, M. Archer, R. Moreira, and S. D. Lucas, “Clickable 4-Oxo- b -lactam-Based Selective Probing for Human Neutrophil Elastase Related Proteomes,” pp. 1–7, 2016. [4] L. R. P. Areias, E. F. P. Ruivo, M. T. Duarte, R. Moreira, S. D. Lucas, and R. C. Guedes, “RSC Advances PAPER A uni fi ed approach toward the rational design of selective low nanomolar human neutrophil elastase,” pp. 51717–51721, 2015. [5] “Uniprot.” [Online]. Available: http://www.uniprot.org/. [Accessed: 16-Aug-2017]. [6] W. Huang, Y. Yamamoto, Y. Li, D. Dou, K. R. Alliston, R. P. Hanzlik, T. D. Williams, and W. C. Groutas, “X-ray Snapshot of the Mechanism of Inactivation of Human Neutrophil Elastase by 1,2,5-Thiadiazolidin-3-one 1,1-Dioxide Derivatives.,” Society, pp. 2003–2008, 2008.

Il nostro gruppo di ricerca lavora da diversi anni nel campo degli inibitori dell'elastasi dei neutrofili umani (HNE), e in questo periodo è stata progettata e sintetizzata un'ampia varietà di strutture bi-eterocicliche azotate, come indazoli, indoli, cinnoline e 7-azaindoli; inoltre, abbiamo anche studiato strutture mono-eterocicliche azotate, come isossazoloni e tiazoloni, ottenendo interessanti risultati. Il primo progetto di questa tesi di dottorato è stata divisa in due sezioni: nella prima parte sono stati progettati nuovi HNE inibitori come ulteriore modifica di indazoli e 7-azaindoli, sintetizzati in precedenza, con l'obiettivo di studiare l'impatto che, sia lo spostamento dell'azoto nell'anello piridinico, sia il numero totale degli azoti nelle molecole, possono avere sull'attività biologica. Quindi abbiamo esplorato inibitori 4-, 5- e 6-azaindolici, come risultato dello spostamento dell'azoto della piridina nel 7-azaindolo, inibitori a struttura 5H-pirrolo[2,3-b]pirazinica, inserendo un atomo di azoto in posizione 4 del nucleo 7-azaindolico, e 5- e 7-azaindazoli (1H-pirazolo[3,4-b]piridina e 1H-pirazolo[4,3-c]piridina) come aza-analoghi dei potenti indazoli. Nella seconda parte di questo primo progetto abbiamo studiato tre nuovi scaffold: 1,5,6,7-tetraidro-4H-indazol-4-one, 5,6-diidrociclopenta[c]pirazolo-4(1H)-one e 5,6,7,8-tetraidrocyclohepta[c]pirazol-4(1H)-one come inibitori di HNE. Inoltre, il nostro gruppo di ricerca ha anche sintetizzato per lungo tempo agonisti dei recettori dei peptidi formilati (FPRs) con scaffold piridazinonico, ottenendo risultati interessanti per attività e selettività; d'altra parte, molti derivati del piridazinone sono noti per esibire un effetto anti-infiammatorio, come ampiamente documentato in letteratura, interagendo con diversi target. Quindi, tenendo conto della nostra esperienza sugli agonisti FPR e delle preziose informazioni riportate in letteratura, nel secondo progetto di questa tesi di dottorato abbiamo selezionato una piccola libreria di agonisti FPR che sono stati ulteriormente studiati per capire se possono presentare un'attività anti-infiammatoria alternativa, che non è correlata alla loro attività FPR. Our research group has been working for several years in the field of human neutrophil elastase (HNE) inhibitors, and over this period a wide variety of nitrogen bi-heterocycle scaffolds have been designed and synthesized, such as indazoles, indoles, cinnolines and 7-azaindoles; moreover, we also investigated nitrogen mono-heterocyclic scaffolds such as isoxazolones and thiazolones obtaining interesting results. The first project of this PhD thesis has been divided in two sections: in the first part new HNE inhibitors have been designed as further modification of both indazoles and 7-azaindoles, synthetized previously, with the goal to investigate the impact that