Binding interactions and in silico ADME prediction of isoconessimine derivatives as potent acetylcholinesterase inhibitors.

In pursuit of new acetylcholinesterase (AChE) inhibitors for treating Alzheimer's disease (AD), a series of ten previously synthesized isoconessimine compounds (7a-7j) was in silico investigated for their binding interactions with AChE and pharmacokinetics based on absorption, distribution, metabolism, and excretion (ADME) properties using molecular docking, ONIOM (Our own N-layered Integrated molecular Orbital and molecular Mechanics) method and SwissADME tools. Docking experiments showed that all compounds bind within the active site gorge of AChE (PDB entry 1C2B), posing its aryloxy-substitutional ethyl group to catalytic site and conessine skeleton to peripheral anionic site. ONIOM interaction energy was used as an ONIOM score to improve docking score, and it ranked 7b as the most potent AChE inhibitor, in agreement with previous experiment. Residues, ASP74, TRP86, GLY122, GLU202, TRP286, GLU292, SER293, ILE294, TYR337, TYR341, and HIS447 were identified as important for the binding of the AChE-isoconessimine complex. The SwissADME investigation suggested that four compounds (7a, 7c, 7d and 7f) agree with the rules of drug-likeness. The steric and electronic effects on the aryloxy-substitutional ethyl group as important factors in the AChE inhibition were also discussed, which brings a better understanding of Alzheimer's disease drug development.

Structural basis for inhibition of a GH116 ß-glucosidase and its missense mutants by GBA2 inhibitors: Crystallographic and quantum chemical study.

The crystal structure of the Thermoanaerobacterium xylanolyticum in glycoside hydrolase family 116 (TxGH116) ß-glucosidase provides a structural model for human GBA2 glucosylceramidase, an enzyme defective in hereditary spastic paraplegia and a potential therapeutic target for treating Gaucher disease. To assess the therapeutic potential of known inhibitors, the X-ray structure of TxGH116 in complex with isofagomine (IFG) was determined at 2.0 Å resolution and showed the IFG bound in a relaxed chair conformation. The binding of IFG and 7 other iminosugar inhibitors to wild-type and mutant enzymes (Asp508His and Arg786His) mimicking GBA2 pathogenic variants was then evaluated computationally by two-layered ONIOM calculations (at the B3LYP:PM7 level). Calculations showed that six charged residues, Glu441, Asp452, His507, Asp593, Glu777, and Arg786 influence inhibitor binding most. His507, Glu777 and Arg786, form strong hydrogen bonds with the inhibitors (∼1.4-1.6 Å). Thus, the missense mutation of one of these residues in Arg786His has a greater effect on the interaction energies for all inhibitors compared to Asp508His. In line with the experimental data for the inhibitors that have been tested, the favorable interaction energy between the inhibitors and the TxGH116 protein followed the trend: isofagomine > 1-deoxynojirimycin > glucoimidazole > N-butyl-deoxynojirimycin ≈ N-nonyl-deoxynojirimycin > conduritol B epoxide ≈ azepane 1 > azepane 2. The obtained structural and energetic properties and comparison to the GBA2 model can lead to understanding of structural requirement for inhibitor binding in GH116 to aid the design of high potency GBA2 inhibitors.

Multiscale QM/MM Simulations Identify the Roles of Asp239 and 1-OH···Nucleophile in Transition State Stabilization in Arabidopsis thaliana Cell-Wall Invertase 1.

