An Experimental Toolbox for Structure‐Based Hit Discovery for P. aeruginosa FabF, a Promising Target for Antibiotics

Abstract

FabF (3-oxoacyl-[acyl-carrier-protein] synthase 2), which catalyses the rate limiting condensation reaction in the fatty acid synthesis II pathway, is an attractive target for new antibiotics. Here, we focus on FabF from P. aeruginosa (PaFabF) as antibiotics against this pathogen are urgently needed. To facilitate exploration of this target we have set up an experimental toolbox consisting of binding assays using bio-layer interferometry (BLI) as well as saturation transfer difference (STD) and WaterLOGSY NMR in addition to robust conditions for structure determination. The suitability of the toolbox to support structure-based design of FabF inhibitors was demonstrated through the validation of hits obtained from virtual screening. Screening a library of almost 5 million compounds resulted in 6 compounds for which binding into the malonyl-binding site of FabF was shown. For one of the hits, the crystal structure in complex with PaFabF was determined. Based on the obtained binding mode, analogues were designed and synthesised, but affinity could not be improved. This work has laid the foundation for structure-based exploration of PaFabF.

Design, quality and validation of the EU-OPENSCREEN fragment library poised to a high-throughput screening collection

“…31 A promising target is beta-ketoacyl-ACP synthase 2 (FabF) which is part of the fatty acid synthesis pathway. 32–34 In an effort to fuel drug discovery, we screened the EFSL against FabF from P. aeruginosa ( Pa FabF) using BLI. BLI is, like SPR, a label-free biosensing technology.…”

New starting points for antibiotics targeting P. aeruginosa FabF discovered by crystallographic fragment screening followed by hit expansion

“…18 This intermediate state can be mimicked by mutating the active site Cys to either Gln or Ala. 18,21 In both enzymes, PaFabF C164A and PaFabF C164Q, the binding site conformation is very similar (Figure S1 and Table S1 in supplementary information) and also binding affinities are comparable, suggestion that these enzyme variants are interchangeable. 16 Here, we initially started working with PaFabF 164Q as the side chain of Gln more closely mimics the intermediate with the fatty acid bound. 18 However, we later discovered that PaFabF C164A yields large, 3D crystals that diffract to a resolutions of up to 1.4 Å and can be used for more than 3 months, compared to PaFabF C164Q crystals that form as very thin plates which are difficult to handle and are only stable for 1 month.…”

“…Platensimycin is the most potent FabF inhibitor known to date with activity in the low nanomolar range. 16,18 . Most of its activity stems from binding to the fatty acid bound intermediate (Figure 1b) while binding to the w.t.…”

“…33 We have discovered a PaFabF ligand using virtual screening but optimization has proven difficult. 16 Further, indazole sulphonamides have been reported as inhibitors of M. tuberculosis KasA (an ortholog of FabB). 34,35 Finally, two B. melitensis FabB crystal structures bound to fragments have been published.…”

Crystal structure of Pseudomonas aeruginosa FabB C161A, a template for structure-based design for new antibiotics

“…As in our hands Pa FabF C164A was more stable than Pa FabF C164Q and thus better suited for biophysical studies, we focused our efforts on Pa FabB C161A. 13 …”