either the shift of the nitrogen in the pyridine ring as well as the total number of nitrogens in the molecules can have on the biological activity. Hence, we explored 4-, 5- and 6-azaindoles as the result of the 7-azaindole pyridine nitrogen shift, 5H-pyrrolo[2,3-b]pyrazines by inserting a nitrogen atom at position 4 of the 7-azaindole nucleus, and 5- and 7-azaindazoles (1H-pyrazolo[3,4-b]pyridine and 1H-pyrazolo[4,3-c]pyridine) as aza-analogues of the potent indazoles. As second part of this first project, we have investigated three new scaffolds: 1,5,6,7-tetrahydro-4H-indazol-4-one, 5,6-dihydrocyclopenta[c]pyrazol-4(1H)-one and 5,6,7,8-tetrahydrocyclohepta[c]pyrazol-4(1H)-one as HNE inhibitors. Furthermore, our research group has also been synthesizing for a long time formyl peptide receptors (FPRs) agonist with pyridazinone scaffold, obtaining interesting results for activity and selectivity; on the other hand, many pyridazinone derivatives are known to exhibit an anti-inflammatory effect, as widely documented in the literature, by interacting with different targets. Thus, taking into account our experience on FPR agonists and the valuable information reported in the literature, in the second project of this PhD thesis we have selected a small library of FPR agonists that were further investigated to understand wether they may present an alternative anti-inflammatory activity, which is not correlated to their FPR activity.

Dissertação de mestrado em Bioengenharia
Human neutrophil elastase, as inflammatory response, has the capacity to degrade collagen and elastin component of extracellular matrix, being extensively involved in wrinkles formation induced by UV radiation damage. In order to develop a cosmetic antiwrinkling emulsion, we incorporated a potent synthetic HNE inhibitor, sivelestat (IC50 = 44 nM, Ki = 0.2 μM), into biopolymeric nanoparticles prepared by high-energy emulsification methods. As nanomaterial was used Bombyx mori silk fibroin protein, an FDA approved natural biocompatible and biodegradable polymer. Silk fibroin nanoparticles (SF-NPs) were produced by two high-energy emulsification methods, ultrasonication and high pressure homogenization (HPH), in oil-in-water (o/w) emulsions using as n-dodecane as oil phase. According to optimized results, best formulations were obtained for 10 g/L of SF at 20/80 of o/w ratio at 22 cycles of homogenization by double-stage HPH (APV-2000™). During the NPs production by HPH emulsification process, the secondary SF structure changed from random-coil conformation to a more stable structure, β -sheets. Stabilizers effect was also studied, namely poloxamer 407, transcutol, tween 80 and sodium dodecyl sulfate (SDS), in which the results suggested that combination of transcutol and SDS would effectively stabilize the SF-NPs over time. To predict the sivelestat incorporation, orange IV was used as a model-drug, being this incorporated into SF-NPs at different concentrations. Release studies of orange IV were evaluated in absence and presence of 0.5 U/mL of HNE. The release of orange IV at 100 μM from SF-NPs (concentration considered as optimal) showed to be controlled at some extent, in which the drug transport mechanism showed to be anomalous (Fickian diffusion and polymer degradation/relaxation) and not affected by 0.5 U/mL of HNE enzyme. Finally, the incorporation of 100 μM of sivelestat in SF-NPs performed with a formation and encapsulation efficiency of 99% and 50%, respectively, successfully inhibited the HNE enzyme, enhancing the potential of this drug delivery system for topical antiwrinkling application.