Arabidopsis thaliana cell-wall invertase 1 (AtCWIN1), a key enzyme in sucrose metabolism in plants, catalyzes the hydrolysis of sucrose into fructose and glucose. AtCWIN1 belongs to the glycoside hydrolase GH-J clan, where two carboxylate residues (Asp23 and Glu203 in AtCWIN1) are well documented as a nucleophile and an acid/base catalyst. However, details at the atomic level about the role of neighboring residues and enzyme-substrate interactions during catalysis are not fully understood. Here, quantum mechanical/molecular mechanical (QM/MM) free-energy simulations were carried out to clarify the origin of the observed decreased rates in Asp239Ala, Asp239Asn, and Asp239Phe in AtCWIN1 compared to the wild type and delineate the role of Asp239 in catalysis. The glycosylation and deglycosylation steps were considered in both wild type and mutants. Deglycosylation is predicted to be the rate-determining step in the reaction, with a calculated overall free-energy barrier of 15.9 kcal/mol, consistent with the experimental barrier (15.3 kcal/mol). During the reaction, the -1 furanosyl ring underwent a conformational change corresponding to 3E ↔ [E2]⧧ ↔ 1E according to the nomenclature of saccharide structures along the full catalytic reaction. Asp239 was found to stabilize not only the transition state but also the fructosyl-enzyme intermediate, which explains findings from previous structural and mutagenesis experiments. The 1-OH···nucleophile interaction has been found to provide an important contribution to the transition state stabilization, with a contribution of ∼7 kcal/mol, and affected glycosylation more significantly than deglycosylation. This study provides molecular insights that improve the current understanding of sucrose binding and hydrolysis in members of clan GH-J, which may benefit protein engineering research. Finally, a rationale on the sucrose inhibitor configuration in chicory 1-FEH IIa, proposed a long time ago in the literature, is also provided based on the QM/MM calculations.

A computational study of the reaction mechanism and stereospecificity of dihydropyrimidinase.

Dihydropyrimidinase (DHPase) is a key enzyme in the pyrimidine pathway, the catabolic route for synthesis of ß-amino acids. It catalyses the reversible conversion of 5,6-dihydrouracil (DHU) or 5,6-dihydrothymine (DHT) to the corresponding N-carbamoyl-ß-amino acids. This enzyme has the potential to be used as a tool in the production of ß-amino acids. Here, the reaction mechanism and origin of stereospecificity of DHPases from Saccharomyces kluyveri and Sinorhizobium meliloti CECT4114 were investigated and compared using a quantum mechanical cluster approach based on density functional theory. Two models of the enzyme active site were designed from the X-ray crystal structure of the native enzyme: a small cluster to characterize the mechanism and the stationary points and a large model to probe the stereospecificity and the role of stereo-gate-loop (SGL) residues. It is shown that a hydroxide ion first performs a nucleophilic attack on the substrate, followed by the abstraction of a proton by Asp358, which occurs concertedly with protonation of the ring nitrogen by the same residue. For the DHT substrate, the enzyme displays a preference for the L-configuration, in good agreement with experimental observation. Comparison of the reaction energetics of the two models reveals the importance of SGL residues in the stereospecificity of catalysis. The role of the conserved Tyr172 residue in transition-state stabilization is confirmed as the Tyr172Phe mutation increases the activation barrier of the reaction by ∼8 kcal mol-1. A detailed understanding of the catalytic mechanism of the enzyme could offer insight for engineering in order to enhance its activity and substrate scope.

Active site dynamics and catalytic mechanism in arabinan hydrolysis catalyzed by GH43 endo-arabinanase from QM/MM molecular dynamics simulation and potential energy surface.

The endo-1,5-α-L-arabinanases, belonging to glycoside hydrolase family 43 (GH43), catalyse the hydrolysis of α-1,5-arabinofuranosidic bonds in arabinose-containing polysaccharides. These enzymes are proposed targets for industrial and medical applications. Here, molecular dynamics (MD), potential energy surface and free energy (potential of mean force) simulations are undertaken using hybrid quantum mechanical/molecular mechanical (QM/MM) potentials to understand the active site dynamics, catalytic mechanism and the electrostatic influence of active site residues of the GH43 endo-arabinanase from G. stearothermophilus. The calculated results give support to the single-displacement mechanism proposed for the inverting GH43 enzymes: first a proton is transferred from the general acid E201 to the substrate, followed by a nucleophilic attack by water, activated by the general base D27, on the anomer carbon. A conformational change (2E ↔E3 ↔ 4E) in the -1 sugar ring is observed involving a transition state featuring an oxocarbenium ion character. Residues D87, K106, H271 are highlighted as potential targets for future mutation experiments in order to increase the efficiency of the reaction. To our knowledge, this is the first QM/MM study providing molecular insights into the glycosidic bond hydrolysis of a furanoside substrate by an inverting GH in solution.Communicated by Ramaswamy H. Sarma.