Identification of small-molecule antagonists of the Pseudomonas aeruginosa transcriptional regulator PqsR: biophysically guided hit discovery and optimization.

The Gram-negative pathogen Pseudomonas aeruginosa produces an intercellular alkyl quinolone signaling molecule, the Pseudomonas quinolone signal. The pqs quorum sensing communication system that is characteristic for P. aeruginosa regulates the production of virulence factors. Therefore, we consider the pqs system a novel target to limit P. aeruginosa pathogenicity. Here, we present small molecules targeting a key player of the pqs system, PqsR. A rational design strategy in combination with surface plasmon resonance biosensor analysis led to the identification of PqsR binders. Determination of thermodynamic binding signatures and functional characterization in E. coli guided the hit optimization, resulting in the potent hydroxamic acid derived PqsR antagonist 11 (IC(50) = 12.5 μM). Remarkably it displayed a comparable potency in P. aeruginosa (IC(50) = 23.6 μM) and reduced the production of the virulence factor pyocyanin. Beyond this, site-directed mutagenesis together with thermodynamic analysis provided insights into the energetic characteristics of protein-ligand interactions. Thus the identified PqsR antagonists are promising scaffolds for further drug design efforts against this important pathogen.

[1]  R. Hartmann,et al.  Discovery of antagonists of PqsR, a key player in 2-alkyl-4-quinolone-dependent quorum sensing in Pseudomonas aeruginosa. , 2012, Chemistry & biology.

[2]  M. Nishio,et al.  The CH/π hydrogen bond in chemistry. Conformation, supramolecules, optical resolution and interactions involving carbohydrates. , 2011, Physical chemistry chemical physics : PCCP.

[3]  S. Chill,et al.  Facile One-Pot Conversion of Aldehydes into Amides , 2010 .

[4]  D. Spring,et al.  Structure-Activity Analysis of the Pseudomonas Quinolone Signal Molecule , 2010, Journal of bacteriology.

[5]  Andrew L Hopkins,et al.  Fragment screening by surface plasmon resonance. , 2010, ACS medicinal chemistry letters.

[6]  Rebecca L. Rich,et al.  Grading the commercial optical biosensor literature—Class of 2008: ‘The Mighty Binders’ , 2009, Journal of molecular recognition : JMR.

[7]  Gerhard Klebe,et al.  Adding calorimetric data to decision making in lead discovery: a hot tip , 2010, Nature Reviews Drug Discovery.

[8]  G. Cuevas,et al.  Enthalpic nature of the CH/pi interaction involved in the recognition of carbohydrates by aromatic compounds, confirmed by a novel interplay of NMR, calorimetry, and theoretical calculations. , 2009, Journal of the American Chemical Society.

[9]  G. Waldrop.  Smaller is better for antibiotic discovery. , 2009, ACS chemical biology.

[10]  Venkataraman Thanabal,et al.  Discovery of antibacterial biotin carboxylase inhibitors by virtual screening and fragment-based approaches. , 2009, ACS chemical biology.

[11]  E. Freire.  Do enthalpy and entropy distinguish first in class from best in class? , 2008, Drug discovery today.

[12]  S. Diggle,et al.  Quorum sensing by 2-alkyl-4-quinolones in Pseudomonas aeruginosa and other bacterial species. , 2008, Molecular bioSystems.

[13]  Anthony M Giannetti,et al.  Surface plasmon resonance based assay for the detection and characterization of promiscuous inhibitors. , 2008, Journal of medicinal chemistry.

[14]  S. Diggle,et al.  A dual biosensor for 2-alkyl-4-quinolone quorum-sensing signal molecules. , 2007, Environmental microbiology.

[15]  A. Clatworthy,et al.  Targeting virulence: a new paradigm for antimicrobial therapy , 2007, Nature Chemical Biology.

[16]  D. Hogan,et al.  Farnesol, a common sesquiterpene, inhibits PQS production in Pseudomonas aeruginosa , 2007, Molecular microbiology.