Elastase de Neutrófilos Humana, como resposta inflamatória, possui a capacidade de degradar os componentes da matriz extracelular, nomeadamente colagénio e elastina, estando envolvida na formação de rugas causadas pela radiação ultravioleta. De forma a desenvolver uma emulsão cosmética antirrugas, decidiu-se incorporar um potente inibidor sintético de elastase, sivelestat (IC50 = 44 nM, Ki = 0,2 μM), em nanopartículas biopoliméricas formadas por métodos de emulsificação de elevada energia. Como nanomaterial usou-se a proteína fibroína de seda extraída do bicho-da-seda Bombyx mori, que é um polímero natural biocompatível e biodegradável aprovado pela FDA. As nanopartículas de fibroína de seda foram produzidas por dois métodos de emulsificação, ultrasonicação e homogeneização de elevada pressão, com emulsões óleo-em-água, tendo-se usado n-dodecano como fase oleosa. De acordo com os resultados otimizados, a melhor formulação foi obtida com 10 g/L de fibroína de seda numa razão óleo/água de 20/80 a 22 ciclos de homogeneização, através do método de duplo-sistema de homogeneização de elevada pressão (APV-2000™). Nestas condições, durante a produção de nanopartículas nestas condições, a estrutura secundária aleatória da proteína de fibroína de seda sofreu uma alteração conformacional para uma estrutura mais estável, em folhas βeta. O efeito de estabilizadores na nanoemulsão foi também estudado, nomeadamente poloxamer 407, transcutol, tween 80 e dodecil sulfato de sódio, em que os resultados obtidos sugerem que a combinação de transcutol e dodecil sulfato de sódio estabilizaria eficientemente as nanopartículas de fibroína de seda ao longo do tempo. Para prever a incorporação de sivelestat, o corante orange IV foi utilizado como composto modelo, tendo sido incorporado nas nanopartículas de fibroína de seda a várias concentrações, e tendo-se estudado a sua libertação na ausência e na presença de elastase (0,5 U/mL). A libertação de orange IV das nanopartículas de fibroína de seda, numa concentração de 100 μM (concentração otimizada), exibiu um mecanismo de transporte controlado, tendo este sido definido como anómalo (difusão de Fickian e degradação/relaxamento do polímero) e não mostrou ser influenciada pela elastase (0,5 U/mL). Finalmente, incorporação de 100 μM de sivelestat nas nanopartículas de fibroína de seda que indicou uma eficiência de formação e de encapsulação de 99% e 50%, respetivamente, inibiu a enzima elastase de forma eficaz, evidenciando o potencial deste nanosistema como cosmético antirrugas para aplicação transdérmica.

碩士
長庚大學
中醫學系天然藥物
106
Acute lung injury (ALI) is clinically characterized as a respiratory failure syndrome. Previous studies have shown that the neutrophils infiltration in lung tissue is one of the pathologic features. It has been reported that human neutrophil elastase (HNE) is highly correlated to the severity of ALI. As a result, the inhibitor of HNE has become an important study interest in ALI. In our previous study, we examined the serine protease activity in 75 traditional Chinese medicines in which we found Prunella vulgaris (PV) an ideal candidate that processes the characteristic of HNE inhibition (IC50, 8.08 ± 1.28 g/ml) .This study focus on isolating the bioactive fraction that significantly inhibit HNE in Prunella vulgaris via the bioactivity-guided fractionation. Its qualitative and quantitative characteristics are also investigated. Our results indicate that PVAP, the acidic insoluble bioactive fraction with the molecular weight is between 100 kDa and 300 kDa extracted from Prunella vulgaris, shows the ability in selective inhibition of HNE (IC50, 2.42 ± 0.19 g/ml). PVAP consists of carbohydrate, protein and uronic acid of 11.94 %, 17.07 % and 15.61 % respectively. In addition, the monosaccharides composition analysis reveals that PVAP contains mannose、rhamnose、galacturonic acid、glucose、xylose、arabinose in molar ratio of 1.000：0.999：1.592：1.605：2.049：0.742：1.040, respectively. In our animal experiments, the results show that PVAP protect mice from lipopolysaccharide-induced ALI by reducing pulmonary edema, myeloperoxidase and elastase activity, neutrophil infiltration, pro-inflammatory cytokines, and neutrophil extracellular traps formation. These results suggested that PVAP alleviate ALI from LPS as a HNE inhibitor. It also demonstrates its potential in pharmaceutical development in prevention of ALI.

