Natural and engineered kallikrein inhibitors: an emerging pharmacopoeia

The kallikreins and kallikrein-related peptidases are serine proteases that control a plethora of developmental and homeostatic phenomena, ranging from semen liquefaction to skin desquamation and blood pressure. The diversity of roles played by kallikreins has stimulated considerable interest in these enzymes from the perspective of diagnostics and drug design. Kallikreins already have well-established credentials as targets for therapeutic intervention and there is increasing appreciation of their potential both as biomarkers and as targets for inhibitor design. Here, we explore the current status of naturally occurring kallikrein protease-inhibitor complexes and illustrate how this knowledge can interface with strategies for rational re-engineering of bioscaffolds and design of small-molecule inhibitors.

Rational design of serine protease inhibitors

Proteases regulate a spectrum of diverse physiological processes, and dysregulation of proteolytic activity drives a plethora of pathological conditions. Understanding protease function is essential to appreciating many aspects of normal physiology and progression of disease. Consequently, development of potent and specific inhibitors of proteolytic enzymes is vital to provide tools for the dissection of protease function in biological systems and for the treatment of diseases linked to aberrant proteolytic activity. The studies in this thesis describe the rational design of potent inhibitors of three proteases that are implicated in disease development. Additionally, key features of the interaction of proteases and their cognate inhibitors or substrates are analysed and a series of rational inhibitor design principles are expounded and tested. Rational design of protease inhibitors relies on a comprehensive understanding of protease structure and biochemistry. Analysis of known protease cleavage sites in proteins and peptides is a commonly used source of such information. However, model peptide substrate and protein sequences have widely differing levels of backbone constraint and hence can adopt highly divergent structures when binding to a protease’s active site. This may result in identical sequences in peptides and proteins having different conformations and diverse spatial distribution of amino acid functionalities. Regardless of this, protein and peptide cleavage sites are often regarded as being equivalent. One of the key findings in the following studies is a definitive demonstration of the lack of equivalence between these two classes of substrate and invalidation of the common practice of using the sequences of model peptide substrates to predict cleavage of proteins in vivo. Another important feature for protease substrate recognition is subsite cooperativity. This type of cooperativity is commonly referred to as protease or substrate binding subsite cooperativity and is distinct from allosteric cooperativity, where binding of a molecule distant from the protease active site affects the binding affinity of a substrate. Subsite cooperativity may be intramolecular where neighbouring residues in substrates are interacting, affecting the scissile bond’s susceptibility to protease cleavage. Subsite cooperativity can also be intermolecular where a particular residue’s contribution to binding affinity changes depending on the identity of neighbouring amino acids. Although numerous studies have identified subsite cooperativity effects, these findings are frequently ignored in investigations probing subsite selectivity by screening against diverse combinatorial libraries of peptides (positional scanning synthetic combinatorial library; PS-SCL). This strategy for determining cleavage specificity relies on the averaged rates of hydrolysis for an uncharacterised ensemble of peptide sequences, as opposed to the defined rate of hydrolysis of a known specific substrate. Further, since PS-SCL screens probe the preference of the various protease subsites independently, this method is inherently unable to detect subsite cooperativity. However, mean hydrolysis rates from PS-SCL screens are often interpreted as being comparable to those produced by single peptide cleavages. Before this study no large systematic evaluation had been made to determine the level of correlation between protease selectivity as predicted by screening against a library of combinatorial peptides and cleavage of individual peptides. This subject is specifically explored in the studies described here. In order to establish whether PS-SCL screens could accurately determine the substrate preferences of proteases, a systematic comparison of data from PS-SCLs with libraries containing individually synthesised peptides (sparse matrix library; SML) was carried out. These SML libraries were designed to include all possible sequence