The Fragment-Based Development of a Benzofuran Hit as a New Class of Escherichia coli DsbA Inhibitors

Abstract

A fragment-based drug discovery approach was taken to target the thiol-disulfide oxidoreductase enzyme DsbA from Escherichia coli (EcDsbA). This enzyme is critical for the correct folding of virulence factors in many pathogenic Gram-negative bacteria, and small molecule inhibitors can potentially be developed as anti-virulence compounds. Biophysical screening of a library of fragments identified several classes of fragments with affinity to EcDsbA. One hit with high mM affinity, 2-(6-bromobenzofuran-3-yl)acetic acid (6), was chemically elaborated at several positions around the scaffold. X-ray crystal structures of the elaborated analogues showed binding in the hydrophobic binding groove adjacent to the catalytic disulfide bond of EcDsbA. Binding affinity was calculated based on NMR studies and compounds 25 and 28 were identified as the highest affinity binders with dissociation constants (KD) of 326 ± 25 and 341 ± 57 µM respectively. This work suggests the potential to develop benzofuran fragments into a novel class of EcDsbA inhibitors.

Structural bioinformatic analysis of DsbA proteins and their pathogenicity associated substrates

“…Secondly, DsbA is present in the periplasm, which is more accessible relative to cytoplasmic targets. Furthermore, all DsbA inhibitors described so far target the hydrophobic groove of DsbA [47] , [66] , [125] , [127] , [128] , [129] , [130] , which is not present in human thioredoxin or PDI. Therefore, despite DsbA belonging to the widespread thioredoxin superfamily, DsbA-tailored inhibitors are less likely to inhibit TRX-like proteins in humans.…”

Application of Fragment-Based Drug Discovery to Versatile Targets

“…MS and X-ray Sijbesma et al, 2019 The RNA-dependent RNA polymerase X-ray Riccio et al, 2019 Apical membrane antigen 1 PRE, NMR Akter et al, 2019 Glyoxalase 1 Computational approach Perez et al, 2019 Focal Adhesion Kinase SPR and NMR Alvarado et al, 2019 E. coli DsbA NMR/X-ray Duncan et al, 2019 PDEδ-RAS (PPI) STD, CMPG-NMR Chen et al, 2019 *This table lists some studies using FBDD. Only a few studies published in 2019 were list for elucidating the application of FBDD to multiple targets.…”

Anti-Virulence Therapeutic Approaches for Neisseria gonorrhoeae

“…Previous studies have identified small-molecule inhibitors, phenylthiazole, benzofuran, and pyridazinone derivatives, against the DsbA/DsbB system in E. coli . Pyridazinone-based compounds [ 161 , 163 , 165 , 169 ] bound to Ec DsbB at the quinone-binding site between the first two transmembrane segments, competing with quinone, or to a segment of the second periplasmic loop that interacts with Ec DsbA [ 164 , 271 ]. Phenylthiazole and benzofuran-based compounds bound to the hydrophobic groove of Ec DsbA, which is required for interaction with Ec DsbB [ 163 , 169 ].…”

“…Pyridazinone-based compounds [ 161 , 163 , 165 , 169 ] bound to Ec DsbB at the quinone-binding site between the first two transmembrane segments, competing with quinone, or to a segment of the second periplasmic loop that interacts with Ec DsbA [ 164 , 271 ]. Phenylthiazole and benzofuran-based compounds bound to the hydrophobic groove of Ec DsbA, which is required for interaction with Ec DsbB [ 163 , 169 ]. Phenylalanine and tyrosine-based phenylthiazole derivatives were also found to selectively inhibit Ec DsbA in in vitro assays, with reduced motility in soft agar and no effect on growth in liquid media [ 163 ].…”