We have demonstrated that the polyethylene glycol (PEG) corona of long-circulating polymeric nanoparticles (NPs) favors interaction with the amyloid-beta (Aβ(1-42)) peptide both in solution and in serum. The influence of PEGylation of poly(alkyl cyanoacrylate) and poly(lactic acid) NPs on the interaction with monomeric and soluble oligomeric forms of Aβ(1-42) peptide was demonstrated by capillary electrophoresis, surface plasmon resonance, thioflavin T assay, and confocal microscopy, where the binding affected peptide aggregation kinetics. The capture of peptide by NPs in serum was also evidenced by fluorescence spectroscopy and ELISA. Moreover, in silico and modeling experiments highlighted the mode of PEG interaction with the Aβ(1-42) peptide and its conformational changes at the nanoparticle surface. Finally, Aβ(1-42) peptide binding to NPs affected neither complement activation in serum nor apolipoprotein-E (Apo-E) adsorption from the serum. These observations have crucial implications in NP safety and clearance kinetics from the blood. Apo-E deposition is of prime importance since it can also interact with the Aβ(1-42) peptide and increase the affinity of NPs for the peptide in the blood. Collectively, our results suggest that these engineered long-circulating NPs may have the ability to capture the toxic forms of the Aβ(1-42) peptide from the systemic circulation and potentially improve Alzheimer's disease condition through the proposed "sink effect".

Glycosyltransferases are sugar-processing enzymes that require a specific metal ion cofactor for catalysis. In the presence of other ions the catalysis is often impaired. Here, for the first time, the enzymatic catalysis in the presence of various metal ions was modeled for a glycosyltransferase using a large enzymatic model. The catalytic mechanism of α-1,2-mannosyltransferase Kre2p/Mnt1p in the presence of Mn(2+) and other ions (Mg(2+), Zn(2+) and Ca(2+)) was modeled at the two hybrid DFT-QM/MM (M06-2X/OPLS2005 and B3LYP/OPLS2005) levels. Kinetic and structural parameters of transition states and intermediates, as well as kinetic isotope effects, were predicted and compared with available experimental and theoretical data. The catalysis in the presence of the metal ions is predicted as a stepwise SNi-like nucleophilic substitution reaction (DNint*AN(‡)DhAxh) via oxocarbenium ion intermediates. In the rate-determining step the leaving phosphate group of the donor substrate plays a role of the base catalyst. The predicted increased enzymatic reactivity (kcat: Zn(2+) ≈ Mg(2+) < Mn(2+) < Ca(2+)) correlated with the metal ion ability to polarize the Kre2p environment (Mg(2+) > Zn(2+) > Mn(2+) > Ca(2+)). The formation of the retained anomeric configuration in the product is controlled by a strict geometry of the active site of Kre2p. The 6-OH group of the attacking acceptor substrate may assist in protection of the anomeric carbon against unwanted hydrolysis by a through-space interaction with the electron deficient C1[double bond, length as m-dash]O5(+) moiety of the oxocarbenium-ion-like transition state.

Hybrid quantum mechanics/molecular mechanics calculations were used to study the catalytic mechanism of the retaining human α-(1,3)-galactosyltransferase (GTBWT) and its E303C mutant (GTBE303C). Both backside (via covalent glycosyl-enzyme intermediate, CGEI) and frontside SNi-like mechanisms (via oxocarbenium-ion intermediate, OCII) were investigated. The calculations suggest that both mechanisms are feasible in the enzymatic catalysis. The nucleophilic attack of the acceptor substrate to the anomeric carbon of OCII is the rate-determining step with an overall reaction barrier (ΔE(‡) = 19.5 kcal mol(-1)) in agreement with an experimental rate constant (kcat = 5.1 s(-1)). A calculated α-secondary kinetic isotope effect (α-KIE) of 1.27 (GTBWT) and 1.26 (GTBE303C) predicts dissociative character of the transition state in agreement with experimentally measured α-KIE of other retaining glycosyltransferases. Remarkably, stable CGEI in GTBE303C compared with its counterpart in GTBWT may explain why the CGEI has been detected by mass spectrometry only in GTBE303C ( Soya N, Fang Y, Palcic MM, Klassen JS. 2011. Trapping and characterization of covalent intermediates of mutant retaining glycosyltransferases. Glycobiology, 21: 547-552).

Inhibition of the biosynthesis of complex N-glycans in the Golgi apparatus influences progress of tumor growth and metastasis. Golgi α-mannosidase II (GMII) has become a therapeutic target for drugs with anticancer activities. One critical task for successful application of GMII drugs in medical treatments is to decrease their unwanted co-inhibition of lysosomal α-mannosidase (LMan), a weakness of all known potent GMII inhibitors. A series of novel N-substituted polyhydroxypyrrolidines was synthesized and tested with modeled GH38 α-mannosidases from Drosophila melanogaster (GMIIb and LManII). The most potent structures inhibited GMIIb (K =50-76 μm, as determined by enzyme assays) with a significant selectivity index of IC (LManII)/IC (GMIIb) >100. These compounds also showed inhibitory activities in in vitro assays with cancer cell lines (leukemia, IC =92-200 μm) and low cytotoxic activities in normal fibroblast cell lines (IC >200 μm). In addition, they did not show any significant inhibitory activity toward GH47 Aspergillus saitoiα1,2-mannosidase. An appropriate stereo configuration of hydroxymethyl and benzyl functional groups on the pyrrolidine ring of the inhibitor may lead to an inhibitor with the required selectivity for the active site of a target α-mannosidase.