combinations of the residues that were suggested to be preferred by a protease using the PS-SCL method. SML screening against the three serine proteases kallikrein 4 (KLK4), kallikrein 14 (KLK14) and plasmin revealed highly preferred peptide substrates that could not have been deduced by PS-SCL screening alone. Comparing protease subsite preference profiles from screens of the two types of peptide libraries showed that the most preferred substrates were not detected by PS SCL screening as a consequence of intermolecular cooperativity being negated by the very nature of PS SCL screening. Sequences that are highly favoured as result of intermolecular cooperativity achieve optimal protease subsite occupancy, and thereby interact with very specific determinants of the protease. Identifying these substrate sequences is important since they may be used to produce potent and selective inhibitors of protolytic enzymes. This study found that highly favoured substrate sequences that relied on intermolecular cooperativity allowed for the production of potent inhibitors of KLK4, KLK14 and plasmin. Peptide aldehydes based on preferred plasmin sequences produced high affinity transition state analogue inhibitors for this protease. The most potent of these maintained specificity over plasma kallikrein (known to have a very similar substrate preference to plasmin). Furthermore, the efficiency of this inhibitor in blocking fibrinolysis in vitro was comparable to aprotinin, which previously saw clinical use to reduce perioperative bleeding. One substrate sequence particularly favoured by KLK4 was substituted into the 14 amino acid, circular sunflower trypsin inhibitor (SFTI). This resulted in a highly potent and selective inhibitor (SFTI-FCQR) which attenuated protease activated receptor signalling by KLK4 in vitro. Moreover, SFTI-FCQR and paclitaxel synergistically reduced growth of ovarian cancer cells in vitro, making this inhibitor a lead compound for further therapeutic development. Similar incorporation of a preferred KLK14 amino acid sequence into the SFTI scaffold produced a potent inhibitor for this protease. However, the conformationally constrained SFTI backbone enforced a different intramolecular cooperativity, which masked a KLK14 specific determinant. As a consequence, the level of selectivity achievable was lower than that found for the KLK4 inhibitor. Standard mechanism inhibitors such as SFTI rely on a stable acyl-enzyme intermediate for high affinity binding. This is achieved by a conformationally constrained canonical binding loop that allows for reformation of the scissile peptide bond after cleavage. Amino acid substitutions within the inhibitor to target a particular protease may compromise structural determinants that support the rigidity of the binding loop and thereby prevent the engineered inhibitor reaching its full potential. An in silico analysis was carried out to examine the potential for further improvements to the potency and selectivity of the SFTI-based KLK4 and KLK14 inhibitors. Molecular dynamics simulations suggested that the substitutions within SFTI required to target KLK4 and KLK14 had compromised the intramolecular hydrogen bond network of the inhibitor and caused a concomitant loss of binding loop stability. Furthermore in silico amino acid substitution revealed a consistent correlation between a higher frequency of formation and the number of internal hydrogen bonds of SFTI-variants and lower inhibition constants. These predictions allowed for the production of second generation inhibitors with enhanced binding affinity toward both targets and highlight the importance of considering intramolecular cooperativity effects when engineering proteins or circular peptides to target proteases. The findings from this study show that although PS-SCLs are a useful tool for high throughput screening of approximate protease preference, later refinement by SML screening is needed to reveal optimal subsite occupancy due to cooperativity in substrate recognition. This investigation has also demonstrated the importance of maintaining structural determinants of backbone constraint and conformation when engineering standard mechanism inhibitors for new targets. Combined these results show that backbone conformation and amino acid cooperativity have more prominent roles than previously appreciated in determining substrate/inhibitor specificity and binding affinity. The three key inhibitors designed during this investigation are now being developed as lead compounds for cancer chemotherapy, control of fibrinolysis and cosmeceutical applications. These compounds form the basis of a portfolio of intellectual property which will be further developed in the coming years.

Non-combinatorial library screening reveals subsite cooperativity and identifies new high-efficiency substrates for kallikrein-related peptidase 14

