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1.  Towards implementation of Quorum Sensing Autoinducers as Biomarkers for Infectious Disease States 
Analytical chemistry  2013;85(6):3355-3362.
The opportunistic bacterial pathogen Pseudomonas aeruginosa causes chronic lung infections in cystic fibrosis (CF) patients. Importantly, virulence factor expression and biofilm formation in P. aeruginosa is coordinated by quorum sensing (QS) and one of the key QS signaling molecules is 3-oxo-C12-HSL. Remarkably, a tetramic acid, (C12-TA), with antibacterial properties is formed spontaneously from 3-oxo-C12-HSL under physiological conditions. Seeking to better understand this relationship we sought to investigate if 3-oxo-C12-HSL and C12-TA may be contributing factors to the overall pathogenicity of P. aeruginosa in CF individuals and their detection and quantitation in sputum samples might be used as an indicator to assess disease states and monitor therapy success in CF patients. To this end, 3-oxo-C12-HSL and C12-TA concentrations were initially analyzed in P. aeruginosa flow cell biofilms using liquid chromatography coupled with mass spectrometry (LC-MS). A liquid chromatography tandem mass spectrometry (LC-MS-MS)-based method was then developed and validated for their detection and quantification in sputa of CF patients. We highlight that this is the first report to show the presence of both the quorum sensing molecule (3-oxo-C12-HSL) and its rearranged product (C12-TA) in human clinical samples such as sputum. A total of 47 sputum samples from 20 CF and 2 non-CF individuals were analyzed: 3-oxo-C12-HSL was detected and quantified in 45 samples with concentrations ranging from 20 nM to >1000 nM; C12-TA was found in 14 samples (13 – 900 nM). Based on our findings, quorum sensing autoinducers merit further investigation as biomarkers for infectious disease states.
doi:10.1021/ac400032a
PMCID: PMC3604138  PMID: 23391272
cystic fibrosis; sputum; quorum sensing; Pseudomonas aeruginosa; 3-oxo-C12-HSL; C12-TA; mass spectrometry
2.  C4-Alkoxy-HPD: a Potent Class of Synthetic Modulators Surpassing Nature in AI-2 Quorum Sensing 
Journal of the American Chemical Society  2012;134(33):13562-13564.
Bacteria have developed cell-to-cell communication mechanisms, termed quorum sensing (QS), which regulate bacterial gene expression in a cell population-dependent manner. Autoinducer-2 (AI-2), a class of QS signaling molecules derived from (4S)-4,5-dihydroxy-2,3-pentanedione (DPD), has been identified in both Gram-negative and Gram-positive bacteria. Despite considerable interest in the AI-2 QS system, the biomolecular communication used by distinct bacterial species still remains shrouded. Herein we report the synthesis and evaluation of a new class of DPD analogs, C4-alkoxy-5-hydroxy-2,3-pentanediones, termed C4-alkoxy-HPDs. Remarkably, two of the analogs were more potent QS agonists than the natural ligand, DPD, in Vibrio harveyi. The findings presented extend insights into ligand-receptor recognition/signaling in the AI-2 mediated QS system.
doi:10.1021/ja305532y
PMCID: PMC3438919  PMID: 22866957
3.  Uncharacterized DPD molecules revealed through NMR: Implications for a greater signaling diversity in bacterial species 
doi:10.1002/anie.201109149
PMCID: PMC3505754  PMID: 22378693
Autoinducer-2; Quorum sensing; bacteria; DPD; NMR spectroscopy
4.  The Fe(III) and Ga(III) coordination chemistry of 3-(1-hydroxymethylidene) and 3-(1-hydroxydecylidene)-5-(2-hydroxyethyl)pyrrolidine-2,4-dione: Novel tetramic acid degradation products of homoserine lactone bacterial quorum sensing molecules 
Journal of Inorganic Biochemistry  2011;107(1):96-103.
Bacteria use small diffusible molecules to exchange information in a process called quorum sensing (QS). An important class of quorum sensing molecules used by Gram-negative bacteria is the family of N-acylhomoserine lactones (HSL). It was recently discovered that a degradation product of the QS molecule 3-oxo-C12-homoserine lactone, the tetramic acid 3-(1-hydroxydecylidene)-5-(2-hydroxyethyl)pyrrolidine-2,4-dione, is a potent antibacterial agent, thus implying roles for QS outside of simply communication. Because these tetramic acids also appear to bind iron with appreciable affinity it was suggested that metal binding might contribute to their biological activity. Here, using a variety of spectroscopic tools, we describe the coordination chemistry of both the methylidene and decylidene tetramic acid derivatives with Fe(III) and Ga(III) and discuss the potential biological significance of such metal binding.
doi:10.1016/j.jinorgbio.2011.10.009
PMCID: PMC3258344  PMID: 22178671
Quorum sensing; tetramic acid; iron binding; Mössbauer spectroscopy
5.  A Multivalent Probe for AI-2 Quorum Sensing Receptors 
Journal of the American Chemical Society  2011;133(40):15934-15937.
