Multi-Target-Directed Ligands and other Therapeutic Strategies in the Search of a Real Solution for Alzheimer's Disease.

The lack of an adequate therapy for Alzheimer's Disease (AD) contributes greatly to the continuous growing amount of papers and reviews, reflecting the important efforts made by scientists in this field. It is well known that AD is the most common cause of dementia, and up-to-date there is no prevention therapy and no cure for the disease, which contrasts with the enormous efforts put on the task. On the other hand many aspects of AD are currently debated or even unknown. This review offers a view of the current state of knowledge about AD which includes more relevant findings and processes that take part in the disease; it also shows more relevant past, present and future research on therapeutic drugs taking into account the new paradigm "Multi-Target-Directed Ligands" (MTDLs). In our opinion, this paradigm will lead from now on the research toward the discovery of better therapeutic solutions, not only in the case of AD but also in other complex diseases. This review highlights the strategies followed by now, and focuses other emerging targets that should be taken into account for the future development of new MTDLs. Thus, the path followed in this review goes from the pathology and the processes involved in AD to the strategies to consider in on-going and future researches.

Industrial biotransformations in the synthesis of building blocks leading to enantiopure drugs.

Due to the growing demand of enantiomerically pure compounds, as well as the increasing strict safety, quality and environmentally requirements of industrial synthetic processes, the development of more sustainable, healthy and economically attractive strategies for the synthesis of chiral biologically active molecules is still an open challenge in the pharmaceutical industry. In this context, the biotransformations field has emerged as a real alternative to traditional synthetic routes, because of the exquisite chemo-, regio- and enantioselectivities commonly displayed by enzymes; thus, biocatalysis is becoming a widespread methodology for the synthesis of chiral compounds, not only at laboratory scale, but also at industrial scale. As hydrolases and oxido-reductases are the most employed enzymes, this review is focused on describing several industrial processes based on the use of these enzymes for obtaining chiral compounds useful for the pharmaceutical industry.

Novel microbial lipases: catalytic activity in reactions in organic media.

2,000 microbial strains were isolated from soil samples and tested to determine their lipolytic activity by employing screening techniques on solid and in liquid media. Culture broths were initially tested with 1,2-O-dilauryl-rac-glycero-3-glutaric acid-resorufinyl ester, a chromogenic substrate specific for lipases. Fourteen lipase-producing microorganisms were selected and their taxonomic identification was carried out. Hydrolysis of tributyrin or olive oil and the esterification of oleic acid with heptanol were selected to preliminary evaluate the catalytic activity of these lipases. All the selected lipases catalysed this esterification reaction with good yields. Resolution of (R,S)-2-(4-isobutylphenyl) propionic acid, (R,S)-1-phenylethanol, (R,S) 1-phenylethylamine and of (R) or (S) glycidol were performed to evaluate the stereoselectivity of these novel enzymes as biocatalysts in reactions in organic media. Lipases from the fungi Fusarium oxysporum and Ovadendron sulphureo-ochraceum gave the best yields and enantioselectivities in the resolution of racemic ibuprofen and 1-phenylethanol. Several lipases displayed a high stereoselectivity in the resolution of chiral amines by an alcoxycarbonylation reaction.

Effect of fermentation conditions in the enzymatic activity and stereoselectivity of crude lipase from Candida rugosa.

Different fed-batch cultures of Candida rugosa were carried out using oleic acid as the only carbon source. The crude lipases obtained under several operational conditions and downstream processes showed different catalytic activity and isoenzymes ratio. This fact implied that the performance of the lipase produced could be modulated by using different operational fermentation conditions. These powders were compared with commercial lipase from Sigma (St. Louis, MO) in hydrolysis and synthesis reactions. Especially interesting was the fact that the enantioselectivity of a crude lipase was higher than that observed with commercial lipase in the resolution of racemic Ketoprofen. In addition, response of both lipases in the presence of water was different.

Influence of the nature of modifier in the enzymatic activity of chemical modified semipurified lipase from Candida rugosa.

Semipurified lipase of Candida rugosa (CRSL) was subjected to chemical modification, and the activities of the modified lipase, in hydrolysis and esterification reactions, were examined. The esterification reactions were carried out in the absence and presence of isooctane. When the enzyme was modified with polyethylene glycol (PEG), two methodologies were studied. The activation of PEG with p-NO(2)-phenylchloroformate gives better biocatalysts than those obtained with cyanuric chloride-PEG. The chemical modification with PEG increases the stability of pure lipases in isooctane at 50 degrees C (extreme conditions). The chemically modified enzymes are useful for biotransformations in organic solvents. In addition the nitration of tyrosines with tetranitromethane was also studied. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 252-260, 1997.

Hydrolysis of used frying palm olein and sunflower oil catalyzed by porcine pancreatic lipase.

