Compounds targeting disulfide bond forming enzyme DsbB of Gram-negative bacteria

Abstract

In bacteria, disulfide bonds confer stability on many proteins exported to the cell envelope or beyond. These proteins include numerous bacterial virulence factors, and thus bacterial enzymes that promote disulfide bond formation represent targets for compounds inhibiting bacterial virulence. Here, we describe a new target- and cell-based screening methodology for identifying compounds that inhibit the disulfide bond-forming enzymes Escherichia coli DsbB (EcDsbB) or Mycobacterium tuberculosis VKOR (MtbVKOR), which can replace EcDsbB, although the two are not homologs. Initial screening of 51,487 compounds yielded six specifically inhibiting EcDsbB. These compounds share a structural motif and do not inhibit MtbVKOR. A medicinal chemistry approach led us to select related compounds, some of which are much more effective DsbB inhibitors than those found in the screen. These compounds inhibit purified DsbB and prevent anaerobic growth of E. coli. Furthermore, these compounds inhibit all but one of the DsbBs of nine other Gram-negative pathogenic bacteria tested.

Figure 1

DsbB pathway and screening basis. E. coli disulfide bond formation pathway with endogenous EcDsbB or exogenous MtbVKOR enzyme. Black arrows indicate the flow of electrons. One DsbA substrate in the screening strain is β-Gal^dbs (see text for details). Contrary to the natural DsbA-substrates, β-Gal^dbs is inactive when oxidized; hence colonies are white when growing on X-Gal agar growth media.

Figure 2

In vivo and in vitro inhibition of EcDsbB by compound 9 and 12. (a) In vitro inhibition of purified EcDsbB enzyme by compounds 9 (left) and 12 (right). Results are average of at least two independent experiments ± SD. (b) In vivo accumulation of reduced DsbA (EcDsbB substrate) caused by compounds 9 (top) and 12 (bottom). Cells were grown aerobically with different concentrations of drug and precipitated proteins were treated with 4-acetamido-4’-maleimidylstilbene-2,2’-disulphonic acid (AMS, 0.5kDa). Samples were run by reducing SDS-PAGE and immunoblotted against anti-DsbA. Dithiothreitol (DTT) was used for reducing disulfide bonds. “ox” refers to the position of the oxidized protein which is the same as that of the reduced protein with no alkylating agent present. “red” refers to bands where the positions of the protein with reduced cysteines are detected because of the alkylation which adds to the molecular weight. Pictures are representative immunoblots of at least two independent experiments. See complete pictures in Supplementary Information. (c) Inhibition of E. coli anaerobic growth with compound 12. Growth curve of wild-type E. coli (black) and dsbB mutant (red) under anaerobic conditions in the absence (solid lines) or presence (dotted lines) of 10 μM compound 12. Results are the average of three independent experiments ± SD.

Figure 3

In vivo inhibition of DsbB enzymes from gram-negative bacteria expressed in E. coli. E. coli dsbB mutant strains expressing β-Gal^dbs and dsbB genes from Salmonella typhimurium (St), Klebsiella pneumoniae (Kp), Vibrio cholerae (Vc), Haemophilus influenzae (Hi), Pseudomonas aeruginosa (Pa), Acinetobacter baumannii (Ab), Francisella tularensis (Ft) as well as two DsbB-homologs of P. aeruginosa (dsbH) and S. typhimurium (dsbI) and a non-homolog vkor from Mycobacterium tuberculosis (Mtb) were tested against pyridazinone-like compounds. Inhibition range from strong to weak is relative to each DsbB-expressing strain and was obtained by dividing the MIC of each compound between the lowest MIC observed for each particular strain. Results are the average of three independent experiments. Compounds that did not inhibit at the highest concentration tested are shown as black. Identity (%) compared to EcDsbB and protein length (amino acid) are indicated.