Multivalency is a common principle in the recognition of cellular receptors, and multivalent agonists and antagonists have played a major role in understanding mammalian cell receptor biology. The study of bacterial cell receptors using similar approaches, however, has lagged behind. Herein we describe our efforts toward the development of a dendrimer-based multivalent probe for studying AI-2 quorum sensing receptors. From these studies, we have discovered a chemical probe specific for Lsr-type AI-2 quorum sensing receptors with the potential for enabling the identification of new bacterial species that utilize AI-2 as a quorum sensing signaling molecule.
doi:10.1021/ja207556d
PMCID: PMC3203215  PMID: 21913711
6.  Probing Autoinducer-2 Based Quorum Sensing: The Biological Consequences of Molecules Unable to Traverse Equilibrium States 
The Journal of organic chemistry  2011;76(17):6981-6989.
Bacteria have developed a cell-to-cell communication system, termed quorum sensing (QS), which allows for the population-dependent coordination of their behavior via the exchange of chemical signals. Autoinducer-2 (AI-2), a class of QS signals derived from 4,5-dihydroxy-2,3-pentandione (DPD), has been revealed as a universal signaling molecule in a variety of bacterial species. In spite of the considerable interest, the study of putative AI-2 based QS systems remains a challenging topic in part due to the rapid interconversion between the linear and cyclic forms of DPD. Herein, we report the design and development of efficient syntheses of carbocyclic analogues of DPD, which are locked in the cyclic form. The synthetic analogues were evaluated for the modulation of AI-2 based QS in Vibrio harveyi and Salmonella typhimurium. No agonists were uncovered in either V. harveyi or S. typhimurium assay, whereas weak to moderate antagonists were found against V. harveyi. Based on NMR analyses and DFT calculations, the heterocyclic oxygen atom within DPD appears necessary to promote hydration at the C3 position of cyclic DPD to afford the active tetrahydroxy species. These results also shed light on the interaction between the heterocyclic oxygen atom and receptor proteins as well as the importance of the linear form and dynamic equilibrium of DPD as crucial requirements for activation of AI-2 based QS circuits.
doi:10.1021/jo200882k
PMCID: PMC3162994  PMID: 21678949
7.  Synthesis of “clickable” acylhomoserine lactone quorum sensing probes: unanticipated effects on mammalian cell activation 
Alkynyl- and azido-tagged 3-oxo-C12-acylhomoserine lactone probes have been synthesized to examine their potential utility as probes for discovering the mammalian protein target of the Pseudomonas aeruginosa autoinducer, 3-oxo-C12-acylhomoserine lactone. Although such substitutions are commonly believed to be quite conservative, from these studies, we have uncovered a drastic difference in activity between the alkynyl- and azido-modified compounds, and provide an example where such structural modification has proved to be much less than conservative.
doi:10.1016/j.bmcl.2010.11.122
PMCID: PMC3081916  PMID: 21190852
Acylhomoserine lactones; Click chemistry; Pseudomonas aeruginosa; Quorum sensing
8.  Synthesis and Molecular Modeling Provide Insight Into a Pseudomonas aeruginosa Quorum Sensing Conundrum 
The triphenyl amide/ester 12 was originally reported to be a potent mimic of the natural 3-oxo-dodecanoyl homoserine lactone quorum sensing molecule in Pseudomonas aeruginosa. However, explicit synthesis/chemical characterization was lacking, and a later report providing protein crystallographic data inferred 12 to be incorrect with 9 now being the surmised structure. Because of these inconsistencies, and our interest in quorum sensing molecules utilized by Gram-negative bacteria, we found it necessary to synthesize 9 and 12 to test for agonistic activity in a P. aeruginosa reporter assay. Despite distinct regiochemical differences, both 9 and 12 were found to have comparable EC50 values. To reconcile these unanticipated findings, modeling studies were conducted and both compounds were revealed to have comparable binding properties to the LasR receptor.
doi:10.1021/ja111138y
PMCID: PMC3060279  PMID: 21348514
10.  Inhibition of tumor metastasis: Functional immune modulation of the CUB Domain Containing Protein 1 
Molecular pharmaceutics  2010;7(1):245.