[17]  A. Zaborin,et al.  Dynorphin Activates Quorum Sensing Quinolone Signaling in Pseudomonas aeruginosa , 2007, PLoS pathogens.

[18]  L. Rahme,et al.  MvfR, a key Pseudomonas aeruginosa pathogenicity LTTR‐class regulatory protein, has dual ligands , 2006, Molecular microbiology.

[19]  M. W. Wong,et al.  Saturated hydrocarbon-benzene complexes: theoretical study of cooperative CH/pi interactions. , 2006, The journal of physical chemistry. A.

[20]  T. Blundell,et al.  Probing hot spots at protein-ligand binding sites: a fragment-based approach using biophysical methods. , 2006, Journal of medicinal chemistry.

[21]  S. Kjelleberg,et al.  A characterization of DNA release in Pseudomonas aeruginosa cultures and biofilms , 2006, Molecular microbiology.

[22]  P. Wagner,et al.  Electronic effects of ring substituents on triplet benzylic biradicals. , 2006, Organic letters.

[23]  J. T. Metz,et al.  Ligand efficiency indices as guideposts for drug discovery. , 2005, Drug discovery today.

[24]  Eric Déziel,et al.  The contribution of MvfR to Pseudomonas aeruginosa pathogenesis and quorum sensing circuitry regulation: multiple quorum sensing‐regulated genes are modulated without affecting lasRI, rhlRI or the production of N‐acyl‐ l‐homoserine lactones , 2004, Molecular microbiology.

[25]  A. Hopkins,et al.  Ligand efficiency: a useful metric for lead selection. , 2004, Drug discovery today.

[26]  Kevin L. Griffith,et al.  Measuring beta-galactosidase activity in bacteria: cell growth, permeabilization, and enzyme assays in 96-well arrays. , 2002, Biochemical and biophysical research communications.

[27]  N. A. Whitehead,et al.  Quorum sensing as a population-density-dependent determinant of bacterial physiology. , 2001, Advances in microbial physiology.

[28]  E. Greenberg,et al.  Quinolone signaling in the cell-to-cell communication system of Pseudomonas aeruginosa. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[29]  Jacques Bille,et al.  Frequency and molecular diversity of Pseudomonas aeruginosa upon admission and during hospitalization: a prospective epidemiologic study. , 1998, Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases.

[30]  J. Reiser,et al.  Autoinducer-mediated regulation of rhamnolipid biosurfactant synthesis in Pseudomonas aeruginosa. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[31]  J. Hearst,et al.  Efflux pumps and drug resistance in gram-negative bacteria. , 1994, Trends in microbiology.

[32]  A Fiechter,et al.  Isolation and characterization of a regulatory gene affecting rhamnolipid biosurfactant synthesis in Pseudomonas aeruginosa , 1994, Journal of bacteriology.

[33]  M. Gambello,et al.  Expression of Pseudomonas aeruginosa virulence genes requires cell-to-cell communication. , 1993, Science.

[34]  N. Høiby,et al.  Pathogenesis of cystic fibrosis , 1993, The Lancet.

[35]  R. Miller,et al.  Enhanced octadecane dispersion and biodegradation by a Pseudomonas rhamnolipid surfactant (biosurfactant) , 1992, Applied and environmental microbiology.

[36]  M. Gambello,et al.  Cloning and characterization of the Pseudomonas aeruginosa lasR gene, a transcriptional activator of elastase expression , 1991, Journal of bacteriology.

[37]  I. Crawford,et al.  Identification and characterization of genes for a second anthranilate synthase in Pseudomonas aeruginosa: interchangeability of the two anthranilate synthases and evolutionary implications , 1990, Journal of bacteriology.

[38]  Douglas E. Conran,et al.  Substituent effects and mechanism in the micellar hydrolysis of hydroxamic acids , 1984 .

[39]  W. Kliegel,et al.  Borchelate von N‐substituierten Hydroxamsäuren , 1983 .

[40]  M. L. Willard,et al.  Optical Constants of Benzamide, its Homologs, and Some Aliphatic Amides1 , 1940 .