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Piperine is a secondary metabolite of black pepper. Its uses in medicine were already studied. However, its derivatives have not gained considerable attention. In the presented study, the Lipoxygenase (LOX) inhibitory activity of piperine and its derivatives, piperonylic acid, piperic acid, and piperonal have been assessed and compared by enzyme kinetics, ITC and molecular modeling experiments. The presented investigations expressed that all the studied compounds inhibited LOX by binding at its active site. The IC(50) values of these compounds were deduced from the kinetics data and found to be 85.79, 43.065, 45.17, and 50.78 μm for piperine, piperonylic acid, piperic acid, and piperonal, respectively. The binding free energies obtained from ITC experiments were -7.47, -8.33, -8.09, and -7.86 kcal/mol for piperine, piperonylic acid, piperic acid, and piperonal, respectively. Similarly, the glide scores obtained for piperine, piperonylic acid, piperic acid, and piperonal were -7.28, -10.32, -10.72, and -9.57 kcal/mol, respectively. The results of ITC and molecular modeling experiments suggested that piperonylic acid and piperonal exhibit stronger binding at the active site than piperine does. From the presented studies, it could be concluded that derivatives of piperine may be of higher significance than piperine for certain medicinal applications, implicating (Ayurvedic) fermented herbal drugs with piperine in them., (© 2014 John Wiley & Sons A/S.)

The novel amides of ketoprofen and its reduced derivatives (5a-f, 4a-n, 6a-g) with aromatic and cycloalkyl amines or hydroxylamines were prepared and screened for their reducing and cytostatic activity as well as for their ability to inhibit soybean lipoxygenase and lipid peroxidation. 1,1-Diphenyl-picrylhydrazyl test for reducing ability revealed that ketoprofen amides were more potent antioxidants than the amides of the reduced ketoprofen derivatives. The most active compound was benzhydryl ketoprofen amide 5f. Lipoxygenase inhibition of the tested compounds varied from strong to very weak. The most potent compound was benzhydryl derivative 6f (IC(50) = 20.5 mum). Aromatic and cycloalkyl amides 4 and 5 were more potent lipoxygenase inhibitors than derivatives with carboxylic group. Aromatic amides of series 4 and 5 showed excellent lipid peroxidation inhibition (92.2-99.9%). On the other hand, the most pronounced cytostatic activity was exerted by O-benzyl derivative 4i, although in general all tested reduced and non-reduced lipophilic derivatives showed similar activity.

Protein-ligand interaction information is captured by determination of interacting fragments (IF) of ligands available in complex X-ray structures. From IF, fingerprints (IF-FP) are calculated for similarity searching. Previously, we have shown that IF-FP often produce higher search performance than general structural fragment- or key-type fingerprints. In this study, we introduce the transfer of target-ligand interaction information from one target to a related one for which no structural information is available. Thus, IFs from a crystallographic target B-ligand complex are incorporated into structural key fingerprints of known ligands for target A. Similarity searching using these IF transfer fingerprints (IF-TFP) is shown to further increase the search performance of conventional ligand fingerprints. Thus, interaction information can be transferred between related targets in order to support ligand-based fingerprint search calculations for targets for which no structural information is currently available.

Lipoxygenases (LOXs) catalyze the regiospecific and stereospecific dioxygenation of polyunsaturated membrane-embedded fatty acids. A Ca(2+)-dependent membrane-binding function was localized to the amino-terminal C2-like domain of 8R-lipoxygenase (8R-LOX) from the soft coral Plexaura homomalla. The 3.2 A crystal structure of 8R-LOX and spectroscopic data suggested that Ca(2+) stabilizes two membrane-insertion loops. Analysis of the protein packing contacts in the crystal lattice indicated that the conformation of one of the two loops complicated efforts to improve the resolution of the X-ray data. A deletion mutant of 8R-LOX in which the corresponding membrane-insertion loop is absent (Delta41-45:GSLOX) was engineered. Removal of the membrane-insertion loop dramatically increases the protein yield from bacterial cultures and the quality of the crystals obtained, resulting in a better than 1 A improvement in the resolution of the diffraction data.

Flash-freezing is a technique that is commonly used nowadays to collect diffraction data for X-ray structural analysis. It can affect both the crystal and molecular structure and the molecule's surface, as well as the internal cavities. X-ray structural data often serve as a template for the protein receptor in docking calculations. Thus, the size and shape of the binding site determines which small molecules could be found as potential ligands in silico, especially during high-throughput rigid docking. Data were analyzed for wild soybean lipoxygenase-3 (MW 97 kDa) at 293 and 93 K and compared with the results from studies of its molecular complexes with known inhibitors, structures published by others for a derivative of the same enzyme (98 K) or a topologically close isozyme lipoxygenase-1 (at ambient temperature and 100 K). Analysis of these data allows the following conclusions. (i) Very small changes in the relative orientation of the molecules in the crystal can cause major changes in the crystal reciprocal lattice. (ii) The volume of the internal cavities can ;shrink' by several percent upon freezing even when the unit-cell and the protein molecular volume show changes of only 1-2%. (iii) Using a receptor structure determined based on cryogenic data as a target for computational screening requires flexible docking to enable the expansion of the binding-site cavity and sampling of the alternative conformations of the crucial residues.