The enzymatic hydrolysis of frying used vegetable oils with different degrees of alteration were measured using porcine pancreatic lipase (acylglycerol acylhydrolase EC 3.1.1.3). Successive frying of potatoes significantly increased the level of total polar lipid content in the palm olein from 9.3 +/- 0.1 mg/100 mg oil to 26.4 +/- 0.3 mg/100 mg oil after 90 fryings, and from 4.0 +/- 0.1 mg/100 mg oil to 27.7 +/- 0.3 mg/100 mg oil in sunflower oil after 60 fryings. Triacylglycerol polymers, triacylglycerol dimers, and oxidized triacylglycerols also increased 37-, 7.9-, and 7.5-times in palm olein, respectively, and 56-, 22-, and 4.7-times in sunflower oil, respectively. However, diacylglycerols and free fatty acid levels related to hydrolytic alteration did not increase with the number of fryings in both oils. The substrate concentration in the reactor was determined by calculating the molecular weight of each oil showing a different degree of alteration. We compared the methodology used by us and that used by other authors. The results show that the methods are reproducible and that the values obtained are in concordance with theoretical values. The kinetic parameters apparent Michaelis-Menten constant (KMapp) and apparent maximum velocity of hydrolysis (Vmaxapp) were different in unused palm olein (5.1 +/- 0.7 and 166 +/- 7.6, respectively) than in sunflower oil (2.2 +/- 0.3 and 62 +/- 2.2, respectively). However, changes in KMapp and Vmaxapp were not related to the degree of alteration of the oils.

Contribution to the study of the alteration of lipase activity of Candida rugosa by ions and buffers.

A semipurified C. rugosa lipase (LS) has been prepared from commercial lipase (LC) using an economical procedure. The presence of sugars and glycopeptides has been detected in LS and LC. Pure lipase only has covalently bonded sugars. The hydrolysis of olive oil catalyzed by LS and commercial lipase (LC) is sensitive to the presence of cations Na(I), Mg(II), Ca(II), and Ba(II) and to the nature of buffer. Highest enzyme activity is obtained with 0.1M Tris/HCl buffers and the combination of NaCl 0.11M and CaCl2 0.11M. Fluorescence spectroscopy analysis of LC, LS, and both pure isoenzymes lipases A and B, was used to analyze the interaction of the lipase with these effectors. Inorganic cations Na or Ca do not interact with pure enzyme LA but do interact with LC and LS and do so slightly with LB. The organic cations (morfolinium or tris) interact with pure lipases. We postulate that the increase in the lipase activity produced by Na(I) or Ca(II) is related with interfacial phenomena, but the increase might be more specific in the hydrolysis of olive oil in the presence of Tris-HCl or morfoline-HCl buffer, owing to enzyme-buffer interaction.

Hydrolysis of nucleic acids in single-cell protein concentrates using immobilized benzonase.

Hydrolysis of nucleic acids for single-cell protein concentrates has been carried out in one step using immobilized benzonase on corn cob. The immobilization is carried out by tosylation of primary alcohols of cellulose of corn cob. The immobilized benzonase is more stable vs pH changes than native benzonase, but the same optimum values of [Mg(II)] and temperature are obtained. The DNase activity is greater than the RNase activity. The percentage of DNA is reduced to 3-6% and that of RNA to 50%. The protein loss is negligible (1%). The enzymatic activity per weight unit of enzyme is greater in the case of benzonase that in reported data for other nucleases insolubilized on corn cob by the same procedure.

Modulation of lipase hydrolysis and synthesis reactions using carbohydrates.

A novel method for modulation of lipase hydrolysis and synthesis lipase was investigated by using carbohydrates in the microenvironment of the Candida rugosa enzyme. The influence of the addition of different sugars to the previously dialysed enzyme was tested on the two reactions. Rates of hydrolysis were lowered by using dialysed enzyme but were increased after sugar addition, regardless of the identity of the added sugar. In contrast, synthesis reaction rates depended on the nature of the carbohydrate. Rates were increased by adding lactose, which is not a water activity depressor, but were lowered by adding fructose, glucose, sucrose or sorbitol, which are all water activity depressors.

New insolubilized derivatives of ribonuclease and endonuclease for elimination of nucleic acids in single cell protein concentrates.

Two derivatives of pancreatic ribonuclease and endonuclease of Staphylococcus aureus, insolubilized on corn cob, have been used to reduce the percentage of nucleic acids in single cell protein (SCP) concentrates from yeasts. These derivatives are thermostable and active at 45 degrees C. At these temperatures the contamination by bacteria is negligible. The thermostability is remarkable, since the native nuclease is deactivated at above 39 degrees C. The hydrolysis of the nucleic acids in SCP is carried out first with the ribonuclease derivative followed by the endonuclease derivative. The catalytic activity of the insolubilized derivatives is similar to that of the native enzymes in the hydrolysis of RNA but not of DNA. The percentage of nucleic acids is reduced from 5-15 to 0.5%, with a loss of protein of 6%. These percentages are lower than those previously reported.

DNase activity of micrococcal endonuclease insolubilized on corn cob.

The endonuclease from S. aureus has been immobilized on ground maize cob, previously activated with tosyl chloride. Pretreatment of the support on acid before tosylation yielded the best insoluble enzyme derivatives. The catalytic activity has been evaluated as percent of total hydrolysis attained in a batch reactor using DNA as a model substrate. The derivatives prepared are very resistant to high temperatures under conditions of catalysis (24 h at 45 degrees C). For these long reaction times, the extent of hydrolysis in the presence of small amounts of organic solvent (dimethyl sulfoxide at 2 percent) is larger than in plain buffer (Tris). This type of derivative could be very useful for the removal of nucleic acids from single-cell protein concentrates.