An array of substrates link the tryptic serine protease, kallikrein-related peptidase 14 (KLK14), to physiological functions including desquamation and activation of signaling molecules associated with inflammation and cancer. Recognition of protease cleavage sequences is driven by complementarity between exposed substrate motifs and the physicochemical signature of an enzyme's active site cleft. However, conventional substrate screening methods have generated conflicting subsite profiles for KLK14. This study utilizes a recently developed screening technique, the sparse matrix library, to identify five novel high-efficiency sequences for KLK14. The optimal sequence, YASR, was cleaved with higher efficiency (k(cat)/K(m)=3.81 ± 0.4 × 10(6) M(-1) s(-1)) than favored substrates from positional scanning and phage display by 2- and 10-fold, respectively. Binding site cooperativity was prominent among preferred sequences, which enabled optimal interaction at all subsites as indicated by predictive modeling of KLK14/substrate complexes. These simulations constitute the first molecular dynamics analysis of KLK14 and offer a structural rationale for the divergent subsite preferences evident between KLK14 and closely related KLKs, KLK4 and KLK5. Collectively, these findings highlight the importance of binding site cooperativity in protease substrate recognition, which has implications for discovery of optimal substrates and engineering highly effective protease inhibitors.

Natural, engineered and synthetic inhibitors of kallikrein-related peptidases

There is a rapidly growing appreciation of the important physiological roles played by kallikreins and kallikrein-related peptidases (KLKs). Recent studies have revealed that these enzymes control key events in processes as diverse as inflammation and skin desquamation. Accordingly, there is considerable interest in developing tools to further dissect kallikrein activity, and a burgeoning effort aimed at producing lead inhibitors for therapeutic development. Indeed, several candidate inhibitors are already in clinical trials. This chapter surveys the naturally occurring kallikrein inhibitors, together with strategies for employing these molecules as bioscaffolds, as well as current progress in the development of small-molecule kallikrein inhibitors.

Prevention and treatment of acute radiation-induced skin reactions : a systematic review and meta-analysis of randomized controlled trials

Background Radiation-induced skin reaction (RISR) is a common side effect that affects the majority of cancer patients receiving radiation treatment. RISR is often characterised by swelling,redness, pigmentation, fibrosis, and ulceration, pain, warmth, burning, and itching of the skin. The aim of this systematic review was to assess the effects of interventions which aim to prevent or manage RISR in people with cancer. Methods We searched the following databases up to November 2012: Cochrane Skin Group Specialised Register, CENTRAL (2012, Issue 11), MEDLINE (from 1946), EMBASE (from 1974), PsycINFO (from 1806), CINAHL (from 1981) and LILACS (from 1982). Randomized controlled trials evaluating interventions for preventing or managing RISR in cancer patients were included. The primary outcomes were development of RISR, and levels of RISR and symptom severity. Secondary outcomes were time taken to develop erythema or dry desquamation; quality of life; time taken to heal, a number of skin reaction and symptom severity measures; cost, participant satisfaction; ease of use and adverse effects. Where appropriate, we pooled results of randomized controlled trials using mean differences (MD) or odd ratios (OR) with 95% confidence intervals (CI). Results Forty-seven studies were included in this review. These evaluated six types of interventions (oral systemic medications; skin care practices; steroidal topical therapies; non-steroidal topical therapies; dressings and other). Findings from two meta-analyses demonstrated significant benefits of oral Wobe-Mugos E for preventing RISR (OR 0.13 (95% CI 0.05 to 0.38)) and limiting the maximal level of RISR (MD −0.92 (95% CI −1.36 to −0.48)). Another meta-analysis reported that wearing deodorant does not influence the development of RISR (OR 0.80 (95% CI 0.47 to 1.37)). Conclusions Despite the high number of trials in this area, there is limited good, comparative research that provides definitive results suggesting the effectiveness of any single intervention for reducing RISR. More research is required to demonstrate the usefulness of a wide range of products that are being used for reducing RISR. Future efforts for reducing RISR severity should focus on promising interventions, such as Wobe-Mugos E and oral zinc.

In vivo and in vitro models demonstrate a role for caveolin-1 in the pathogenesis of ischaemic acute renal failure

