In order to provide a standard inhibitor for quality control

In order to provide a standard inhibitor for quality control a panel of typical protein kinase inhibitors, eg staurosporine, purvalanol and kenpaullone was evaluated but none of the compounds showed any EcIspE inhibition at 100 mM. Therefore, compounds 3 and 4, which we identifed by virtual screening were used to monitor the assay performance. An average signal to noise ratio of 2.1 and an average Z9 value of 0.62 were obtained for the screen. Initial hits were re-assayed in duplicate at the same concentrations as used for the primary screen. This resulted in confirmed activity for two compounds. The compounds were repurchased to determine their IC50 values. They inhibit EcIspE in the low micromolar range and have 1092351-67-1 ligand efficiencies comparable to the virtual screening hits. Unfortunately, extensive co-crystallisation experiments and soaking of preformed AaIspE crystals with the hits identified by the virtual and high-throughput screening approaches did not provide any structural information. Therefore, SAR for the virtual screening hits were derived based on the modelled binding modes. Only compounds with Hill coefficients close to one were followed up for hit expansion. Higher Hill coefficients are not consistent with a binding model for competitive binding to a single binding site and are possibly indicative of compound aggregation, solubility issues, an assay artefact, or more than a single class of binding sites in the assay solution. The remaining compounds were therefore disregarded. In the predicted binding mode, compound 3 is positioned between Tyr24 and Tyr175 and forms three hydrogen bonds to His25 in the cytidine binding pocket and an additional hydrogen bond to Asp130. Three commercially available analogues with the same core fragment predicted to interact with His25 but with different substituents on the amino group were selected for testing against EcIspE. All proved less Hexyl 5-aminolevulinate hydrochloride biological activity potent than the screening hit. Compounds 9 and 11 are lacking a functional group that can interact with Asp130 and the hydroxyl group of 10 Figure 10c) is not in the right orientation required for a hydrogen bond with this amino acid. This might explain the loss in affinity of these ligands compared to the initial hit. In the modelled binding mode of ligand 4 the pyrrolopyridine scaffold is sandwiched between Tyr24 and Tyr175 and potentially interacts with His25 via three hydrogen bonds. In addition, the charged amino group in the tetrahydropyrimidinium ring may form a salt bridge interaction with Asp130. Commercially available analogues of this compound were selected to probe possible interactions with Asp130, His25 and the backbone of Lys145. Compounds 12 and 13 were chosen because of substituents o