Colin A. Lowery

An unexpected switch in the modulation of AI-2-based quorum sensing discovered through synthetic 4,5-dihydroxy-2,3-pentanedione analogues.

Quorum sensing (QS) has traditionally referred to a mechanism of communication within a species of bacteria. However, emerging research implicates QS in interspecies communication and competition, and such 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 proposed 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 treatment of bacterial infections and the study of unknown AI-2-based QS systems. Toward this end, we have designed and synthesized a panel of 4,5-dihydroxy-2,3-pentanedione/AI-2 analogues and evaluated their effects on the AI-2 QS of various bacteria. The panel of compounds exhibited differential effects in the bacterial cell lines examined, providing a platform for the development of broad-spectrum modulators of AI-2-based QS.

Defining the Mode of Action of Tetramic Acid Antibacterials Derived from Pseudomonas aeruginosa Quorum Sensing Signals

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 (C(12)-TA), derived from one of its quorum sensing molecules. Here, we present a comprehensive study of the expanded spectrum of C(12)-TA antibacterial activity against microbial competitors encountered by P. aeruginosa in nature as well as significant human pathogens. The mechanism of action of C(12)-TA was also elucidated, and C(12)-TA was found to dissipate both the membrane potential and the 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-C(12)-HSL), neither activation of cellular stress pathways nor cytotoxicity was observed in human cells treated with C(12)-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 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 C(12)-TA scaffold may also serve as a new lead compound for the development of antimicrobial therapeutics.

Medicinal chemistry as a conduit for the modulation of quorum sensing.

The evolutionary development of bacterial cell-to-cell communication, a process termed “quorum sensing” (QS), enabled coordinated behavioral efforts of bacterial populations, thus providing for microbial interaction with higher organisms. QS signaling defines the process of bacteria secreting and responding to small diffusible chemical signals, or autoinducers, in a cell density-dependent process.1 As the number of cells, and thus autoinducer concentrations, increase, bacteria synchronize their gene expression to behave as a unified group. These concerted efforts are beneficial to the bacterial population, but often come at the expense of human health, as QS has been shown to regulate functions such as biofilm formation and the expression of virulence factors. Consequently, the modulation of QS has emerged as a prophylactic and therapeutic anti-virulence target of considerable interest.2,3 QS signaling is mediated by autoinducing molecules that can be categorized into three major classes:4–7: i) N-acyl homoserine lactones (AHLs), ii) oligopeptides, and iii) autoinducer-2 (AI-2).8,9 AHLs are produced by many Gram-negative bacterial species, and signals are differentiated by acyl chain length and oxidation patterns. Likewise, oligopeptides, such as staphylococcal auto-inducing peptides (AIPs), are the signals employed by Gram-positive bacteria, and are often chemically posttranslationally modified. Lastly, the AI-2 class of signals is derived from the precursor 4,5-dihydroxy-2,3-pentanedione (DPD) and has been shown to signal in both Gram-positive and –negative bacteria. Because of the shared DPD core of the known AI-2 signaling molecules, and the ubiquity of the DPD synthase in the bacterial kingdom, AI-2 has been suggested as a language of interspecies communication. In addition to these three classes, other small molecules including the Pseudomonas quinolone signal (PQS)10, 3-hydroxypalmitic acid methyl ester11, bradyoxetin12, and (S)-3-hydroxytridecan-4-one13 have also been identified. In general, individual species rely on chemically distinct signals to avoid interspecies cross-talk or interference. However, as discussed in this Perspective, the signals of one species often exert agonistic or antagonistic effects on the QS systems of other species. Three main paradigms have been explored for the development of QS modulators as potential therapeutics: i) interference with the signal synthase, ii) sequestration of the autoinducer, and iii) antagonism of the receptor, with receptor antagonism having received the most attention to date for the discovery of QS modulators. Additionally, other modes of actions, such as prevention of signal secretion or inhibition of downstream signaling events, have also been examined. In this Perspective, we will focus on the third approach towards QS modulators, and review their development from a medicinal chemical perspective with a focus on the methods and rationale used for their discovery and/or design and synthesis. We will discuss agonists as well as antagonists of QS systems, and have included relative potencies (EC50values for agonists and IC50values for antagonists) where given in the original literature. In consideration of these values, it is important to state that strict comparisons may only be applied within a set of analogs examined in a particular assay, largely due to variations in reporter assays used for analog evaluation. Finally, we will discuss studies that have strived to establish QS as a viable target for the development of antimicrobial therapies.

Toward Implementation of Quorum Sensing Autoinducers as Biomarkers for Infectious Disease States

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 whether 3-oxo-C12-HSL and C12-TA may be contributing factors to the overall pathogenicity of P. aeruginosa in CF individuals and if 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 the sputa of CF patients. To the best of our knowledge, 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 to >1000 nM; C12-TA was found in 14 samples (13-900 nM). On the basis of our findings, quorum sensing autoinducers merit further investigation as biomarkers for infectious disease states.

Probing autoinducer-2 based quorum sensing: the biological consequences of molecules unable to traverse equilibrium states.

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 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. On the basis of 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.

Uncharacterized 4,5-Dihydroxy-2,3-Pentanedione (DPD) Molecules Revealed Through NMR Spectroscopy: Implications for a Greater Signaling Diversity in Bacterial Species†

Chemical communication among bacteria, termed quorum sensing (QS), is a phenomenon that has attracted considerable interest over the last three decades. In this process, the exchange of small chemical signals enables bacterial populations to act together as an ensemble rather than single organisms.[1] This allows bacteria to achieve functions beneficial to an entire population and promotes coexistence with higher organisms. For example, several bacterial species use QS to regulate biofilm formation, resulting in an increased survival rate due to a higher tolerance to antibiotics.[1] Thus, interference of this communication could lead to improved control of bacterial infections and contaminations in health care settings. Importantly, this approach presents advantages over traditional antimicrobials because of a presumed diminished selective pressure to develop resistance.[2] Therefore, a sound understanding of this communication system at a molecular level could be vital for new anti-microbial therapeutics.

A Multivalent Probe for AI-2 Quorum Sensing Receptors

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.

C4-Alkoxy-HPD: A Potent Class of Synthetic Modulators Surpassing Nature in AI-2 Quorum Sensing

Bacteria have developed cell-to-cell communication mechanisms, termed quorum sensing (QS), that 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 analogues, C4-alkoxy-5-hydroxy-2,3-pentanediones, termed C4-alkoxy-HPDs. Remarkably, two of the analogues 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.

Inhibition of tumor metastasis: functional immune modulation of the CUB domain containing protein 1.

Despite significant progress and notable successes in tumor therapy, malignant disease remains an extremely difficult problem in todays 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.

Synthesis of “clickable” acylhomoserine lactone quorum sensing probes: unanticipated effects on mammalian cell activation

Alkynyl- and azido-tagged 3-oxo-C(12)-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-C(12)-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.