Both GDC-0879 Raf inhibitor compounds had slightly improved affinities compared to the screening hit suggesting that the additional interactions may compensate for the loss of the salt bridge. In order to accommodate 14 in the binding site in a similar binding mode as 4, structural rearrangements are required to avoid a steric clash of the cyclo-propyl moiety with the backbone carbonyl group of Lys145. An alternative binding mode for this Adriamycin Topoisomerase inhibitor Compound is also possible in which the core is flipped by 180u compared to 4 but still forms hydrogen bonds with His25. In this orientation, both substituents are solvent exposed. Since this ligand had a 5.8-fold weaker IC50 value than the screening hit, either of the two alternatives appear to be less favourable than the interactions formed by the screening hit. Compounds 15-17 carry substitutions that prevent the same placement in the binding site with respect to His25 as suggested for the hit compound. None of these compounds displayed any inhibition of EcIspE even when tested up to their solubility limit adding confidence to the proposed binding mode of 4. Initially, we were unable to model plausible binding modes for the HTS hit compounds 7 and 8 in either the ATP or cytidine pocket. No analogues of 7 containing an indole moiety were present in the screening library. For compound 8, 44 analogues with a quinazolinone core were found. Three of these showed.40% inhibition in the initial screens but such activity was not confirmed in the subsequent potency assay. Therefore, to establish initial SAR further analogues of the screening hits were identified using the similarity search method FTrees. Our inhouse library of commercially available compounds was screened using the HTS hits as query molecules and finally three analogues of 7 and ten of 8 were purchased for biochemical evaluation. In the case of compound 7, the analogues displayed a one to two order loss in affinity for EcIspE. Common to all three analogues was the deletion of a hydroxyl group at R1 suggesting therefore that this group plays an important role for molecular recognition. Based on this observation, a potential binding mode for the S enantiomer of this compound which is a racemic mixture could be modelled in the cytidine pocket after manually adjusting some side chains. In the proposed pose, the pyridinyl substituent is stacked between the two aromatic residues in the cytidine binding site and additionally forms hydrogen bonds with His25 while the indolyl moiety is buried in a hydrophobic cleft. Further, the hydroxyl group of R1 is involved in a hydrogen bond with Asp130. The later interaction was already suggested to be important for binding of inhibitors 3 and 4. Consistent with this hypothesis, compounds 18 and 19, which cannot form this interaction and, in the case of 19 would even lead to a steric clash with Asp130, displayed markedly reduced affinity compared to the screening hit 7. Compound 20 bears a chlorophenyl group instead of the pyridinyl moiety and accordingly, favourable interactions with His25 are no longer possible. This is in agreement with the 130-fold reduced potency of this inhibitor compared to the hit compound. All purchased analogues of 8 proved less active than the screening hit and no plausible binding modes could be modelled for any of these compounds. Due to availability issues, most of the selected compounds contain more than one change compared to the hit compound or to each other therefore compromising the derivation of unambiguous SAR. However, it appears that a nitrogen atom at R1, preferably in the meta position, is beneficial for affinity. Replacement of the bromopyridinyl moiety of 8 with a methoxyphenyl group is tolerated with a 11-fold loss in affinity.