ToxR activates the Vibrio cholerae virulence genes by tethering DNA to the membrane through versatile binding to multiple sites.

ToxR, a Vibrio cholerae transmembrane one-component signal transduction factor, lies within a regulatory cascade that results in the expression of ToxT, toxin coregulated pilus, and cholera toxin. While ToxR has been extensively studied for its ability to activate or repress various genes in V. cholerae, here we present the crystal structures of the ToxR cytoplasmic domain bound to DNA at the toxT and ompU promoters. The structures confirm some predicted interactions, yet reveal other unexpected promoter interactions with implications for other potential regulatory roles for ToxR. We show that ToxR is a versatile virulence regulator that recognizes diverse and extensive, eukaryotic-like regulatory DNA sequences, that relies more on DNA structural elements than specific sequences for binding. Using this topological DNA recognition mechanism, ToxR can bind both in tandem and in a twofold inverted-repeat-driven manner. Its regulatory action is based on coordinated multiple binding to promoter regions near the transcription start site, which can remove the repressing H-NS proteins and prepares the DNA for optimal interaction with the RNA polymerase.

Naphthoquinone derivatives exert their antitrypanosomal activity via a multi-target mechanism.

BACKGROUND AND METHODOLOGY: Recently, we reported on a new class of naphthoquinone derivatives showing a promising anti-trypanosomatid profile in cell-based experiments. The lead of this series (B6, 2-phenoxy-1,4-naphthoquinone) showed an ED(50) of 80 nM against Trypanosoma brucei rhodesiense, and a selectivity index of 74 with respect to mammalian cells. A multitarget profile for this compound is easily conceivable, because quinones, as natural products, serve plants as potent defense chemicals with an intrinsic multifunctional mechanism of action. To disclose such a multitarget profile of B6, we exploited a chemical proteomics approach. PRINCIPAL FINDINGS: A functionalized congener of B6 was immobilized on a solid matrix and used to isolate target proteins from Trypanosoma brucei lysates. Mass analysis delivered two enzymes, i.e. glycosomal glycerol kinase and glycosomal glyceraldehyde-3-phosphate dehydrogenase, as potential molecular targets for B6. Both enzymes were recombinantly expressed and purified, and used for chemical validation. Indeed, B6 was able to inhibit both enzymes with IC(50) values in the micromolar range. The multifunctional profile was further characterized in experiments using permeabilized Trypanosoma brucei cells and mitochondrial cell fractions. It turned out that B6 was also able to generate oxygen radicals, a mechanism that may additionally contribute to its observed potent trypanocidal activity. CONCLUSIONS AND SIGNIFICANCE: Overall, B6 showed a multitarget mechanism of action, which provides a molecular explanation of its promising anti-trypanosomatid activity. Furthermore, the forward chemical genetics approach here applied may be viable in the molecular characterization of novel multitarget ligands.