This work describes the application of the N-demethylase activity of immobilized soybean lipoxygenase to the oxidative degradation of xenobiotics. Previously (1) we have shown that immobilized lipoxygenase produces the oxidative degradation of CPZ in the presence of hydrogen peroxide. As a continuation of this work, here we studied the N-demethylation of CPZ by the hydroperoxidase activity of covalent immobilized soybean lipoxygenase. The obtained results clearly reveal that the immobilized system produces the N-demethylation of CPZ in the presence of hydrogen peroxide, maintaining a high level of activity in comparison with free enzyme. Additionally, the immobilized lipoxygenase shows stability higher than that of free enzyme, making feasible its use in a bioreactor operating in continuous or discontinuous mode. The results obtained in this work, together with those obtained previously by us for the oxidation of CPZ, suggest that hydroperoxidase activity of immobilized lipoxygenase may constitute a valuable tool for oxidative xenobiotics degradation or for application to synthetic processes in which a N-demethylation reaction is involved.

4-Nitrocatechol (4NC) is a known inhibitor of lipoxygenase. This work presents the X-ray structure of soybean lipoxygenase-3 in complex with 4NC refined at 2.15 A resolution. The X-ray analysis shows 4NC near iron with partial occupancy, blocking access to Fe but not covalently bound to it. The two hydroxyl groups interact with the C-terminus (4-OH) and His523 ND1 (3-OH). The residues surrounding the iron cofactor, His518*, His523, His709, Ile857* COO(-) and water, form a trigonal bipyramid with the residues marked with asterisks in the axial positions. The water bound to iron and the presence of the inhibitor seem to be responsible for the rearrangements and changes in the geometry of the ligand distribution and confirm the displacement of His518 from iron coordination. A description of the catechol binding contributes to the understanding of lipoxygenase inhibition and the participation of its co-oxidative activity in the utilization of natural flavonoids.

The application of the hydroperoxidase activity of immobilized lipoxygenase to xenobiotic metabolization is reported in this work. Soya bean lipoxygenase has been immobilized by covalent coupling to oxirane acrylic beads. This immobilized system has been applied to the oxidation of chlorpromazine (CPZ), a phenothiazine of wide pharmacological use which in some cases presents toxicity. Immobilized lipoxygenase produces the oxidation of CPZ in the presence of hydrogen peroxide at acidic pH, maintaining a high level of activity and stability. In comparison with free lipoxygenase, the immobilized enzyme system shows higher catalytic efficiency and protection against enzymic inactivation produced by the presence of H(2)O(2). When the system of immobilized enzyme was loaded into a bioreactor operating in continuous mode, the level of CPZ oxidation was higher than obtained using a discontinuous procedure or the free enzyme. The results obtained in this work suggest that a system of covalent immobilized lipoxygenase operating in continuous mode may constitute a valuable tool for xenobiotic detoxification or metabolization studies.

The objective of this investigation was to study kinetically the effect of pressure and temperature either as a single, as a combined, or as a sequential action on lipoxygenase (LOX) inactivation in crude green beans extract. The LOX isozymes in green beans extract had a different heat sensitivity but a similar pressure stability: two fractions following apparent first-order reactions, i.e., a heat-labile fraction and a heat-stable fraction, were observed in studies on its thermostability, whereas only one fraction following a first-order reaction was noticed in studies on its pressure stability. At ambient pressure, irreversible LOX inactivation was studied in a temperature range from 55 to 70 degrees C. At room temperature, pressures around 500 MPa were required in order to inactivate LOX in green beans extract. The effect of a pressure or a thermal pretreatment on LOX thermo- or barostability, respectively, was also investigated but no significant differences in inactivation kinetics due to the pretreatment were observed.

Lipoxygenase (LOX) is an enzyme that regioselectively introduces the hydroperoxide functionality into polyunsaturated fatty acids, such as linoleic acid (LA). Hydroperoxide derivatives of polyunsaturated fatty acids are of interest because they can serve as important intermediates in the synthesis of chemical and pharmaceutical compounds. In this study, LOX was immobilized in dispersed phyllosilicate layers that were cross-linked with silicate polymers formed by the hydrolysis of tetramethyl orthosilicates. The effects of substrate concentration, reaction temperature and solvent participation were studied on the oxidation of LA by LOX. The temperature optimum for the oxidation of LA by immobilized LOX was 25 degrees C and values of Km and Vmax for this reaction were 1.7 mM and 0.023 micromol/min respectively. Enzymic activity was stimulated by the addition of 10% (v/v) iso-octane to the reaction mixture. The immobilized LOX preparation showed a degree of substrate preference that demonstrated that 1,3-dilinolein was a better substrate than LA in the oxidation reaction, followed in order by 1-monolinolein, methyl oleate and trilinolein. In general, LOX immobilized in cross-linked phyllosilicates retained the physical and chemical characteristics of free LOX.