Golgi α-mannosidase II (GM) is a pharmaceutical target for the design of inhibitors with anticancer activity. The known potent GM inhibitors undergo complex interactions with Zn ions and the active-site amino acids, many of which contain ionisable functional groups. Herein, the physical insight into the ligandreceptor interactions has been provided based on energy decomposition techniques: SAPT (symmetry adapted perturbation theory) and FMO-PIEDA (fragment molecular orbital-pair interaction energy decomposition analysis) for a large GM active-site cluster. Protonation-dependent molecular recognition in Golgi α-mannosidase was demonstrated for five inhibitors, mannose, and its transition state. Zn ion and Asp472 induce the key interactions with the deprotonated inhibitors (bearing an amino group in the neutral state), followed by Asp92 and Asp341. This interaction pattern is consistent for all the studied inhibitors and is similar to the interaction pattern of the enzyme native substrate - mannose. The interactions with Zn ion become repulsive for the protonated states of the inhibitors (bearing an amino group with +1 charge) and the mannosyl transition state. The importance of Asp92 and Asp204 considerably increases, while the interactions with Asp472 and Asp341 are slightly modified. The interaction pattern for the protonated ligands seems to have an oxocarbenium transition state-like character, rather than a Michaelis complex of GM. The electrostatic interactions with amino acids coordinating zinc ion are of key importance for both the neutral and protonated states of the inhibitors. The ligand's diol group has a dual role as an electron donor, coordinating zinc ion, and as an electron acceptor, interacting with Asp92 and Asp472 via strong hydrogen bonds. This interaction pattern is an essential structural feature of the potent GM inhibitors, which is consistent with the experimental findings. Based on the calculations, either the protonated or deprotonated state of the ligand may be the active form of the GM inhibitor, exhibiting different interacting patterns.

Ab initio [Hartree-Fock (HF)] and density functional theory (B3LYP) ECPcalculations on intermediates and transition states of the reduction of phenylseleninic acid with hydrogen sulfide and of seleninic acid with benzenethiol as models for the species involved in the redox cycle of selenoenzymes are presented. Selenenyl sulfide is found to be the final product of the reduction reaction with sulfides. Further reduction to the selenol requires surmounting a substantial barrier (∼50 kcal mol −1 ). The final step of the redox cycle-oxidation of the selenol by hydrogen peroxide-is strongly exothermic. In case of the unsubstituted derivative H 2 Se three mechanisms for this oxidation are found [concerted and stepwise 1,2-hydrogen and oxygen shift (TS6A, TS6B, resp.) and a four-membered transition state TS6C]. For the oxidation of phenylselenol only TS6A and TS6B are obtained. For nearly all calculated structures the HF wave functions show a triplet instability. In contrast, at the B3LYP level only TS6C has a significant triplet instability. On the basis of QCISD(T)//QCISD results a mechanism involving TS6C seems highly unlikely.

Three new triazole conjugates derived from D-mannose were synthesized and assayed in in vitro assays to investigate their ability to inhibit α-mannosidase enzymes from the glycoside hydrolase (GH) families 38 and 47. The triazole conjugates were more selective for a GH47 α-mannosidase (Aspergillus saitoi α1,2-mannosidase), showing inhibition at the micromolar level (IC 50 values of 50-250 μM), and less potent towards GH38 mannosidases (IC 50 values in the range of 0.5-6 mM towards jack bean α-mannosidase or Drosophila melanogaster lysosomal and Golgi α-mannosidases). The highest selectivity ratio [IC 50 (GH38)/IC 50 (GH47)] of 100 was exhibited by the triazole conjugate 6. To understand structure-activity properties of synthesized compounds, 3-D complexes of inhibitors with α-mannosidases were built using molecular docking calculations.

Three approaches of computational chemistry [quantum mechanics (QM) calculations, docking, and molecular dynamics (MD) simulations] were used to investigate the redox cycle of bovine erythrocyte glutathione peroxidase from class 1 (GPx1, EC 1.11.1.9). The pKa calculations for two redox states of the active-site selenocysteine of GPx1 (selenol, Sec45-SeH, and selenenic acid, Sec45-SeOH) were estimated using a bulk solvent model (B3LYP-IEFPCM and B3LYP-CPCM-COSMO-RS). The calculated pKa values of Sec45-SeH and Sec45-SeOH were corrected via a simple linear fit to a training set of organoselenium compounds, which consisted of aliphatic selenols and aromatic selenenic acids with available experimental pKa values. Based on docking calculations, binding sites for both molecules of the cofactor glutathione (GSH) are described. MD simulations on the dimer of GPx1 have been performed for all chemical states of the redox cycle: without GSH and with one or two molecules of GSH bound at the active site. Conformational analyses of MD trajectories indicate high mobility of the Arg177 and His79 residues. These residues can approach the vicinity of Sec45 and take part in the catalytic mechanism. On the basis of the calculated data, new atomistic details for a generally accepted mechanism of GPx1 are proposed.