Caveolae and their proteins, the caveolins, transport macromolecules; compartmentalize signalling molecules; and are involved in various repair processes. There is little information regarding their role in the pathogenesis of significant renal syndromes such as acute renal failure (ARF). In this study, an in vivo rat model of 30 min bilateral renal ischaemia followed by reperfusion times from 4 h to 1 week was used to map the temporal and spatial association between caveolin-1 and tubular epithelial damage (desquamation, apoptosis, necrosis). An in vitro model of ischaemic ARF was also studied, where cultured renal tubular epithelial cells or arterial endothelial cells were subjected to injury initiators modelled on ischaemia-reperfusion (hypoxia, serum deprivation, free radical damage or hypoxia-hyperoxia). Expression of caveolin proteins was investigated using immunohistochemistry, immunoelectron microscopy, and immunoblots of whole cell, membrane or cytosol protein extracts. In vivo, healthy kidney had abundant caveolin-1 in vascular endothelial cells and also some expression in membrane surfaces of distal tubular epithelium. In the kidneys of ARF animals, punctate cytoplasmic localization of caveolin-1 was identified, with high intensity expression in injured proximal tubules that were losing basement membrane adhesion or were apoptotic, 24 h to 4 days after ischaemia-reperfusion. Western immunoblots indicated a marked increase in caveolin-1 expression in the cortex where some proximal tubular injury was located. In vitro, the main treatment-induced change in both cell types was translocation of caveolin-1 from the original plasma membrane site into membrane-associated sites in the cytoplasm. Overall, expression levels did not alter for whole cell extracts and the protein remained membrane-bound, as indicated by cell fractionation analyses. Caveolin-1 was also found to localize intensely within apoptotic cells. The results are indicative of a role for caveolin-1 in ARF-induced renal injury. Whether it functions for cell repair or death remains to be elucidated.

Métodos biofísicos não invasivos para avaliação da eficácia de cosméticos

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Histopatologia do intestino de Pseudoplatystoma fasciatum (osteichthyes, pimelodidae) parasitados com cestodas proteocefalídeos e nematodas

This work describes intestine histopathology of Pseudoplatystoma fasciatum infected by endohelminths. Nineteen specimens were caught from Aquidauna River, Mato Grosso do Sul State, in October 2005 and submitted to necropsy, when intestine of each specimen was fixed with AFA solution during 12 h. Soon after, samples were immersed in buffered formalin and 24 h after, transferred to alcohol 70%. Study of histology sections was made to 5; m and stained with haematoxylin and eosin. It showed structures displaying helminths encapsulated by connective tissue in intestinal muscle with fibroplasy in several stages of capsule development. Were observed focus of necrosis in intestinal mucous and sub-mucous and helminthes cut obliquely in intestinal lumen. In addition, there was discreet mucous desquamation, associated to basal membrane thickness.

Prevalence and histopathology of Neoechinorhynchus curemai Noronha, 1973 (Acanthocephala: Neoechinorhynchidae) in Prochilodus lineatus Valenciennes, 1836 from Volta Grande Reservoir, MG, Brazil

O presente trabalho estudou a prevalência e a histopatologia de Neoechinorhynchus curemai Noronha, 1973 (Acanthocephala: Neoechinorhynchidae) em curimbatá, Prochilodus lineatus Valenciennes, 1836. Dezoito peixes com comprimento total médio de 46,7 + 1,1 cm e peso médio de 1.674,8 + 75,6 g foram coletados com rede, bimestralmente, de dezembro de 1995 a dezembro de 1996 na usina hidrelétrica do Reservatório de Volta Grande (Cemig), Minas Gerais, Brasil. Dos peixes analisados, 15 estavam infectados com acantocéfalos no intestino (prevalência de 83,3%). A maior intensidade média ocorreu em agosto de 1996, com 66,5 (16 a 208) parasitos. A análise histopatológica revelou completa descamação do epitélio intestinal com severa hiperplasia e hipertrofia das células caliciformes. Observou-se, ainda, forte reação inflamatória na submucosa, deslocamento de feixes, associado a edemas, bem como infiltração mononuclear e eosinofílica.

