BackgroundMany bacteria, including Vibrio spp., regulate virulence gene expression in a cell-density dependent way through a communication process termed quorum sensing (QS). Hence, interfering with QS could be a valuable novel antipathogenic strategy. Cinnamaldehyde has previously been shown to inhibit QS-regulated virulence by decreasing the DNA-binding ability of the QS response regulator LuxR. However, little is known about the structure-activity relationship of cinnamaldehyde analogs.Methodology/Principal FindingsBy evaluating the QS inhibitory activity of a series of cinnamaldehyde analogs, structural elements critical for autoinducer-2 QS inhibition were identified. These include an α,β unsaturated acyl group capable of reacting as Michael acceptor connected to a hydrophobic moiety and a partially negative charge. The most active cinnamaldehyde analogs were found to affect the starvation response, biofilm formation, pigment production and protease production in Vibrio spp in vitro, while exhibiting low cytotoxicity. In addition, these compounds significantly increased the survival of the nematode Caenorhabditis elegans infected with Vibrio anguillarum, Vibrio harveyi and Vibrio vulnificus.Conclusions/SignificanceSeveral new and more active cinnamaldehyde analogs were discovered and they were shown to affect Vibrio spp. virulence factor production in vitro and in vivo. Although ligands for LuxR have not been identified so far, the nature of different cinnamaldehyde analogs and their effect on the DNA binding ability of LuxR suggest that these compounds act as LuxR-ligands.

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We present a scalable synthesis of a versatile MTX reagent with an azide ligation handle that allows rapid γ-selective conjugation to yield MTX fusion compounds (MFCs) appropriate for MASPIT, a three-hybrid system that enables the identification of mammalian cytosolic proteins that interact with a small molecule of interest. We selected three structurally diverse pharmacologically active compounds (tamoxifen, reversine, and FK506) as model baits. After acetylene functionalization of these baits, MFCs were synthesized via a CuAAC reaction, demonstrating the general applicability of the MTX reagent. In analytical mode, MASPIT was able to give concentration-dependent reporter signals for the established target proteins. Furthermore, we demonstrate that the sensitivity obtained with the new MTX reagent was significantly stronger than that of a previously used non-regiomeric conjugate mixture. Finally, the FK506 MFC was explored in a cellular array screen for targets of FK506. Out of a pilot collection of nearly 2000 full-length human ORF preys, FKBP12, the established target of FK506, emerged as the prey protein that gave the highest increase in luciferase activity. This indicates that our newly developed synthetic strategy for the straightforward generation of MFCs is a promising asset to uncover new intracellular targets using MASPIT cellular array screening.

To expand the structure-activity relationships of fosmidomycin and FR900098, two potent antimalarials interfering with the MEP-pathway, we decided to replace a methylene group in β-position of the phosphonate moiety of these leads by an oxygen atom. β-oxa-FR900098 (11) proved equally active as the parent compound.When applied to 4-[hydroxyl(methyl)amino]-4-oxobutyl phosphonic acid, featuring a hydroxamate instead of the retrohydroxamate moiety, a β-oxa modification yielded a derivative (13) with superior activity against a 3D7 P. falciparum strain than fosmidomycin, while a γ-oxa modification resulted in less active derivatives.A bis(pivaloyloxymethyl)ester of phosphonate 13 proved twice as active in inhibiting cultured parasites than a similar prodrug of FR900098.

Activity-based protein profiling (ABPP) research is directed towards the development of tools and techniques that report on enzyme activity in complex biological samples.[1-4] With the aid of activity-based probes (ABPs)-small molecules designed to react specifically, covalently, and irreversibly with the active site residues of an enzyme or enzyme family-enzymatic activity levels are detected, rather than the protein expression levels that are measured by means of conventional proteomics techniques. A typical ABP consists of three parts: 1) a "warhead", the reactive group that binds covalently and irreversibly to the enzyme active site, 2) a recognition element targeting the ABP to a certain enzyme (family), and 3) an affinity tag or a fluorophore for visualization and/or enrichment purposes. In most ABPs that report on enzyme activity, the reporter group is directly attached to the probe, with obvious advantages with respect to experimental design. Incorporation of, for instance, a biotin or large fluorophore in an ABP, however, might have a detrimental effect either on bioavailability (cell permeability) or on enzyme reactivity of the probe, or on both. With the aim of alleviating these problems, the two-step labeling approach is an important alternative in ABPP. We and Cravatt and co-workers simultaneously reported that this approach is also versatile in the profiling of enzyme families: namely the proteasome and serine hydrolases, respectively.[5,6] In two-step ABPP approaches a small biocompatible reactive group, normally an azide or an acetylene, is introduced into an ABP. After covalent modification of a target protein (family), a reporter group is introduced in a chemoselective manner, by means either of Staudinger-Bertozzi ligation[6-8] or of Huisgen [2+3] cycloaddition (the "click reaction", of which both copper(I)-catalyzed[5,9-13] and copper-free[14,15] versions exist). Key to the success of such two-step ABPP experiments are the selectivity (in terms of cross-reactivity towards endogenous functional groups in a biological sample) and efficiency (in terms of chemical yields with which the azide-or acetylene-modified proteins are converted) of the chemoselective ligation step by which the reporter group is attached to the modified proteins. There are several reports on the selectivity of both Staudinger-Bertozzi and click ligations.[11,14] Here we describe a compatible set of one-step and two-step proteasome

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