Despite significant progress and notable successes in tumor therapy, malignant disease remains an extremely difficult problem in today's health care setting. There is, however, an increasing application of new therapies targeting proteins specifically upregulated on tumor cells. These innovative therapeutic approaches are aimed at molecules that contribute to malignant development and progression but spare normal tissues. The CUB Domain Containing Protein 1 (CDCP1) is such a tumor-associated protein and thus, a potential candidate for targeted cancer immunotherapy. Herein, we describe the generation of function-blocking human antibodies against CDCP1 that were obtained from human scFv phage display libraries using subtractive panning protocols on CDCP1 expressing cancer cells and immunopurified CDCP1 protein. One of the isolated anti-CDCP1 antibodies, namely C20Fc, efficiently blocked experimental metastasis of human carcinoma cells, including HeLa cells stably transfected with CDCP1 and prostate carcinoma cells PC-hi/diss naturally expressing CDCP1, in both chick embryo and mouse model systems. The C20Fc antibody also reduced colony formation of CDCP1 expressing cells in a soft agar assay for anchorage-independent cell growth. Specific targeting of CDCP1 by C20Fc mediated the delivery of a toxin-conjugated antibody complex, thus, providing evidence for antibody internalization and specific killing of CDCP1-positive tumor cells. Our findings indicate a functional role for CDCP1 in human cancer and underscore the therapeutic potential of function-blocking anti-CDCP1 antibodies targeting both primary and metastatic carcinoma cells.
doi:10.1021/mp900236t
PMCID: PMC2815031  PMID: 19916495
Immunotherapy; CDCP-1; metastasis; human antibodies; carcinoma
11.  Revisiting AI-2 quorum sensing inhibitors: Direct comparison of alkyl-DPD analogs and a natural product fimbrolide 
Journal of the American Chemical Society  2009;131(43):15584-15585.
Quorum sensing (QS) systems have been proposed in a wide variety of bacteria. The AI-2-based QS system represents the most studied of these proposed interspecies systems, and has been shown to regulate diverse functions such as bioluminescence, expression of virulence factors, and biofilm formation. As such, the development of modulatory compounds, both agonists and antagonists, is of great interest for the study of unknown AI-2 based QS systems and the potential treatment of bacterial infections. The fimbrolide class of natural products has exhibited excellent inhibitory activity against AI-2-based QS, and as such may be considered the “gold-standard” of AI-2 inhibitors. Thus, we sought to include a fimbrolide as a control compound for our recently developed alkyl-DPD panel of AI-2 modulators. Herein, we present a revised synthesis of a commonly studied fimbrolide, as well as a direct comparison between the fimbrolide and alkyl-DPD analogs. We demonstrate that our alkyl-DPD analogs are more potent inhibitors of QS in both Vibrio harveyi and Salmonella typhimurium, the two organisms with defined AI-2 systems, and in doing so, call into question the widely accepted use of fimbrolide-derived compounds as the “gold standard” of AI-2 inhibition.
doi:10.1021/ja9066783
PMCID: PMC2784249  PMID: 19824634
12.  Defining the Mode of Action of Tetramic Acid Antibacterials Derived from Pseudomonas aeruginosa Quorum Sensing Signals 
Journal of the American Chemical Society  2009;131(40):14473-14479.
In Nature, bacteria rarely exist as single, isolated entities, but rather as communities comprised of many other species including higher host organisms. To survive in these competitive environments, microorganisms have developed elaborate tactics such as the formation of biofilms and the production of antimicrobial toxins. Recently, it was discovered that the Gram-negative bacterium Pseudomonas aeruginosa, an opportunistic human pathogen, produces an antibiotic, 3-(1-hydroxydecylidene)-5-(2-hydroxyethyl)pyrrolidine-2,4-dione (C12-TA), derived from one of its quorum sensing molecules. Here, we present a comprehensive study of the expanded spectrum of C12-TA antibacterial activity against microbial competitors encountered by P. aeruginosa in Nature as well as significant human pathogens. The mechanism of action of C12-TA was also elucidated and C12-TA was found to dissipate both the membrane potential and pH gradient of Gram-positive bacteria, correlating well with cell death. Notably, in stark contrast to its parent molecule 3-oxo-dodecanoyl homoserine lactone (3-oxo-C12-HSL), neither activation of cellular stress pathways nor cytotoxicity was observed in human cells treated with C12-TA. Our results suggest that the QS machinery of P. aeruginosa has evolved for a dual-function, both to signal others of the same species, and also to defend against both host immunity and competing bacteria. Because of the broad-spectrum antibacterial activity, established mode of action, lack of rapid resistance development, and tolerance by human cells, the C12-TA scaffold may also serve as a new lead compound for the development of antimicrobial therapeutics.
doi:10.1021/ja9056079
PMCID: PMC2760024  PMID: 19807189

Results 1-13 (13)