Like compound substitution on compound contains amino and ca
Like compound substitution on compound () contains amino and carboxamide groups attached to adjacent ring positions. These groups have similar interactions as compound with the protein backbone and Glu114. The triazole core is coordinated with Lys291. The triazole ring core π-stacks with Tyr226. The furan remains in the same plane as the triazole occupying a similar position as occupied by many of the inhibitors discussed here and fills the entry to the hydrophobic pocket. A high-concentration fragment screen designed to find novel starting points for inhibitors and new interactions for LigA inhibition was run with and prioritized (see ). The hit evaluation included a 2D NMR screen to determine where the compounds were. Chemical shift perturbations observed in the protein NMR experiments are shown in for compounds and . The 2D NMR confirmation offers the advantage of screening libraries with higher capacity than X-ray crystallography. If mapped through the use of known inhibitors the chemical shift changes observed in protein NMR can be used to confirm interactions with specific residues, but not the exact binding mode which would require more laborious full structural determination. As a further confirmation of binding to the target and to determine their binding modes to LigA X-ray co-crystal structures were determined. Crystal structures of the fragments bound to LigA are shown below. In the case of compound the phenolic OH hydrogen bonds with Lys291 and the methoxy group occupies the entry to the ribose binding site. The ring nitrogen adjacent to the phenolic OH also H-bonds with Lys291 (a). Compound binds similarly to compound . The carboxylic Gefitinib hydrochloride is positioned in a hydrophobic pocket (b). Compound shares a core with compound but has a different substitution that could be combined to make new analogs. Replacing the carboxylate with a small lipophilic group would build into the hydrophobic pocket as in compound or , and adding a carboxamide at the 6 position of the core to form H-bonds as in compound . The amino group of compound hydrogen bonds with Glu114, and the alkyl ether occupies in the hydrophobic pocket. The ring nitrogen hydrogen bonds to Lys291 and the methyl group are directed into the ribose binding site, although with a slightly different vector than compound (c). X-ray crystal structures of compounds , , and are shown in below. We focused our design efforts on the 6,6-bicyclic core in an attempt to further improve the fragment hits by hybridization with more potent inhibitors and the knowledge of specific ligand–protein interactions. A virtual screen of our corporate collection led to compound (). We independently evaluated the impact of both methoxy groups on the right hand side of compounds and found hydrophobic interaction by the upper methoxy group with the hydrophobic pocket to be a major source of potency, whereas the lower methoxy is likely to interfere with binding (data not shown). We next replaced the methoxy group with a -butyl to occupy the hydrophobic pocket with amino and nitrile groups for hinge binding, as reported previously, leading to compound (). These changes resulted in a dramatic improvement in potency for enzyme inhibition, especially for Gram-positive isozymes, and resulted in improved in vitro bacterial growth inhibition (). The compounds were evaluated for antibacterial activity against three pathogens; ARC12 (CLSI QC reference strain, ATCC 29213), ARM158 (acrB gene deletion based on ARC446), and ARC446 (ATCC 51907, wild type). ARC2349 (ATCC 10813) was included to better understand the potential spectrum of these inhibitors. Additionally, if you compare the IC/MIC ratio of the evolved leads in with compound , with the assumption of a consistent mode of action for the wild type MIC’s ( ARC446) there is a significantly improved ability to access the target due to improved permeability and efflux balances. The efficiency of in this species may be estimated by comparing the wild type to a pump deficient species (). The molecular weight, Log, and plasma protein binding properties of this analog were still in a desirable range which are. The binding mode of compound (not shown) maintains the expected interactions of the nitrile with Leu117, amine to Glu114, and heterocyclic nitrogens to Lys291.