Integridade tissular de pacientes com úlceras venosas: um estudo baseado na Classificação dos Resultados de Enfermagem

The venous ulcer is an epidemiological problem of high prevalence, causing disability and dependence. Assess the tissue impairment level of patients with venous lesions, within a nursing referential, is relevant for the implementation of a directed assistance to specific clientele. Thus, this work aims to characterize the health status regarding the integrity the lower limbs skin of patients with venous ulcers, according to the of tissue integrity outcome indicators from the Nursing Outcomes Classification. A cross-sectional study conducted in a university hospital in Natal - Rio Grande do Norte. The sample consisted of 50 participants, selected through consecutive sampling. Data collection occurred through a interview and physical examination form and a operational definitions tool for indicators of the nursing Tissue Integrity outcome directed to patients with venous ulcer, applied from February to June 2012. Data analysis was done by descriptive statistics and nonparametric tests (Spearman, Kruskal-Wallis and Mann-Whitney tests). The project was approved by the Research Ethics Committee with protocol 608/11 and Presentation Certificate to Ethical Consideration No. 0038.0.294.000-11. The results were presented using three scientific articles derivatives of research. It was found that the indicators show moderate impairment, light and not impaired, as the median. The respondents had an average of 59.72 years, 66% female, 50% were retired, 60% with a partner, 44% had arterial hypertension, 26% allergies, 20% diabetes mellitus, 96% were sedentary, 14% drank alcohol and 6% were smokers. There was a statistically significant correlation of low intensity between age and hydration (p=0.032; rs=-0.304) and skin desquamation (p=0.026; rs=-0.316), family income and necrosis (p=0.012; rs=-0.353); Ankle Brachial Index and tissue perfusion (p=0,044; rs=-0,329); Diabetes Mellitus and texture (p=0.015) and tissue perfusion (p=0.026); allergy and texture (p=0.034), physical activity and hydration (p=0.034), smoking and thickness (p=0.018), and alcohol consumption and exudate (p=0.045). We conclude that the patients had light to moderate impairment, indicating a good state of health on the integrity of the skin of the lower limbs, according to the indicators of the outcome of tissue integrity Classification Nursing Outcomes valued in the present study. It is believed that the evaluation of impairment tissue using a self-nursing system and its relation with socioeconomic, clinical and risk factors are unique tools in the care planning and in the wound healing

High laryngeal mask airway pressures resulting from nitrous oxide do not increase pharyngeal mucosal injury in dogs

Purpose: During general anesthesia, nitrous oxide (N2O) diffuses rapidly into the air-filled laryngeal mask airway (LMA) cuff, increasing intracuff pressure. There is no clear correlation between LMA intracuff pressure and pressure on the pharynx. We have studied the effects of high LMA intracuff pressures secondary to N2O on the pharyngeal mucosa of dogs.Methods: Sixteen mongrel dogs were randomly allocated to two groups: G1 (intracuff volume, 30 mL; n = 8) breathed a mixture of O-2 (1 L.min(-1)) and air (1 L.min(-1)) and G2 (intracuff volume, 30 mL; n=8) a mixture of O-2 (1 L.min(-1)) and N2O (1 L.min(-1)). Anesthesia was induced and maintained with pentobarbitone. LMA cuff pressure was measured at zero (control), 30, 60, 90 and 120 min after #4 LMA insertion. The dogs were sacrificed, and biopsy specimens from seven predetermined areas of the pharynx in contact with the LMA cuff were collected for light (LM) and scanning electron microscopic (SEM) examination by a blinded observer.Results: LMA intracuff pressure decreased with time in G1 (P < 0.001) and increased in G2 (P < 0.001). There was a significant difference between the groups (P < 0.001). In both groups, the LM study showed a normal epithelium covering the pharyngeal mucosa and mild congestion in the subepithelial layer There were no differences between the groups (P > 0.10) or among the areas sampled (P > 0.05). In both groups, the SEM study showed a normal pharyngeal mucosa with mild superficial desquamation. Few specimens in G1 and G2 showed more intense epithelial desquamation.Conclusion: High LMA intracuff pressures produced by N2O do not increase pharyngeal mucosal injury in dogs.

Papilomatose laríngea: análise morfológica pela microscopia de luz e eletrônica do HPV-6

Papilomatose laríngea é neoplasia benigna mais freqüente nas crianças, causada pelo HPV, principalmente subtipos 6 e 11 e caracteriza-se pela presença de lesões proliferativas exofíticas e recidivantes sobre a mucosa das vias aérea, em especial na laringe. Forma de Estudo: Clínico prospectivo. OBJETIVOS: Demonstrar alterações epiteliais morfológicas (pela microscopia de luz e eletrônica) em lesões papilíferas casadas pelo HPV-6. MATERIAL E MÉTODOS: Fragmentos de lesões de papilomatose laríngea, colhidos durante procedimento cirúrgico de quatro crianças (1 masculino, 3 femininas), foram submetidos à tipagem do HPV (por método de PCR), análise pela microscopia de luz e microscopia eletrônica (varredura e transmissão). RESULTADOS: Na tipagem, todos os papilomas eram do subtipo 6. A microscopia de varredura identificou projeções epiteliais de vários tamanhos, com células superficiais em descamação. A microscopia de luz demonstrou lesões exofíticas, revestidas por epitélio hiperplásico com coilócitos e binucleações, característicos do HPV. A membrana basal e o córion adjacente estavam íntegros. À microscopia eletrônica de transmissão identificou-se vacuolização perinuclear e alargamento das junções intercelulares. CONCLUSÕES: As alterações morfológicas apresentadas pelo HPV-6 demonstram o caráter não-invasivo da lesão, sendo necessário estudos morfológicos adicionais relacionando os outros tipos de HPV, considerados mais agressivos, com os achados ultra-estruturais.

Aspectos clínicos, histológicos e de microscopia eletrônica dos granulomas de intubação das pregas vocais

Granulomas são lesões bilaterais e pediculadas das apófises vocais. Etiologias: intubação, refluxo, traumatismos, fonotraumatismo e idiopática. OBJETIVO: Analisar aspectos clínicos e morfológicos dos granulomas de intubação. MATERIAL E MÉTODOS: Estudo retrospectivo dos pacientes submetidos à microcirurgia por granulomas de intubação, atendidos na Instituição onde foi realizado, a partir de 2002. Analisaram-se: idade, sexo, indicação e tempo da intubação, sintomas, laudos de videolaringoscopia e número de biópsia. Realizou-se estudo histológico em todos os casos e de microscopia eletrônica em três deles. RESULTADOS: 10 pacientes (7 F e 3 M), idade entre 2 anos e 72 anos e tempo de intubação entre 4 horas e 21 dias. Rouquidão foi o principal sintoma. A histologia mostrou hiperplasia epitelial, intenso inflamação importante no corion e proliferação vascular. Na MEV observou-se epitélio escamoso com escassa descamação. À MET, junções intercelulares alargadas e desmossomos alterados. No corion havia lagos sanguíneos, intensa inflamação, e fibroblastos com alterações estruturais como núcleos irregulares e cisternas dilatadas. CONCLUSÕES: Granulomas pós-intubação aparecem em qualquer idade, mesmo em intubação por curto período, e causam rouquidão precocemente. As principais alterações morfológicas são observadas no corion, como proliferação vascular, inflamação e alterações estruturais em fibroblastos indicando disfunção e dano celular.

Is Reinke's Edema a Precancerous Lesion? Histological and Electron Microscopic Aspects

The objectives of this study were to evaluate morphologic alterations and precancerous lesions in Reinke's edema. Patients included were 54 smokers with bilateral Reinke's edema submitted to surgery in the Otolaryngology Department, Botucatu Medical School, São Paulo State University, Brazil, between 2002 and 2006. All specimens were evaluated by light microscopy and five contralateral lesions were also evaluated by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The main histological alterations were edema (100%), inflammation (81.48%), basal membrane (bm) thickening (77.77%), and vessel proliferation (75.92%). Epithelium alterations were classified as grade 0 (11.11%), grade 1 (70.37%), grade 2 (14.81%), and grade 3 (3.70%). In the case included in grade 3 classification, microinvasive carcinoma was detected. SEM showed epithelial surface with some cellular desquamation, few microridges, and a polished and impermeable surface aspect. TEM showed epithelial hyperplasia, basal cells with different sizes, widening of the intercellular spaces, abnormal desmosome architecture, thickening of the bm, some electron-dense vesicles, and points of interruption. The morphological alterations presented in this study are not specific to Reinke's edema but this lesion can be the site of different grades of dysplasia and carcinoma, justifying the importance of periodic laryngeal endoscopic exams and meticulous histological analysis.