Apoferritin encapsulation of cysteine protease inhibitors for cathepsin L inhibition in cancer cells

Cysteine proteases play a key role in tumorigenesis causing protein degradation and promoting invasive tumour growth. Cathepsin L is overexpressed in cancer cells and could provide a specific target for delivery of anticancer agents. We encapsulated novel dipeptidyl nitrile based cysteine protease inhibitors (Neq0551, Neq0554 and Neq0568) into biocompatible apoferritin (AFt) protein nanocages to achieve specific delivery to tumours and pH-induced drug release. AFt-encapsulated Neq0554 demonstrated $3-fold enhanced in vitro activity (GI 50 ¼ 79 mM) compared to naked agent against MiaPaCa-2 pancreatic carcinoma cells. Selectivity for cancer cells was confirmed by comparing their activity to non-tumourigenic human fibroblasts (GI 50 > 200 mM). Transferrin receptor (TfR-1) expression, detected only in lysates prepared from carcinoma cells, may contribute to the cancer-selectivity. The G 1 cell cycle arrest caused by AFt-Neq0554 resulting in cytostasis was corroborated by clonogenic assays. Superior and more persistent inhibition of cathepsin L up to 80% was achieved with AFt-encapsulated agent in HCT-116 cells following 6 h exposure to 50 mM agent. The selective anticancer activity of AFt-encapsulated cysteine protease inhibitor Neq0554 reported here warrants further preclinical in vivo evaluation

Molecular design, trypanosomicidal and anticancer activity of reversible covalent inhibitors of cysteine proteases

A atividade de cisteíno proteases (CP) tem sido relacionada a diferentes patologias, como no caso da leishmaniose, doença de Chagas de alguns tipos de câncer. Devido a homologia entre as cisteíno proteases presentes em altos níveis nesses sistemas celulares, foi investigada aqui a importância dessas enzimas para o desenvolvimento e estabelecimento dessas doenças a partir da atividade biológica in vitro de novos inibidores reversíveis de cisteíno proteases. De maneira geral, as substâncias apresentaram relevante atividade inibitória de cisteíno proteases expressas pelos diferentes sistemas celulares, com máximo de inibição de 42% para o Neq0554 em relação à atividade de CP expressas por Leishmania spp. e 76% em relação a atividade de CP expressas por células de câncer de pâncreas. Diferentes níveis de atividade biológica foram observados entre os sistemas celulares, porém todos apresentaram supressão em relação aos parâmetros citostáticos após a inibição da atividade de CP. Quando testados em Leishmania spp. o crescimento celular foi suprimido em pelo menos 67%, com máximo de inibição de 95% para o Neq0551 a 10 μM. Da mesma maneira, em células de câncer de pâncreas, alterações no ciclo celular e supressão dos processos de migração e formação de colônias foram os resultados mais evidentes, comretenção de 50% da capacidade de formação de colônias das células Mia-Paca2 pelo Neq0554 a 10 μM. Já em relação aos protozoários da capa Y de Trypanosoma cruzi os inibidores testados apresentaram interessante seletividade contra os parasitos, em relação à célula hospedeira LLC-MK2, além de promoverem a supressão de cerca de 80% do processo de invasão celular in vitro quando a célula hospedeira foi previamente tratada com 10 μM do inibidor Neq0662 por 2 h antes do processo de infecção. Por fim, a encapsulação do Neq0554 em apoferritina promoveu um incremento na atividade anticancerígena para células de câncer de pâncreas, com IC50 de 79 μM contra > 200 μM em relação às células de fibroblasto, aumentando sua seletividade. De maneira geral, os resultados corroboram a hipótese de a inibição de cisteíno proteases nos sistemas celulares é eficiente para promover efeitos citostáticos, podendo ser utilizada com controle e supressão do desenvolvimento das patologias. Além disso, a atividade de CP nas células de protozoários e câncer de pâncreas apresentou perfil semelhante de ação, no qual inibidores de CP não promoveram a morte em nível significativo das células, mas ressaltaram os efeitos citostáticos em relação ao crescimento celular.Cysteine proteases (CP) activity has been related to different pathologies, such as leishmaniasis, Chagas disease and some types of cancer. Due to the homology between cysteine proteases expressed by these cellular systems, it was investigated here the importance of these enzymes for the development and establishment of these diseases based on the in vitro biological activity of novel reversible cysteine protease inhibitors. In general, the inhibitor showed a significant inhibitory activity of cysteine proteases expressed by the different cellular systems, with a maximum inhibition of 42% for Neq0554 concerning the CP activity expressed by Leishmania spp. and 76% to CP activity expressed by pancreatic cancer cells. Different profiles of biological activity were observed between the cellular systems, but all substances had significant CP activity suppression, in cytostatic levels after the inhibition of CPA. When the inhibitors were tested against Leishmania spp., the cell growth was suppressed by at least 67%, with maximum inhibition of 95% for Neq0551 at 10 μM. Similarly in pancreatic cancer cells, changes in the cell cycle profile were the most evident results, as well as the suppression of migration and colony formation ability, with 50% retention of the colony development of Mia-Paca2 cells by Neq0554 at 10 μM. In contrast, to protozoa from Trypanosoma cruzi Y strain, the inhibitors tested showed an interesting selectivity against the parasites concerning the host cell LLC-MK2, also promoting the in vitro cell invasion suppression in about 80% when the host cell was pre-treated with Neq0662 10 μM for 2 h. Finally, the encapsulation of Neq0554 promoted an increase in its anticancer activity against pancreatic cancer cells, with IC50 of 79 μM alongside > 200 μM to fibroblast cells, besides increasing its selectivity. In general, the results corroborate the hypothesis that the inhibition of cysteine proteases in the cellular systems is efficient to promote cytostatic effects, being an interesting tool to be used as control and development suppression of some pathologies. Also, CP activity in protozoa cells and pancreatic cancer showed a similar profile of action, in which cysteine protease inhibitors did not promote death at a significant level for the cells, but emphasized cytostatic effects about cell growth

