Design and synthesis of a biotinylated chemical probe for detecting the molecular targets of an inhibitor of the production of the Pseudomonas aeruginosa virulence factor pyocyanin

Pseudomonas aeruginosa is a human pathogen associated with a variety of life-threatening nosocomial infections. This organism produces a range of virulence factors which actively cause damage to host tissues. One such virulence factor is pyocyanin, known to play a crucial role in the pathogenesis of P. aeruginosa infections. Previous studies had identified a novel compound capable of strongly inhibiting the production of pyocyanin. It was postulated that this inhibition results from modulation of an intercellular communication system termed quorum sensing, via direct binding of the compound with the LasR protein receptor. This raised the possibility that the compound could be an antagonist of quorum sensing in P. aeruginosa, which could have important implications as this intercellular signaling mechanism is known to regulate many additional facets of P. aeruginosa pathogenicity. However, there was no direct evidence for the binding of the active compound to LasR (or any other targets). Herein we describe the design and synthesis of a biotin-tagged version of the active compound. This could potentially be used as an affinity-based chemical probe to ascertain, in a direct fashion, the active compound’s macromolecular biological targets, and thus better delineate the mechanism by which it reduces the level of pyocyanin production

Identification of new quorum sensing autoinducer binding partners in Pseudomonas aeruginosa using photoaffinity probes

Many bacterial species, including the human pathogen Pseudomonas aeruginosa, employ a mechanism of intercellular communication known as quorum sensing (QS), which is mediated by signalling molecules termed autoinducers. The Pseudomonas Quinolone Signal (PQS) and 2-Heptyl-3H-4-Quinolone (HHQ) are autoinducers in P. aeruginosa, and they are considered important factors in the progress of infections by this clinically relevant organism. Herein, we report the development of HHQ and PQS photoaffinity-based probes for chemical proteomic studies. Application of these probes led to the identification of previously unsuspected putative HHQ and PQS binders, thereby providing new insights into QS at a proteomic level and revealing potential new small molecule targets for virulence attenuation strategies. Notably, we found evidence that PQS binds RhlR, the cognate receptor in the Rhl QS sub-system of P. aeruginosa. This is the first indication of interaction between the Rhl and PQS systems at the protein/ligand level, which suggests that RhlR should be considered a highly attractive target for antivirulence strategies

Chemogenomics approaches for receptor deorphanization and extensions of the chemogenomics concept to phenotypic space

Chemogenomic approaches, which link ligand chemistry to bioactivity against targets (and, by extension, to phenotypes) are becoming more and more important due to the increasing number of bioactivity data available both in proprietary databases as well as in the public domain. In this article we review chemogenomics approaches applied in four different domains: Firstly, due to the relationship between protein targets from which an approximate relation between their respective bioactive ligands can be inferred, we investigate the extent to which chemogenomics approaches can be applied to receptor deorphanization. In this case it was found that by using knowledge about active compounds of related proteins, in 93% of all cases enrichment better than random could be obtained. Secondly, we analyze different chemin-formatics analysis methods with respect to their behavior in chemogenomics studies, such as subgraph mining and Baye-sian models. Thirdly, we illustrate how chemogenomics, in its particular flavor of 'proteochemometrics', can be applied to extrapolate bioactivity predictions from given data points to related targets. Finally, we extend the concept of 'chemoge-nomics' approaches, relating ligand chemistry to bioactivity against related targets, into phenotypic space which then falls into the area of 'chemical genomics' and 'chemical genetics'; given that this is very often the desired endpoint of approaches in not only the pharmaceutical industry, but also in academic probe discovery, this is often the endpoint the experimental scientist is most interested in. © 2011 Bentham Science Publishers