Tese de Doutoramento em Engenharia Química e Biológica
The inflammatory response is an important step in wound healing, however if there is an imbalance between the immune response and tissue regeneration, a delay on the healing process will occur, resulting in a chronic wound. Chronic wounds are a significant problem for health care system, accounting for almost 4% of its total budget, and currently increasing. Early detection of incipient wound infection and chronic inflammation reduces the severity of the disease and decreases health care expenses. Currently, it is difficult, expensive and time consuming to accurately assess a wound status, hence, there is an urgent need for a new diagnostic that would give the medical staff a fast and reliable method to determine inflammation/infection of a chronic wound at an early stage. Human neutrophil elastase (HNE) is one of the most abundant proteinases in wounds and its activity was found to be elevated in case of infection and chronic inflammation, making this enzyme a suitable marker for impaired healing. This PhD thesis intends to explore new strategies for the detection of HNE through the development of chromogenic and fluorogenic sensors that when embedded in a dressing may provide an in situ and real-time assessment of the wound status. For that, a specific cleavage sequence for HNE was designed by molecular docking studies and then confirmed in vitro. Then, this substrate was associated to chromogenic or fluorogenic detection systems. Regarding the chromogenic detection systems, two strategies were followed using ultramarine (UM), a GFP-like chromoprotein. The rationale of this approach was to use this protein’s ability to regenerate its dark blue colour (hysteresis) upon HNE cleavage, turning it into a switch-on sensor, or to lose its colour upon proteolysis, thus making a switch-off sensor. In a first attempt to convert ultramarine into a HNE substrate switch was based on a split protein strategy which would signal as a gain of colour sign. We were able to detect an absorbance increase upon HNE cleavage, but this effect was not long lasting due to an instability of the interaction between the signalling and sensing fragments. Indeed, this strategy proved it to be extremely challenging. The main motivation to pursue this work resided in its innovative aspect, as GFP-like chromogenic sensors were never reported. Then, a second strategy was investigated through sitedirected mutagenesis of ultramarine. In this latter strategy we were able to obtain sensors with an opposite signal, switch-off. To provide a better understanding regarding UM’s chromogenic environment, molecular dynamics studies coupled to protein expression were implemented to each of the seven produced UM-mutants. Our findings provided new information regarding the effect of certain mutations in UM protein conformation and allowed us to explore its application as a switch sensor. Here, we explored for the first time the development of a colour switch-on sensor based on chromogenic GFP-like proteins. Even though, we were unable to fully accomplish the regeneration of a stable chromophore environment for colour appearance, this work may serve as a starting point for advances in switch-on chromogenic sensors using non-fluorescent GFP-like proteins. For the fluorogenic detection system, the strategy studied resulted in the successful development of a FRET (Förster resonance energy transfer) peptide to monitor HNE. Upon HNE action, this peptide proved to be more effective than the traditional peptides coupled to fluorophores. Its visual detection was possible using an UV-portable light source. The embedding of this sensor in dressings would provide advantages for its application as a wearable sensor. The methods here used for the FRET-peptide immobilization onto a dressing compromised its detection. However, we envision that with the proper method of integration into a dressing, this FRET peptide could be used as a new strategy of smart materials. The strategies explored in this thesis can be further investigated and possibly implemented as point-of-care medical devices for wound control. As wound care tools, these devices would allow a prompt detection of chronic wounds, contributing to their proper and effective care and, consequently, granting a better outcome for the millions of people that suffer from non-healing wounds.
