Insights into Glucose-6-phosphate Allosteric Activation of ß-Glucosidase A.

Second-generation ethanol production involves the use of agricultural and forestry waste as feedstock, being an alternative to the first-generation technology as it relies on low-cost abundant residues and does not affect food agriculture. However, the success of second-generation biorefineries relies on energetically efficient processes and effective enzyme cocktails to convert cellulose into fermentable sugars. ß-glucosidases catalyze the last step on the enzymatic hydrolysis of cellulose; however, they are often inhibited by glucose. Previous studies demonstrated that glucose-6-phosphate (G6P) is a positive allosteric modulator of Bacillus polymyxa ß-glucosidase A, improving enzymatic efficiency, providing thermoresistance, and imparting glucose tolerance. However, the precise molecular details of G6P-ß-glucosidase A interactions have not yet been described so far. We investigated the molecular details of G6P binding into B. polymyxa ß-glucosidase A through in silico docking using the site identification by ligand competitive saturation technology followed by site-directed mutagenesis studies, from which an allosteric binding site for G6P was identified. In addition, a mechanistic shift toward the transglycosylation reaction as opposed to hydrolysis was observed in the presence of G6P, suggesting a new role of G6P allosteric modulation of the catalytic activity of ß-glucosidase A.

Cyclopropyl-containing positive allosteric modulators of metabotropic glutamate receptor subtype 5.

Positive allosteric modulators (PAMs) binding to the transmembrane (TM) domain of metabotropic glutamate receptor 5 (mGluR5) are promising therapeutic agents for psychiatric disorders and traumatic brain injury (TBI). Novel PAMs based on a trans-2-phenylcyclopropane amide scaffold have been designed and synthesized. Facilitating ligand design and allowing estimation of binding affinities to the mGluR5 TM domain was the novel computational strategy, site identification by ligand competitive saturation (SILCS). The potential protective activity of the new compounds was evaluated using nitric oxide (NO) production in BV2 microglial cell cultures treated with lipopolysaccharide (LPS), and the toxicity of the new compounds tested using a cell viability assay. One of the new compounds, 3a, indicated promising activity with potency of 30 µM, which is 4.5-fold more potent than its lead compound 3,3'-difluorobenzaldazine (DFB), and showed no detectable toxicity with concentrations as high as 1000 µM. Thus this compound represents a new lead for possible development as treatment for TBI and related neurodegenerative disorders.

Acyl-2-aminobenzimidazoles: a novel class of neuroprotective agents targeting mGluR5.

Positive allosteric modulators (PAMs) of the metabotropic glutamate receptor 5 (mGluR5) are promising therapeutic agents for treating traumatic brain injury (TBI). Using computational and medicinal methods, the structure-activity relationship of a class of acyl-2-aminobenzimidazoles (1-26) is reported. The new compounds are designed based on the chemical structure of 3,3'-difluorobenzaldazine (DFB), a known mGluR5 PAM. Ligand design and prediction of binding affinities of the new compounds have been performed using the site identification by ligand competitive saturation (SILCS) method. Binding affinities of the compounds to the transmembrane domain of mGluR5 have been evaluated using nitric oxide (NO) production assay, while the safety of the compounds is tested. One new compound found in this study, compound 22, showed promising activity with an IC50 value of 6.4 µM, which is ∼20 fold more potent than that of DFB. Compound 22 represents a new lead for possible development as a treatment for TBI and related neurodegenerative conditions.

Inclusion of multiple fragment types in the site identification by ligand competitive saturation (SILCS) approach.

The site identification by ligand competitive saturation (SILCS) method identifies the location and approximate affinities of small molecular fragments on a target macromolecular surface by performing molecular dynamics (MD) simulations of the target in an aqueous solution of small molecules representative of different chemical functional groups. In this study, we introduce a set of small molecules to map potential interactions made by neutral hydrogen bond donors and acceptors and charged donor and acceptor fragments in addition to nonpolar fragments. The affinity pattern is obtained in the form of discretized probability or, equivalently, free energy maps, called FragMaps, which can be visualized with the target surface. We performed SILCS simulations for four proteins for which structural and thermodynamic data is available for multiple diverse ligands. Good overlap is shown between high affinity regions identified by the FragMaps and the crystallographic positions of ligand functional groups with similar chemical functionality, thus demonstrating the validity of the qualitative information obtained from the simulations. To test the ability of FragMaps in providing quantitative predictions, we calculate the previously introduced ligand grid free energy (LGFE) metric and observe its correspondence with experimentally measured binding affinity. LGFE is computed for different conformational ensembles and improvement in prediction is shown with increasing ligand conformational sampling. Ensemble generation includes a Monte Carlo sampling approach that uses the GFE FragMaps directly as the energy function. The results show that some but not all experimental trends are predicted and warrant improvements in the scoring methodology. In addition, the potential utility of atom-based free energy contributions to the LGFE scores and the use of multiple ligands in SILCS to identify displaceable water molecules during ligand design are discussed.