Gaucher disease (GD), the most prevalent lysosomal storage disease, is caused by a deficiency of glucocerebrosidase (GCase). The identification of small molecules acting as agents for enzyme enhancement therapy is an attractive approach for treating different forms of GD. A thermal denaturation assay utilizing wild type GCase was developed to screen a library of 1,040 Food and Drug Administration-approved drugs. Ambroxol (ABX), a drug used to treat airway mucus hypersecretion and hyaline membrane disease in newborns, was identified and found to be a pH-dependent, mixed-type inhibitor of GCase. Its inhibitory activity was maximal at neutral pH, found in the endoplasmic reticulum, and undetectable at the acidic pH of lysosomes. The pH dependence of ABX to bind and stabilize the enzyme was confirmed by monitoring the rate of hydrogen/deuterium exchange at increasing guanidine hydrochloride concentrations. ABX treatment significantly increased N370S and F213I mutant GCase activity and protein levels in GD fibroblasts. These increases were primarily confined to the lysosome-enriched fraction of treated cells, a finding confirmed by confocal immunofluorescence microscopy. Additionally, enhancement of GCase activity and a reduction in glucosylceramide storage was verified in ABX-treated GD lymphoblasts (N370S/N370S). Hydrogen/deuterium exchange mass spectrometry revealed that upon binding of ABX, amino acid segments 243-249, 310 -312, and 386 -400 near the active site of GCase are stabilized. Consistent with its mixedtype inhibition of GCase, modeling studies indicated that ABX interacts with both active and non-active site residues. Thus, ABX has the biochemical characteristics of a safe and effective enzyme enhancement therapy agent for the treatment of patients with the most common GD genotypes.

Understanding antigen-antibody interactions at the sub-molecular level is of particular interest for scientific, regulatory, and intellectual property reasons, especially with increasing demand for monoclonal antibody therapeutic agents. Although various techniques are available for the determination of an epitope, there is no widely applicable, high-resolution, and reliable method available. Here, a combination approach using amide hydrogen/deuterium exchange coupled with proteolysis and mass spectrometry (HDX-MS) and computational docking was applied to investigate antigen-antibody interactions. HDX-MS is a widely applicable, medium-resolution, medium-throughput technology that can be applied to epitope identification. First, the epitopes of cytochrome c-E8, IL-13-CNTO607, and IL-17A-CAT-2200 interactions identified using the HDX-MS method were compared with those identified by X-ray co-crystal structures. The identified epitopes are in good agreement with those identified using high-resolution X-ray crystallography. Second, the HDX-MS data were used as constraints for computational docking. More specifically, the non-epitope residues of an antigen identified using HDX-MS were designated as binding ineligible during computational docking. This approach, termed HDX-DOCK, gave more tightly clustered docking poses than stand-alone docking for all antigen-antibody interactions examined and improved docking results significantly for the cytochrome c-E8 interaction.

Virtual screening (VS), if applied appropriately, could significantly shorten the hit identification and hit-to-lead processes in drug discovery. Recently, the version of VS that is based upon similarity to a pharmacophore has received increased attention. This is due to two major factors: first, the public availability of the ZINC1 conformational database has provided a large selection pool with high-quality and purchasable small molecules; second, new technology has enabled a more accurate and flexible definition of pharmacophore models coupled with an efficient search speed. The major goal of this study was to achieve improved specificity and sensitivity of pharmacophore-based VS by optimizing the variables used to generate conformations of small molecules and those used to construct pharmacophore models from known inhibitors or from inhibitor-protein complex structures. By using human immunodeficiency virus protease and its inhibitors (PIs) as a case study, the impact of the key variables, including the selection of chemical features, involvement of excluded volumes (EV), the tolerance radius of excluded volumes, energy windows, and the maximum number of conformers in conformation generation, was explored. Protein flexibility was simulated by adjusting the sizes of EV. Our best pharmacophore model, combining both chemical features and excluded volumes, was able to correctly identify 60 out of 75 structurally diverse known PIs, while misclassifying only 5 out of 75 similar compounds that are not inhibitors. To evaluate the specificity of the model, 1193 oral drugs on the market were screened, and 25 original hits were identified, including 5 out of 6 known PI drugs.

Analogs of the flexible dopamine reuptake inhibitor, GBR 12909 (1), may have potential utility in the treatment of cocaine abuse. As a first step in the 3D-QSAR modeling of the dopamine transporter (DAT)/serotonin transporter (SERT) selectivity of these compounds, we carried out conformational analyses of two analogs of 1: a piperazine (2) and a related piperidine (3). Ensembles of conformers consisting of local minima on the potential energy surface of the molecule were generated in the vacuum phase and in implicit solvent by random search conformational analysis using the Tripos and MMFF94 force fields. Some differences were noted in the conformer populations due to differences in the treatment of the tertiary amine nitrogen and ether oxygen atom types by the force fields. The force fields also differed in their descriptions of internal rotation around the C(sp³)-O(sp³) bond proximal to the bisphenyl moiety. Molecular orbital calculations at the HF/6-31G(d) and B3LYP/6-31G(d) levels of C-O internal rotation in model compound (5), designed to model the effect of the proximity of the bisphenyl group on C-O internal rotation, showed a broad region of low energy between -60° to 60° with minima at both -60° and 30° and a low rotational barrier at 0°, in closer agreement with the MMFF94 results than the Tripos results. Molecular mechanics calculations on model compound (6) showed that the MMFF94 force field was much more sensitive than the Tripos force field to the effects of the bisphenyl moiety on C-O internal rotation.