A resposta inflamatória é um importante passo no processo de cicatrização de feridas. No entanto, desequilíbrios entre a resposta do sistema imunitário e a regeneração celular podem desencadear um atraso significativo no processo de cicatrização, resultando numa ferida crónica. As feridas crónicas apresentam um problema significativo para o sistema de saúde, contabilizando 4% do orçamento total de saúde, sendo que se estima um aumento destes números nas próximas décadas. A deteção antecipada de infeção e inflamação crónica numa ferida reduz a severidade da doença, consequentemente diminuindo o seu impacto nas despesas de saúde. Atualmente, a avaliação do estado de uma ferida de forma exata e precisa é um processo difícil, dispendioso e demoroso. Por esta razão, existe uma necessidade urgente de desenvolvimento de novas ferramentas de diagnóstico que possibilitem a deteção precoce de infeção/inflamação em feridas crónicas de forma rápida e eficaz. A elastase neutrofílica humana (HNE) é uma das proteinases mais abundantes em feridas, estando os seus elevados níveis de atividade em feridas correlacionados com a ocorrência de infeção e inflamação crónica. De facto, esta enzima é considerada um excelente biomarcador para feridas cujo processo de cicatrização se encontra comprometido. O objetivo principal desta tese de doutoramento foi e exploração de novas estratégias para a deteção da HNE através do desenvolvimento de sensores cromogénicos e fluorogénicos que possam ser incorporados em pensos para feridas, possibilitando uma avaliação in situ e em temporeal do estado da ferida. Para tal, primeiramente desenharam-se sequências de clivagem específicas para HNE cuja afinidade para a enzima foi analisada através de estudos de acoplamento molecular e posteriormente foram confirmados com ensaios in vitro. Depois, o substrato foi associado a sistemas de deteção cromogénico ou fluorogénico. Relativamente aos sistemas de deteção cromogénicos, desenvolveram-se duas estratégias usando a ultramarina (UM), uma cromoproteína do tipo GFP, como substrato sinalizador. A competência desta proteína em regenerar a sua cor azul (devido à capacidade de histerese) permitiu transformá-la num sensor que se liga (switch-on) ou que se desliga (switch-off) após a sua clivagem com a HNE. A primeira tentativa para converter a proteína ultramarina num substrato comutador de sinal para a HNE consistiu na produção de uma proteína segmentada em duas partes que quando em interação resultam num sinal com ganho de cor (sensor switch-on). Após clivagem com HNE do fragmento substrato, conseguiu-se obter um aumento da absorvância, embora transiente, devido à interação com o fragmento sinalizador. De facto, esta estratégia foi muito desafiante. No entanto, a principal motivação em seguir esta linha de trabalho prendeu-se principalmente com o seu carácter inovador, visto que nunca tinham sido reportados sensores usando proteínas cromogénicas do tipo GFP. Neste seguimento, explorou-se uma segunda estratégia para o desenvolvimento de substratos sensores switch-on para a HNE através da mutagénese direcionada da proteína ultramarina. Esta abordagem permitiu também obter sensores com o sinal oposto – switch-off. Com o objetivo de melhor compreender as condições que proporcionam o ambiente cromogénico da proteína UM, foram implementados estudos de simulação de dinâmica molecular complementares à expressão das proteínas para cada um dos sete mutantes produzidos. A partir deste trabalho conseguiu-se recolher novos dados acerca do efeito de certas mutações na conformação da proteína UM e possibilitou-nos também explorar a sua aplicação como sensor comutador de sinal. Nestes trabalhos investigou-se pela primeira vez o desenvolvimento de sensores cromogénicos switch-on baseados em proteínas do tipo GFP. Embora não se tenha conseguido obter a regeneração estável do cromóforo que permite o aparecimento de cor, estes resultados poderão servir como ponto de partida para outros trabalhos em sensores cromogénicos switch-on usando proteínas não fluorescentes do tipo GFP. Em relação aos sistemas de deteção fluorogénicos, a abordagem explorada resultou no desenvolvimento bem-sucedido de um péptido FRET para monitorização de HNE. Após proteólise, este péptido provou ser mais eficaz do que os tradicionais péptidos fluorescentes (acoplados a fluoróforos). A deteção visual da fluorescência foi possível recorrendo a uma luz UV portátil. A incorporação deste sensor em pensos de feridas trará vantagens relativamente à sua aplicação como sensores wearable. Os métodos aqui descritos para a imobilização do péptido FRET em têxteis usados como material de penso comprometeram a deteção do seu sinal. No entanto, prevêse que a utilização de um método de integração mais apropriado possa permitir a sua aplicação em novas estratégias no desenvolvimento de tecidos inteligentes. A implementação da investigação aqui descrita em dispositivos médicos para a monitorização do processo de cicatrização de feridas poderá complementar o leque de ferramentas usadas no cuidado das feridas. Estes dispositivos possibilitarão a deteção atempada da cronicidade de uma ferida, contribuindo para o seu cuidado apropriado e eficaz. Consequentemente irá garantir uma melhor qualidade de vida para milhões de doentes que sofrem de feridas de difícil cicatrização.