Imobilização de lipases por adsorção e ligação covalente em derivados de agarose e quitosana e sua aplicação em biocatálise

In the biocatalysis is necessary their immobilization on inert supports for further reuse, stabilization, purification and hyperactivaction of the biocatalyst. This study aimed to immobilization of different lipases using different supports (ionic, hydrophobic and covalent) and to evaluate the activity, selectivity, stability and purification of these immobilized proteins. The chapter 1 of the work describes some techniques of immobilization and pre-purification of crude lipases from Acremonium sp by ultrafiltration showed higher activity and yield of immobilization on octyl- and phenyl-agarose. The activity of these immobilized enzymes was hyperactived by incubation at 0.01 m∙L-1 NaCl and higher concentrations of this salt was observed a decrease on the enzyme activity. The hydrolytic activity of the immobilized lipase was influenced by the vegetable oil source. In Chapter 2 we present the chemical modification and characterization of chitosan previously activated with glycidol, epichlorohydrin and glutaraldehyde for subsequent use as support for immobilization of lipases, but the low surface area of chitosan determined by BET (4 cm²∙g-1) negatively affected the immobilization process. The use of ultrasound radiation in biotransesterification of soybean oil catalyzed by immobilized lipases (TLL and L435) by hydrophobic interaction was studied in Chapter 3, and the yield of total production of esters by soybean, corn and sunflower oils. The soybean and corn oils showed highest transesterification and an increased ester production of 400 % obtained was when sonicated. The last chapter of this search presents the results obtained in the Instituto de Catálisis y Petroleoquímica de Madrid during the research stage, which focused on the improvement of the methods of immobilization of lipases and applications in the enantioselective hydrolysis and production of omega-3 from fish oil, with lipase B from Candida antarctica showing ...Na biocatálise faz-se necessária a imobilização destas enzimas em suportes inertes ao meio reacional, a fim de reutilizar, estabilizar, purificar e muitas vezes melhorar a atividade enzimática, levando à uma hiperativação. O presente trabalho teve como objetivo principal utilizar técnicas de imobilização de enzimas sob diferentes suportes (iônicos, hidrofóbicos e covalentes) e aplicá-los na imobilização de distintas lipases para avaliar o efeito da imobilização na atividade, seletividade, estabilidade e purificação dessas proteínas. O Capítulo 1 do trabalho relata algumas técnicas de imobilização e pré-purificação das lipases brutas obtidas a partir do fungo Acremonium sp, no qual as enzimas pré-purificadas por ultrafiltração apresentaram maior atividade e rendimento de imobilização sob os suportes hidrofóbicos octil- e fenil-agarose. Essas enzimas imobilizadas foram hiperativadas na presença de 0,01 mol∙L-1 de NaCl e concentrações maiores desse sal causaram a diminuição da atividade enzimática. A atividade hidrolítica das lipases imobilizadas foi avaliada utilizando diferentes fontes de óleos vegetais, com maior atividade para enzima imobilizada do que a enzima solúvel em 40% dos óleos testados. No Capítulo 2 é apresentada a modificação química e caracterização da quitosana ativada com glicidol, epicloridrina e glutaraldeído para posterior aplicação como suporte para imobilização de lipases por ligação covalente, porém tal procedimento não foi possível devido à baixa área superficial do polímero (cerca de 4cm²·g-1) tanto para reagir com os agentes modificantes quanto para o carregamento proteico. A utilização da radiação do ultrassom na biotransesterificação do óleo de soja catalisada por lipases imobilizadas (TLL e L435) por interação hidrofóbica foi estudada no Capítulo 3, bem como o rendimento de produção total de ésteres dos óleos de soja, milho e girassol ...Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Modulation of the activity and selectivity of the immobilized lipases by surfactants and solvents

