Since no binding constants could be determined using BLI for the selected XChem hits, the binding behaviour of fragments mimicking the benzoic acid part of platensimycin/platencin (Figure ) was assessed. These fragments displayed super-stochiometric behaviour in the BLI assay. The crystal structure of one of the fragments (P3, Figure ) in complex with FabF could be determined and the obtained binding mode was identical with that of the corresponding moieties in platencin (Figure and, Figure ). These results indicate that the bulk of affinity of platencin and platensimycin (2.75 and 0.52 µM, respectively) stems from the interactions between the apolar tail and the receptor opening, rather than the interactions close to the catalytic residues within the active site. This is in line with previous structureactivity relationships (SAR) studies, where exchanging the ketolide tail of platensimycin with short alkyl chains abolished antibacterial activity and resulted in only weak binding compounds in vitro (KD = 650 µM for the ethyl analogue binding to E. coli FabFC163Q). Consequently, it is reasonable to believe that the discovered hits can be optimized into potent inhibitors, despite their low affinities. Therefore, we continued with exploring analogues of these fragment hits.