Fundação para a Ciência e Tecnologia (FCT) e à Tecminho pelo financiamento da minha bolsa de doutoramento (SFRH/BD/113247/2015). Ao projeto da comissão europeia INFACT (FP7-NMP-2013-SME-7 - Grant agreement no. 604278)

碩士
長庚大學
中醫學系天然藥物
100
In chronic inflammation, neutrophil elastase is a major secreted product of stimulated neutrophils and a major contributor to the destruction of tissue in chronic inflammatory disease. Therefore, NE may prove a potential target in the development of new anti-inflammatory agents. In previous study, we synthesis a series anthranilate analogs and evaluate their activities on modulating neutrophil function. The results showed anthranilate analogs exhibiting high selectively inhibitory effects on NE release induced by formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) in human neutrophil. In attempt to generate more potency anti-inflammatory agents. 30 anthranilate analogs were synthesized and evaluated on to their inhibitory effects on superoxide anion generation and NE release in FMLP-activated human neutrophils. Herein, we describe the synthesis, bioactive data, and structure–activity relationship (SAR) of the anti-inflammatory effects related to anthranilate analogs. Among them, compounds WWH d1, d2, j1 and j2 have most inhibitory effect on neutrophil elastase with IC50 value of 0.09, 0.04, 0.05 and 0.05 μM, respectively. The results of SARs to anthranilate analogs revealed sulfonamide group is favorable for pharmaceutical activity. Likewise, modification of aniline at ortho-substituent demonstrated that ketone or the groups that contain hydroxyl are the most active.

博士
臺北醫學大學
醫學科學研究所
97
Neutrophils are recruited to the airways and play a role in the pathogenesis of chronic airway diseases, such as COPD, cystic fibrosis and more severe form of asthma. Elastase released by neutrophils is implicated in such chronic conditions. ASM has long been regarded as having mainly contractile properties in response to many proinflammatory mediators and neurotransmitters. Studies now show that ASM is also a source of inflammatory mediators through its synthetic functions, through which may regulate the development and progression of airway inflammation. NF-κB repressing factor (NRF), a nuclear inhibitor of NF-κB, is constitutively expressed and is implicated in the basal silencing of specific NF-κB targeting genes, including IFN-β, IL-8/CXCL8, and iNOS. Little is known about the regulation of itself and its role in response to stimuli. We have previously reported that neutrophil elastase (NE) activates NF-κB in primary human ASM (hASM), leading to induction of TGF-β1. It is describe here that, instead of inducing the NF-κB response gene IL-8/CXCL8, NE suppressed IL-8/CXCL8 release and mRNA expression in hASM cells. Transcriptional blockade studies using Actinomycin D revealed a similar degradation rate of IL-8/CXCL8 mRNA in the presence or absence of NE, suggesting an involvement at the transcription level. Mechanistically, the NE repressive effect was mediated by inducing NRF, as shown by RT-PCR and Western blotting, which was subsequently recruited to the native IL-8/CXCL8 promoter leading to removal of RNA polymerase II (RNA Pol 2) from the promoter, as demonstrated by chromatin-immunoprecipitation (ChIP) assays. Knockdown of NRF by siRNA prevented NE-induced suppression of IL-8/CXCL8 expression. In contrast, NE did not induce NRF expression in A549 and Beas-2B cells, where NE only stimulates NF-κB activation and IL-8/CXCL8 induction. Forced expression of NRF in A549 cells by a NRF expression plasmid suppressed IL-8/CXCL8 expression. The consequence of NF-κB activation is cell and context dependent and sometimes Janus-like. This is in part due to involvement of variable subunits which have distinct and non-overlapping functions. It is therefore possible that activation of different NF-κB subunits confer the differential effect of NE between ASM and airway epithelial cells. Here, it is demonstrated that NE specifically inhibits the expression of the proinflammatory chemokine IL-8/CXCL8 in primary hASM cells indirectly by utilising RelB-associated dimers. In contrast, NE stimulates IL-8/CXCL8 expression in lung epithelial cells through activation of p65-associated dimers. A similar effect is also seen in IL-1β-stimulated primary hASM cells. Mechanistically, the RelB repressive is mediated by inducing NRF which is subsequently recruited to the native IL-8/CXCL8 promoter leading to removal of RNA pol 2 from the promoter. Knockdown of NRF by siRNA prevents NE-induced suppression of IL-8/CXCL8 expression. NE-activated RelB is recruited to the native NRF promoter and knockdown of RelB by siRNA attenuates NRF induction by NE. Hence we describe a novel negative regulatory mechanism of RelB-induced NRF which has a potential anti-inflammatory effect. In conclusion, this report has described a novel negative regulatory mechanism of NE-induced NRF which is restricted to hASM and mediates RelB-activated NRF up-regulation the suppression of IL-8/CXCL8 expression.