Fragment Screening Hit Draws Attention to a Novel Transient Pocket Adjacent to the Recognition Site of the tRNA-Modifying Enzyme TGT.

Fragment-based lead discovery was applied to tRNA-guanine transglycosylase, an enzyme modifying post-transcriptionally tRNAs in Shigella, the causative agent of shigellosis. TGT inhibition prevents translation of Shigella's virulence factor VirF, hence reducing pathogenicity. One discovered fragment opens a transient subpocket in the preQ1-recognition site by pushing back an aspartate residue. This step is associated with reorganization of further amino acids structurally transforming a loop adjacent to the recognition site by duplicating the volume of the preQ1-recognition pocket. We synthesized 6-carboxamido-, 6-hydrazido-, and 4-guanidino-benzimidazoles to target the opened pocket, including a dihydro-imidazoquinazoline with a propyn-1-yl exit vector pointing into the transient pocket and displacing a conserved water network. MD simulations and hydration-site analysis suggest water displacement to contribute favorably to ligand binding. A cysteine residue, exclusively present in bacterial TGTs, serves as gatekeeper of the transient subpocket. It becomes accessible upon pocket opening for selective covalent attachment of electrophilic ligands in eubacterial TGTs.

Fragments as Novel Starting Points for tRNA-Guanine Transglycosylase Inhibitors Found by Alternative Screening Strategies.

Crystallography provides structural information crucial for fragment optimization, however several criteria must be met to screen directly on protein crystals as soakable, well-diffracting specimen must be available. We screened a 96-fragment library against the tRNA-modifying enzyme TGT using crystallography. Eight hits, some with surprising binding poses, were detected. However, the amount of data collection, reduction and refinement is assumed substantial. Therefore, having a reliable cascade of fast and cost-efficient methods available for pre-screening before embarking to elaborate crystallographic screening appears beneficial. This allows filtering of compounds to the most promising hits, available to rapidly progress from hit-to-lead. But how to ensure that this workflow is reliable? To answer this question, we also applied SPR and NMR to the same screening sample to study whether identical hits are retrieved. Upon hit-list comparisons, crystallography shows with NMR and SPR, only one overlapping hit and all three methods shared no common hits. This questions a cascade-type screening protocol at least in the current example. Compared to crystallography, SPR and NMR detected higher percentages of non-active-site binders suggesting the importance of running reporter ligand-based competitive screens in SPR and NMR, a requirement not needed in crystallography. Although not specific, NMR proved a more sensitive method relative to SPR and crystallography, as it picked up the highest numbers of binders.

Mining ZINC Database to Discover Potential Phosphodiesterase 9 Inhibitors Using Structure-Based Drug Design Approach.

In view of the emerging clinical indications for Phosphodiesterase 9 inhibitors e.g. treatment of Alzheimer, diabetes, cancer, and the limited number of its selective inhibitors which possess a single chemical scaffolds, a structure-based approach was undertaken to mine the ZINC database by virtual screening to identify novel PDE9 inhibitors. The database, which was never reported to have been used before for discovery of PDE9 inhibitors, was screened against the ligand binding pocket of the PDE9 complex (PDB:4GH6) using molecular docking programs, MOE and AutoDock Vina in PyRx. Three different scoring functions were used to evaluate the docking poses and scores of the compounds, and the compounds were selected through consensus selection, thus reducing the margin of error in docking. The highest scoring compounds were then selected and purchased for in vitro testing as PDE9 inhibitors and cancer growth inhibitory agents. This led to the discovery of three previously unreported potent PDE 9 inhibitory compounds with two unique chemical scaffolds. Consistent with the role of PDE9 in cancer cell growth, the compounds also inhibited the growth of breast tumor cell lines, MCF-7 and MDA-468 at concentrations comparable to those that inhibited PDE9.