Identification of a Fragment-Based Scaffold that Inhibits the Glycosyltransferase WaaG from Escherichia coli

Abstract

WaaG is a glycosyltransferase that is involved in the biosynthesis of lipopolysaccharide in Gram-negative bacteria. Inhibitors of WaaG are highly sought after as they could be used to inhibit the biosynthesis of the core region of lipopolysaccharide, which would improve the uptake of antibiotics. Herein, we establish an activity assay for WaaG using 14C-labeled UDP-glucose and LPS purified from a ∆waaG strain of Escherichia coli. We noted that addition of the lipids phosphatidylglycerol (PG) and cardiolipin (CL), as well as the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) increased activity. We then use the assay to determine if three molecular scaffolds, which bind to WaaG, could inhibit its activity in vitro. We show that 4-(2-amino-1,3-thiazol-4-yl)phenol inhibits WaaG (IC50 1.0 mM), but that the other scaffolds do not. This study represents an important step towards an inhibitor of WaaG by fragment-based lead discovery.

Synergy between Group 2 capsules and lipopolysaccharide underpins serum resistance in Extra-intestinal Pathogenic Escherichia coli

“…As seen in Figure 10, the addition of L1 to cultures results in a concentration-dependent reduction in capsule (A), in addition to changes in LPS quantity and electrophoretic mobility (B). Taken together, the above results show that L1 can reduce capsule levels through LPS inhibition, and also displays its inhibitory activity on live bacterial cells and not just purified LPS, as had been explored in (39).…”

“…Published works by Muheim et al identified a WaaG inhibitor with in vitro activity towards purified LPS, 4-(2-amino-1,3-thiazol-4-yl) (known as L1) with IC 50 = 1.0 mM (39). Due to the role for WaaG in capsule retention identified in this study (by virtue of its contribution to LPS composition and surface charge), it was hypothesised that L1 could be used therapeutically to reduce surface association of K2 capsule with the cell and increase the sensitivity of CFT073 to antibiotics.…”

Structural and functional insights into the Pseudomonas aeruginosa glycosyltransferase WaaG and the implications for lipopolysaccharide biosynthesis

“…A limitation of our solution NMR experiments is that they only measure donor substrate hydrolysis, and while this can indicate whether a donor sugar is correctly bound and accessible to a water molecule that is activated as a nucleophile, it does not measure in vivo GT activity. A GT assay for EcWaaG has been published ( 29 ); however, it is not immediately adaptable to PaWaaG as the acceptor substrate is different for the two enzymes ( Fig. 1 B ).…”

“…Our previous studies have focused on identifying small molecules that bind and inhibit WaaG activity in E. coli ( 28 , 29 , 30 ). For these studies the crystal structures of WaaG from E. coli have been extremely useful as a reference ( 31 ).…”

Extraction of ADP-Heptose and Kdo2-Lipid A from E. coli Deficient in the Heptosyltransferase I Gene

“…Kdo 2 -Lipid A is not fully water soluble and will require the use of detergent for experimental purposes. Chaps and Triton X-100 are two commercially available detergents that have been shown to work well with HepI and similar proteins that will solubilize the Kdo 2 -Lipid A [34]. Experimentally, it has been shown that selective deacylation generates a hydrophilic lipid A moiety that does not differ greatly from the fully acylated version in it is binding and kinetic interactions with the protein [25].…”