Phytochemical analysis with the antioxidant and aldose reductase inhibitory capacities of Tephrosia humilis aerial parts’ extracts

The aerial parts of Tephrosia humilis were tested about their antioxidant potential, their ability to inhibit the aldose/aldehyde reductase enzymes and their phenolic content. The plant material was exhaustively extracted with petroleum ether, dichloromethane and methanol, consecutively. The concentrated methanol extract was re-extracted, successively, with diethyl ether, ethyl acetate and n-butanol. All extracts showed significant antioxidant capacity, but the most effective was the ethyl acetate extract. As about the aldose reductase inhibition, all fractions, except the aqueous, were strong inhibitors of the enzyme, with the n-butanolic and ethyl acetate fractions to inhibit the enzyme above 75%. These findings provide support to the ethnopharmacological usage of the plant as antioxidant and validate its potential to act against the long-term diabetic complications. The phytochemical analysis showed the presence of 1,4-dihydroxy-3,4-(epoxyethano)-5-cyclohexene(1), cleroindicin E(2), lupeol(3), methyl p-coumarate(4), methyl 4-hydroxybenzoate(5), prunin(6), 5,7,2?,5?-tetrahydroxyflavanone 7-rutinoside(7), protocatechuic acid(8), luteolin 7-glucoside(9), apigenin(10), naringin(11), rhoifolin(12) and luteolin 7-glucuronate(13).     

Solvent as a Probe of Active Site Motion and Chemistry during the Hydrogen Tunnelling Reaction in Morphinone Reductase

The reductive half‐reaction of morphinone reductase involves a hydride transfer from enzyme‐bound β‐nicotinamide adenine dinucleotide (NADH) to a flavin mononucleotide (FMN). We have previously demonstrated that this step proceeds via a quantum mechanical tunnelling mechanism. Herein, we probe the effect of the solvent on the active site chemistry. The pK_a of the reduced FMN N1 is 7.4±0.7, based on the pH‐dependence of the FMN midpoint potential. We rule out that protonation of the reduced FMN N1 is coupled to the preceding H‐transfer as both the rate and temperature‐dependence of the reaction are insensitive to changes in solution pH above and below this pK_a. Further, the solvent kinetic isotope effect is ～1.0 and both the 1° and 2° KIEs are insensitive to solution pH. The effect of the solvent’s dielectric constant is investigated and the rate of H‐transfer is found to be unaffected by changes in the dielectric constant between ～60 and 80. We suggest that, while there is crystallographic evidence for some water in the active site, the putative promoting motion involved in the H‐tunnelling reaction is insensitive to such changes.     

Modeling the inhibition of quadruple mutant <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> dihydrofolate reductase by pyrimethamine derivatives

Modeling studies were performed on known inhibitors of the quadruple mutant Plasmodium falciparum dihydrofolate reductase (DHFR). GOLD was used to dock 32 pyrimethamine derivatives into the active site of DHFR obtained from the x-ray crystal structure 1J3K.pdb. Several scoring functions were evaluated and the Molegro Protein-Ligand Interaction Score was determined to have one of the best correlation to experimental pK _i . In conjunction with Protein-Ligand Interaction scores, predicted binding modes and key protein-ligand interactions were evaluated and analyzed in order to develop criteria for selecting compounds having a greater chance of activity versus resistant strains of Plasmodium falciparum. This methodology will be used in future studies for selection of compounds for focused screening libraries.     

The importance of the long type 1 copper-binding loop of nitrite reductase for structure and function

The long 15-residue type 1 copper-binding loop of nitrite reductase has been replaced with that from the cupredoxin amicyanin (7 residues). This sizable loop contraction does not have a significant effect on the spectroscopy, and therefore, the structures of both the type 1 and type 2 Cu(II) sites. The crystal structure of this variant with Zn(II) at both the type 1 and type 2 sites has been determined. The coordination geometry of the type 2 site is almost identical to that found in the wild-type protein. However, the structure of the type 1 centre changes significantly upon metal substitution, which is an unusual feature for this class of site. The positions of most of the coordinating residues are altered of which the largest difference was observed for the coordinating His residue in the centre of the mutated loop. This ligand moves away from the active site, which results in a more open metal centre with a coordinating water molecule. Flexibility has been introduced into this region of the protein. The 200 mV increase in the reduction potential of the type 1 copper site indicates that structural changes upon reduction must stabilise the cuprous form. The resulting unfavourable driving force for electron transfer between the two copper sites, and an increased reorganisation energy for the type 1 centre, contribute to the loop variant having very little nitrite reductase activity. The extended type 1 copper-binding loop of this enzyme makes a number of interactions that are important for maintaining quaternary structure.     

A Minimal Functional Complex of Cytochrome P450 and FBD of Cytochrome P450 Reductase in Nanodiscs

Structural interactions that enable electron transfer to cytochrome‐P450 (CYP450) from its redox partner CYP450‐reductase (CPR) are a vital prerequisite for its catalytic mechanism. The first structural model for the membrane‐bound functional complex to reveal interactions between the full‐length CYP450 and a minimal domain of CPR is now reported. The results suggest that anchorage of the proteins in a lipid bilayer is a minimal requirement for CYP450 catalytic function. Akin to cytochrome‐b₅ (cyt‐b₅), Arg 125 on the C‐helix of CYP450s is found to be important for effective electron transfer, thus supporting the competitive behavior of redox partners for CYP450s. A general approach is presented to study protein–protein interactions combining the use of nanodiscs with NMR spectroscopy and SAXS. Linking structural details to the mechanism will help unravel the xenobiotic metabolism of diverse microsomal CYP450s in their native environment and facilitate the design of new drug entities.     

