Juraj Kóňa

Comparative DFT study on the α-glycosidic bond in reactive species of galactosyl diphosphates

Correct prediction of the structure and energetics along the reaction pathway of the formation or dissociation of the glycosidic bond in sugar phosphates is crucial for the understanding of catalytic mechanism and for the determination of transition state structures of sugar-phosphate processing enzymes. The performance of seven density functional theory (DFT) methods (BLYP, B3LYP, MPW1PW91, MPW1K, MPWB1K, M05 and M05-2X) and two wave function methods (HF and MP2) was tested using four structural models with the activated sugar-phosphate α-glycosidic linkage. The models were chosen based on the crystal structure of the retaining glycosyltransferase LgtC complex with methyl α-d-galactopyranose diphosphate and its 2-fluoro derivative. Results of the MP2 method were used as a benchmark for the other methods. Two structural trends were observed in the calculations: predicted length of the activated C1-O1 glycosidic bond of 1.49–1.63 Å was significantly larger than values of a standard C1-O1 glycosidic bond in crystal structures of carbohydrates (1.39–1.48 Å), and the calculated value depended on the DFT method used. The MPW1K, M05 and M05-2X functionals provided results in closest agreement with those from the MP2 method, the difference being less than 0.02 Å in the calculated glycosidic bond lengths. On the contrary, the BLYP and B3LYP functionals failed to predict sugar diphosphate in the (-sc) conformation as a stable structure. Instead, the only stationary points localized along the C1-O1 dissociation coordinate were oxocarbenium ions at the distance of approximately 2.8 Å. The M05-2X, MPW1K and MPWB1K functionals gave the most reasonable prediction of the thermochemical kinetic parameters, where the formation of the oxocarbenium ion has a slightly endothermic character (0.4–1.7 kJ mol−1) with an activation barrier of 7.9–9.2 kJ mol−1.

α-d-Mannose derivatives as models designed for selective inhibition of Golgi α-mannosidase II

Human Golgi α-mannosidase II (hGM) is a pharmaceutical target for the design of inhibitors with anti-tumor activity. Nanomolar inhibitors of hGM exhibit unwanted co-inhibition of the human lysosomal α-mannosidase (hLM). Hence, improving specificity of the inhibitors directed toward hGM is desired in order to use them in cancer chemotherapy. We report on the rapid synthesis of D-mannose derivatives having one of the RS-, R(SO)- or R(SO(2))- groups at the α-anomeric position. Inhibitory properties of thirteen synthesized α-D-mannopyranosides were tested against the recombinant enzyme Drosophila melanogaster homolog of hGM (dGMIIb) and hLM (dLM408). Derivatives with the sulfonyl [R(SO(2))-] group exhibited inhibitory activities at the mM level toward both dGMIIb (IC(50) = 1.5-2.5 mM) and dLM408 (IC(50) = 1.0-2.0 mM). Among synthesized, only the benzylsulfonyl derivative showed selectivity toward dGMIIb. Its inhibitory activity was explained based on structural analysis of the built 3-D complexes of the enzyme with the docked compounds.

Theoretical study of enzymatic catalysis explains why the trapped covalent intermediate in the E303C mutant of glycosyltransferase GTB was not detected in the wild-type enzyme

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).

'Click chemistry' synthesis of 1-(α-D-mannopyranosyl)-1,2,3-triazoles for inhibition of α-mannosidases.

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 (IC50 values of 50-250 μM), and less potent towards GH38 mannosidases (IC50 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 [IC50(GH38)/IC50(GH47)] of 100 was exhibited by the phenyltriazole conjugate. To understand structure-activity properties of synthesized compounds, 3-D complexes of inhibitors with α-mannosidases were built using molecular docking calculations.

Using DFT methodology for more reliable predictive models: Design of inhibitors of Golgi α-Mannosidase II.

Human Golgi α-mannosidase II (GMII), a zinc ion co-factor dependent glycoside hydrolase (E.C.3.2.1.114), is a pharmaceutical target for the design of inhibitors with anti-cancer activity. The discovery of an effective inhibitor is complicated by the fact that all known potent inhibitors of GMII are involved in unwanted co-inhibition with lysosomal α-mannosidase (LMan, E.C.3.2.1.24), a relative to GMII. Routine empirical QSAR models for both GMII and LMan did not work with a required accuracy. Therefore, we have developed a fast computational protocol to build predictive models combining interaction energy descriptors from an empirical docking scoring function (Glide-Schrödinger), Linear Interaction Energy (LIE) method, and quantum mechanical density functional theory (QM-DFT) calculations. The QSAR models were built and validated with a library of structurally diverse GMII and LMan inhibitors and non-active compounds. A critical role of QM-DFT descriptors for the more accurate prediction abilities of the models is demonstrated. The predictive ability of the models was significantly improved when going from the empirical docking scoring function to mixed empirical-QM-DFT QSAR models (Q(2)=0.78-0.86 when cross-validation procedures were carried out; and R(2)=0.81-0.83 for a testing set). The average error for the predicted ΔGbind decreased to 0.8-1.1kcalmol(-1). Also, 76-80% of non-active compounds were successfully filtered out from GMII and LMan inhibitors. The QSAR models with the fragmented QM-DFT descriptors may find a useful application in structure-based drug design where pure empirical and force field methods reached their limits and where quantum mechanics effects are critical for ligand-receptor interactions. The optimized models will apply in lead optimization processes for GMII drug developments.

N-Benzyl Substitution of Polyhydroxypyrrolidines: The Way to Selective Inhibitors of Golgi α-Mannosidase II

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 (Ki=50–76 μm, as determined by enzyme assays) with a significant selectivity index of IC50(LManII)/IC50(GMIIb) >100. These compounds also showed inhibitory activities in in vitro assays with cancer cell lines (leukemia, IC50=92–200 μm) and low cytotoxic activities in normal fibroblast cell lines (IC50>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.