One of the most common methods to treat the electrostatic effect of the environment in QM/MM calculations is to include the MM atoms as point charges in the QM Hamiltonian. In this case, a microiterative geometry optimization ignoring the QM contributions to the forces in the relaxation of the environment cannot yield exact stationary points. One solution that has been suggested in the literature is based on using a constant additive correction to the MM gradient during the microiterations, determined in the preceding macroiteration. Here, we analyze the convergence properties of the gradient correction method and point out that a smooth relaxation is not ensured if the curvature of the approximate, MM-based description of the potential energy surface of the environment is too small in comparison with the exact one. We suggest a computationally cheap second-order correction that uses an estimated Hessian from the Davidon-Fletcher-Powell method to tackle the problems caused by the too small curvature. Test calculations on four metalloenzymatic systems (∼100 QM atoms, ∼2000 relaxed MM atoms, ∼20,000 atoms in total) show that our approach efficiently restores the convergence where gradient correction alone would lead to oscillations.

• MeSH
• Quantum Theory MeSH
• Molecular Dynamics Simulation MeSH
• Software MeSH
• Computational Biology methods   MeSH
• Publication type
• Journal Article MeSH

The glycosylation of cell surface proteins plays a crucial role in a multitude of biological processes, such as cell adhesion and recognition. To understand the process of protein glycosylation, the reaction mechanisms of the participating enzymes need to be known. However, the reaction mechanism of retaining glycosyltransferases has not yet been sufficiently explained. Here we investigated the catalytic mechanism of human isoform 2 of the retaining glycosyltransferase polypeptide UDP-GalNAc transferase by coupling two different QM/MM-based approaches, namely a potential energy surface scan in two distance difference dimensions and a minimum energy reaction path optimisation using the Nudged Elastic Band method. Potential energy scan studies often suffer from inadequate sampling of reactive processes due to a predefined scan coordinate system. At the same time, path optimisation methods enable the sampling of a virtually unlimited number of dimensions, but their results cannot be unambiguously interpreted without knowledge of the potential energy surface. By combining these methods, we have been able to eliminate the most significant sources of potential errors inherent to each of these approaches. The structural model is based on the crystal structure of human isoform 2. In the QM/MM method, the QM region consists of 275 atoms, the remaining 5776 atoms were in the MM region. We found that ppGalNAcT2 catalyzes a same-face nucleophilic substitution with internal return (SNi). The optimized transition state for the reaction is 13.8 kcal/mol higher in energy than the reactant while the energy of the product complex is 6.7 kcal/mol lower. During the process of nucleophilic attack, a proton is synchronously transferred to the leaving phosphate. The presence of a short-lived metastable oxocarbenium intermediate is likely, as indicated by the reaction energy profiles obtained using high-level density functionals.

• MeSH
• Algorithms MeSH
• Models, Chemical * MeSH
• Glycosylation MeSH
• Glycosyltransferases chemistry   ultrastructure   MeSH
• Catalysis MeSH
• Kinetics MeSH
• Protein Conformation MeSH
• Models, Molecular * MeSH
• Computer Simulation MeSH
• Polysaccharides chemistry   ultrastructure   MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

A quantum mechanics (QM)-based scoring function has been applied to complexes of cyclin-dependent kinase 2 (CDK2) and thirty-one pyrazolo[1,5-a]pyrimidine-based inhibitors and their bioisosteres. A hybrid three-layer QM/MM setup (DFT-D/PM6-D3H4X/AMBER in generalized Born solvent) was used here for the first time as an extension of our previous full QM and SQM/MM (SQM means semiempirical QM) approaches. Two approaches to obtain the structures of the CDK2/inhibitor complexes were examined: i) building the modifications from one X-ray structure available coupled with a conformational search and ii) docking the compounds into CDK2. The QM-based scoring entailed a QM/SQM/MM optimization followed by calculations of the binding scores which were subsequently correlated with the experimental binding free energies. The correlation for the building protocol was good (r(2) = 0.64, predictive index = 0.81), whereas the docking approach failed. A decomposition of the interaction energies to ligand fragments enabled us to rationalize the differences in the binding affinities. In conclusion, we have developed and refined a QM-based scoring protocol and successfully applied it to reproduce the binding affinities in congeneric series of CDK2 inhibitors and to rationalize their potency. We thus propose that such a tool can be used in computer-aided rational drug design.

• MeSH
• Cyclin-Dependent Kinase 2 antagonists & inhibitors   chemistry   metabolism   MeSH
• Quantum Theory MeSH
• Humans MeSH
• Pyrazoles chemistry   pharmacology   MeSH
• Pyrimidines chemistry   pharmacology   MeSH
• Molecular Dynamics Simulation MeSH
• Molecular Docking Simulation MeSH
• Thermodynamics MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

A series of carbamate-based inhibitors of glutamate carboxypeptidase II (GCPII) were designed and synthesized using ZJ-43, N-[[[(1S)-1-carboxy-3-methylbutyl]amino]carbonyl]-l-glutamic acid, as a molecular template in order to better understand the impact of replacing one of the two nitrogen atoms in the urea-based GCPII inhibitor with an oxygen atom. Compound 7 containing a C-terminal 2-oxypentanedioic acid was more potent than compound 5 containing a C-terminal glutamic acid (2-aminopentanedioic acid) despite GCPII's preference for peptides containing an N-terminal glutamate as substrates. Subsequent crystallographic analysis revealed that ZJ-43 and its two carbamate analogs 5 and 7 with the same (S,S)-stereochemical configuration adopt a nearly identical binding mode while (R,S)-carbamate analog 8 containing a d-leucine forms a less extensive hydrogen bonding network. QM and QM/MM calculations have identified no specific interactions in the GCPII active site that would distinguish ZJ-43 from compounds 5 and 7 and attributed the higher potency of ZJ-43 and compound 7 to the free energy changes associated with the transfer of the ligand from bulk solvent to the protein active site as a result of the lower ligand strain energy and solvation/desolvation energy. Our findings underscore a broader range of factors that need to be taken into account in predicting ligand-protein binding affinity. These insights should be of particular importance in future efforts to design and develop GCPII inhibitors for optimal inhibitory potency.