Synthesis of quaternary 8-(1-acylethene-1-yl)-13-methylcoptisine chlorides and their selective growth inhibitory activity between human cancer cell lines and normal intestinal epithelial cell-6

Quaternary 8-(1-acylethene-1-yl)-13-methylcoptisine chlorides targeting TrxRs were designed and synthesized. An in vitro evaluation for the growth inhibitory activities against cancer cell lines and for the viability of the normal intestinal epithelial cell-6 cell line is reported.     

Heme Fe?SO2− intermediates in sulfite reduction: Contrasts with Fe?OO2− species from oxygen–oxygen bond activating systems

Sulfite reductase (SiR) catalyzes a six electron and six proton reduction of sulfite to sulfide. Similarly to the cytochrome P450 (cytP450) family, the active site in SiR contains a (partially reduced) heme bound axially to a cysteinate ligand—though with an extra Fe₄S₄ cluster. Fe(III) SO²⁻, Fe(III) SOH⁻, and Fe(III) SO(H₂) intermediates have been proposed for the catalytic cycle of SiR, leading to a formally Fe(V)S species—akin to the widely accepted reaction mechanism in cytP450. Here, density functional theory (DFT) data is reported for of such FeSO(H₂) intermediates. The Fe(III) SO²⁻ models display relatively high energies for homolytic bond breaking compared to their isomeric oxygen‐bound Fe(III) OS²⁻ models, and thus offer a better alternative in terms of avoiding radical side products able to induce enzyme suicide. This could be due to the fact that the (iron‐bound) sulfur is more active from a redox standpoint compared to oxygen, thus permitting the departing oxygen to maintain a redox‐inert state. Di‐protonation of the oxygen is computed to lead to a compound I type Fe(IV)S coupled to a porphyrin radical anion—consistent with an intermediate previously observed by x‐ray crystallography.     

Synthesis and anticancer activity of novel NHC-gold(I)-sugar complexes

Gold(I) complexes containing stabilising ligands such as phosphines or N-heterocyclic carbenes (NHCs) are known to be inhibitors of the enzyme thioredoxin reductase (TrxR) and therefore act as potential apoptosis-inducing anticancer drug candidates. The conjugation of biomolecules overexpressed in cancer cells to the gold complexes makes them semi-targeted metabolites. Auranofin, an anti-arthritis agent, encompasses this property and exhibits anti-tumour activities. The synthesis, characterization and biological evaluation of four novel N-heterocyclic carbene-gold(I)-thiosugar complexes derived from glucose, lactose and galactose is reported. The reactions of 1,3-dibenzyl-4,5-diphenyl-imidazol-2-ylidene gold(I) chloride (NHC1-Au-Cl) with pre-synthesized glycosyl thiols under mildly basic conditions gave the desired NHC-Au-thiosugar complexes in high to excellent yields (79–91%). The complexes retain the strong and redox-active Au-S bond contained in Auranofin. All complexes showed good solubility in biological media and were tested against the NCI 60 cancer cell panel for cytotoxicity. The synthesized NHC1-Au derivatives showed good activity in the medium to low micromolar region, while complex 2 showed activity in the low micromolar to nanomolar region against the tested cell lines. To provide a theoretical structure of 4, computational calculations were carried out based on the crystal structures of NHC-Au-SCN and NHC-Au-S-C₆H₄OMe.     

The production and properties of a new xylose reductase from fungus

Neurospora crassa XI was found to ferment xylose and glucose simultaneously. Xylose was the appropriate inducer for the production of xylose reductase that had two isoenzymes designated as EI and EII. Both EI and EII, which were purified by affinity chromatography, had NADPH-dependent xylose reductase activities. EII also had NADH-dependent activity, and EI is the only xylose reductase found so far without any NADH-dependent activity. EI and EII had MWs of 30 kDa and 27 kDa, and pIs of 5.6 and 5.2, respectively. The specifities of EI and EII against triose, pentoses, and hexoses were studied. The K_ms against xylose for EI and EII were 2.3 mM and 1.1 mM respectively, which were much lower than those of the xylose reductase from yeast.     

FLOG: A system to select ‘quasi-flexible’ ligands complementary to a receptor of known three-dimensional structure

Summary We present a system, FLOG (Flexible Ligands Oriented on Grid), that searches a database of 3D coordinates to find molecules complementary to a macromolecular receptor of known 3D structure. The philosophy of FLOG is similar to that reported for DOCK [Shoichet, B.K. et al., J. Comput. Chem., 13 (1992) 380]. In common with that system, we use a match center representation of the volume of the binding cavity and we use a clique-finding algorithm to generate trial orientations of each candidate ligand in the binding site. Also we use a grid representation of the receptor to assess the fit of each orientation. We have introduced a number of novel features within this paradigm. First, we address ligand flexibility by including up to 25 explicit conformations of each structure in our databases. Nonhydrogen atoms in each database entry are assigned one of seven atom types (anion, cation, donor, acceptor, polar, hydrophobic and other) based on their local bonded chemical environments. Second, we have devised a new grid-based scoring function compatible with this heavy atom representation of the ligands. This includes several potentials (electrostatic, hydrogen bonding, hydrophobic and van der Waals) calculated from the location of the receptor atoms. Third, we have improved the fitting stage of the search. Initial dockings are generated with a more efficient clique-finding algorithm. This new algorithm includes the concept of essential points, match centers that must be paired with a ligand atom. Also, we introduce the use of a rapid simplex-based rigid-body optimizer to refine the orientations. We demonstrate, using dihydrofolate reductase as a sample receptor, that the FLOG system can select known inhibitors from a large database of drug-like compounds.