Most of lipases are in equilibrium between a majority inactive closed form and a minority active open form in aqueous media. Perhaps, a certain stabilization of these open forms of lipases could be achieved in the presence of cosolvents or surfactants in the reaction medium. Three commercial lipases were studied (from Thermomyces lanuginosa (TLL), Candida Antarctica fraction B (CALB) and Lecitase (LEC)). Different derivatives were tested: TLL and LEC were adsorbed on an anionic exchanger and their activity strongly depends on the equilibrium between their open and closed form and CALB was adsorbed on a hydrophobic support when the open form was already stabilized by the support. Derivatives ionically adsorbed were hyperactivated by surfactans as well as by cosolvents: the activity of LEC increased 12 times in the presence of 15-20% of ethanol. CALB adsorbed on hydrophobic supports was hardly hyperactivated and even it was inhibited. The modification of the rate of covalent modification of the catalytic Ser seems to confirm that the observed hyperactivations were due to a stabilization of the open form of the adsorbed lipases (TLL and LEC). The hydrolysis of sardine oil was also studied in the presence or absence of surfactants and cosolvents. An interesting improvement in the ability of derivatives to discriminate the release of eicosipentaenic acid (EPA) and docosahexaenicacid (DHA) was found.The authors thank the financial support of Coordination for the Improvement of Higher Level -or Education Personnel (CAPES) and São Paulo Research Foundation (FAPESP), grant 2013/00530-0.Peer Reviewe

Ethyl esters production catalyzed by immobilized lipases is influenced by n-hexane and ter-amyl alcohol as organic solvents

Lipase stability in organic solvent is crucial for its application in many biotechnological processes as biocatalyst. One way to improve lipase’s activity and stability in unusual reaction medium is its immobilization on inert supports. Here, lipases from different sources and immobilized through weak chemical interactions on hydrophobic and ionic supports had their transesterification ability dramatically dependent on the support and also on the solvent that had been used. The ethanolysis of sardine oil was carried out at the presence of cyclohexane and tert-amyl alcohol, in which Duolite A568-Thermomyces lanuginosa lipase derivative achieved 49% of ethyl esters production after 24 h in cyclohexane. The selectivity of immobilized lipases was also studied and, after 3 h of synthesis, the reaction with Duolite A568-Thermomyces lanuginosa derivative in cyclohexane produced 24% ethyl ester of eicosapentaenoic acid and 1.2% ethyl ester of docosahexaenoic acid, displaying a selectivity index of 20 times the ethyl ester of eicosapentaenoic acid. Different derivatives of Candida antarctica lipases fraction B (CALB) and phospholipase Lecitase® Ultra (Lecitase) were also investigated. Along these lines, a combination between these factors may be applied to improve the activity and selectivity of immobilized lipases, decreasing the total cost of the process.The authors thank the financial support from the São Paulo Research Foundation (FAPESP – grants 2012/09054-3 and 2013/00530-0), and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil (CAPES – grant number 3894/13-4).Peer reviewe