• MeSH
• Cell Line MeSH
• Drosophila genetics   MeSH
• Enzyme Assays MeSH
• Glutamate Carboxypeptidase II antagonists & inhibitors   chemistry   metabolism   MeSH
• Protease Inhibitors chemical synthesis   chemistry   metabolism   MeSH
• Carbamates chemical synthesis   chemistry   metabolism   MeSH
• Catalytic Domain MeSH
• Quantum Theory MeSH
• Humans MeSH
• Urea analogs & derivatives   chemical synthesis   chemistry   metabolism   MeSH
• Models, Molecular MeSH
• Stereoisomerism MeSH
• Protein Binding MeSH
• Hydrogen Bonding MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH

Cyclic dinucleotides are second messengers in the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, which plays an important role in recognizing tumor cells and viral or bacterial infections. They bind to the STING adaptor protein and trigger expression of cytokines via TANK binding kinase 1 (TBK1)/interferon regulatory factor 3 (IRF3) and inhibitor of nuclear factor-κB (IκB) kinase (IKK)/nuclear factor-κB (NFκB) signaling cascades. In this work, we describe an enzymatic preparation of 2'-5',3'-5'-cyclic dinucleotides (2'3'CDNs) with use of cyclic GMP-AMP synthases (cGAS) from human, mouse, and chicken. We profile substrate specificity of these enzymes by employing a small library of nucleotide-5'-triphosphate (NTP) analogues and use them to prepare 33 2'3'CDNs. We also determine affinity of these CDNs to five different STING haplotypes in cell-based and biochemical assays and describe properties needed for their optimal activity toward all STING haplotypes. Next, we study their effect on cytokine and chemokine induction by human peripheral blood mononuclear cells (PBMCs) and evaluate their cytotoxic effect on monocytes. Additionally, we report X-ray crystal structures of two new CDNs bound to STING protein and discuss structure-activity relationship by using quantum and molecular mechanical (QM/MM) computational modeling.

• MeSH
• Biological Assay MeSH
• Cytokines metabolism   MeSH
• HEK293 Cells MeSH
• Protein Conformation MeSH
• Leukocytes, Mononuclear drug effects   MeSH
• Humans MeSH
• Membrane Proteins chemistry   metabolism   MeSH
• Nucleotides, Cyclic chemical synthesis   pharmacology   MeSH
• Computer Simulation MeSH
• Gene Expression Regulation drug effects   MeSH
• Structure-Activity Relationship MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Inhibition of the human O-linked β-N-acetylglucosaminidase (hOGA, GH84) enzyme is pharmacologically relevant in several diseases such as neurodegenerative and cardiovascular disorders, type 2 diabetes, and cancer. Human lysosomal hexosaminidases (hHexA and hHexB, GH20) are mechanistically related enzymes; therefore, selective inhibition of these enzymes is crucial in terms of potential applications. In order to extend the structure-activity relationships of OGA inhibitors, a series of 2-acetamido-2-deoxy-d-glucono-1,5-lactone sulfonylhydrazones was prepared from d-glucosamine. The synthetic sequence involved condensation of N-acetyl-3,4,6-tri-O-acetyl-d-glucosamine with arenesulfonylhydrazines, followed by MnO2 oxidation to the corresponding glucono-1,5-lactone sulfonylhydrazones. Removal of the O-acetyl protecting groups by NH3/MeOH furnished the test compounds. Evaluation of these compounds by enzyme kinetic methods against hOGA and hHexB revealed potent nanomolar competitive inhibition of both enzymes, with no significant selectivity towards either. The most efficient inhibitor of hOGA was 2-acetamido-2-deoxy-d-glucono-1,5-lactone 1-naphthalenesulfonylhydrazone (5f, Ki = 27 nM). This compound had a Ki of 6.8 nM towards hHexB. To assess the binding mode of these inhibitors to hOGA, computational studies (Prime protein-ligand refinement and QM/MM optimizations) were performed, which suggested the binding preference of the glucono-1,5-lactone sulfonylhydrazones in an s-cis conformation for all test compounds.

• MeSH
• Antigens, Neoplasm chemistry   metabolism   MeSH
• beta-Hexosaminidase beta Chain chemistry   metabolism   MeSH
• Histone Acetyltransferases chemistry   metabolism   MeSH
• Hyaluronoglucosaminidase chemistry   metabolism   MeSH
• Hydrazones chemical synthesis   chemistry   pharmacology   MeSH
• Enzyme Inhibitors chemical synthesis   chemistry   pharmacology   MeSH
• Lactones chemistry   MeSH
• Humans MeSH
• Molecular Conformation MeSH
• Models, Molecular MeSH
• Oxides chemistry   MeSH
• Manganese Compounds chemistry   MeSH
• Structure-Activity Relationship MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH