In poses in which polar groups of the ligands do not form hydrogenbonding interactions with the receptor or vice versa and are therefore likely to be false positive predictions. These can often be removed by using a pharmacophore to filter the docking solutions and such improve the results. Therefore, all docking poses were post processed. The successful application of similar strategies to other targets gave us confidence in this approach. To consider the presence and absence of the cofactor and the potential tautomers of His25, four different setups for docking were prepared. While compounds from all setups were chosen for testing, for the most promising hit compounds only one of the possible tautomers for His25 was found to be important. In this representation, a protonated NE of His25 is required, which is different from the substrate-bound state of the pocket. Coincidently, this is the same tautomer that was used for modelling the binding mode of the biochemical screening hit 7. The virtual screening library contained a mix of fragment and lead-like compounds. To favour compounds that were predicted to bind with high ligand efficiency we normalized the scores by the number of heavy atoms. Both, compounds with a high total score and a high normalized score were carried forward for visual inspection. Interestingly, all compounds that showed any IspE inhibition were selected based on the latter criteria and were in the fragment-like space making this exercise yet another success story of fragment-based virtual screening. In silico and in vitro screening retrieved chemically distinct hits. On the basis of structural considerations and for reasons of cost efficiency, it was decided to use a small, focussed library containing about 6,000 compounds for in vitro screening. Despite the limited size, the scaffolds of both virtual screening hits were contained in this library. With just five examples, chemical space around hit 4 was MCE Company Pefabloc FG poorly represented. It is therefore unsurprising, that this compound class was not retrieved using in vitro screening. In contrast, 185 compounds containing aminothiazoles were part of the screening library yet this compound class did not appear among the HTS hits. A reason for this might be that only aminothiazoles were unsubstituted in the 5-position as in the screening hit and all of them had purchase CY7 additional functionalities that were predicted to lead to a steric clash in the binding site and/or unfavourable interactions with Asp130. It is an on-going debate as to how many analogues should be contained in a screening l
