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

ARTICLE

PUBLISHED ONLINE: 16 FEBRUARY 2015 | http://www.nature.com/doifinder/10.1038/nchembio.1752

Web End =DOI: 10.1038/NCHEMBIO.1752

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

Cristina Landeta1,5, Jessica L Blazyk1,5, Feras Hatahet1,5, Brian M Meehan1, Markus Eser1, Alissa Myrick2, Ludmila Bronstain3, Shoko Minami2, Holly Arnold1,4, Na Ke1,4, Eric J Rubin2, Barbara C Furie3, Bruce Furie3, Jon Beckwith1*, Rachel Dutton1,4 & Dana Boyd1

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 bondforming 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.

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Disulfide bonds between pairs of cysteines contribute substantially to the folding and stability of many proteins. In bacteria, this advantageous modification is generally

limited to proteins that are exported to the cell envelope or beyond. Many proteins involved in bacterial virulence (such as toxins, adhesins, flagella, fimbriae, pili, and types II and III secretion systems) require disulfide bonds for their stability and activity1.

Thus, inactivation of enzymes that make protein disulfide bonds interferes with the activity of multiple bacterial virulence factors. Inhibitors of these enzymes could thus have profound effects on pathogen virulence.

In Gram-negative bacteria, disulfide bonds are introduced into substrate proteins as they cross through the cytoplasmic membrane into the cell envelope2,3. The periplasmic enzyme DsbA, a member of the thioredoxin family, oxidizes pairs of cysteines in substrate proteins through its Cys-X-X-Cys active site4. The resulting reduced DsbA is reoxidized by the membrane protein DsbB, regenerating DsbAs activity. DsbB itself is reoxidized by...