Extended Data Fig. 4: Drug binding assays, structure–activity relationship, testing prodrug potency with different carrier molecules and determining prodrug cleavage and E. coli IspH inhibition by LC–MS.
From: RETRACTED ARTICLE: IspH inhibitors kill Gram-negative bacteria and mobilize immune clearance
a, SPR signals (resonance units (RU)) from different concentrations of HMBPP, C23.20 and C23.21 run on E. coli IspH crosslinked NTA chip, plotted against concentrations to calculate K D and R max (the amount of ligand (in RU) to be immobilized) values (n = 3 biological and 2 technical replicates). b, Structure–activity guided analogue design reduced the IC50 values of multiple C23 analogues compared with the parent compound. Structures are shown in Supplementary Fig. 2. c, d, Prodrug ester forms of analogue C23.47 obtained by linking ethanol, TPP or dimethylaminopropanol (synthetic reactions are shown in Supplementary Fig. 3) were tested for E. coli killing by dynamic growth curves (c) and by resazurin blue assay (d). For c, d, n = 3 biological and 8 technical replicates. e, Escherichia coli cells treated with 5 μM C23.28–TPP for 30 min were lysed and the lysates analysed by LC–MS to quantify the relative abundance of C23.28–TPP (prodrug), TPP (carrier molecule) and C23.28 (active drug). Respective molecules were identified by their respective retention times (RT) and mass:charge (m/z) ratios. Area under the respective peaks is measured in arbitrary units (AU) and is directly proportional to the abundance of the molecules. f, Relative abundances of C23.28–TPP (prodrug), TPP (carrier molecule) and C23.28 (active drug) found within E. coli treated with different concentrations (10–5,000 nM) of C23.28–TPP (n = 3 technical and 2 biological replicates). g, Methyl viologen assay performed by treating 1 mM HMBPP with 50 nM E. coli IspH pre-treated with 5 μM C23.28 or TPP for 30 min. Samples analysed by LC–MS to quantify relative conversion of HMBPP (IspH substrate) to DMAPP and IPP (IspH products). Respective molecules were identified by their respective retention times and mass:charge (m/z) ratios. Area under the respective peaks is measured in AU and is directly proportional to the abundance of the molecules. h, Conversion of 1 mM HMBPP (black) to DMAPP and IPP (grey) in 30 min by 50 nM E. coli IspH in the presence of different concentrations (10–5,000 nM) of TPP (dotted lines) or C23.28 (solid lines) (n = 3 technical and 2 biological replicates). For f, h, data are mean of 3 independent experiments ± s.e.m.
