This journal is c The Royal Society of Chemistry 2013 Chem. Commun., 2013, 49, 1515--1517 1515

Cite this: Chem. Commun.,2013,49, 1515

Immunomodulation and the quorum sensing molecule3-oxo-C12-homoserine lactone: the importance ofchemical scaffolding for probe development†

Amanda L. Garner,a Jing Yu,a Anjali K. Struss,a Gunnar F. Kaufmann,a

Vladimir V. Kravchenko*b and Kim D. Janda*a

As a guide for chemical probe design, focused analogue synthetic

studies were undertaken upon the lactone ring of 3-oxo-C12-homo-

serine lactone. We have concluded that hydrolytic instability of the

heterocyclic ring is pivotal for its ability to modulate immune signaling

and probe preparation was aligned with these findings.

The immune system relies on innate and adaptive componentsthat are essential for protection against microbial assault. Uponinfection, the innate immune system triggers an inflammatoryresponse through microbial product-mediated activation of Toll-likereceptor (TLR) signaling effectors, such as the transcription factorNF-kB and protein kinase p38, and the production of immunemodulators, including tumor necrosis factor (TNF) among othercytokines.1,2 The goal of this inflammatory response is not onlypathogen elimination but also activation of the adaptive immunesystem via cytokine- and T- and B-cell receptor-driven inductionof similar effector mechanisms, thereby triggering lymphocyteproliferation essential for the generation of efficient immunity.1,2

Certain microbes, however, including the human opportunisticpathogen Pseudomonas aeruginosa,3 are armed with specific factorsthat can impair signaling within both components of the immunesystems, often leading to pathological complications accompanyingacute or persistent infections.4

P. aeruginosa is a nosocomial Gram-negative bacteria responsiblefor a number of infections, particularly in immunocompromisedindividuals such as those suffering from cystic fibrosis.5–7 In orderto promote such persistent infections, P. aeruginosa secretes a varietyof virulence factors8 and signaling molecules,9 including the

quorum sensing signaling molecule N-(3-oxododecanoyl)-L-homo-serine lactone (acylhomoserine lactone (AHL), C12, Fig. 1).9,10

In contrast to other pathogenicity-promoting factors produced byP. aeruginosa,11 C12 has been demonstrated to exert a broadspectrum of biological activities in mammalian cells.11–19 Withrespect to innate and adaptive immunities, C12 suppresses bothTLR-mediated cytokine expression in macrophages12,19 andproliferation of activated lymphocytes,16 two key responses ofimmune cells to invading pathogens.1,2,4 Our laboratory hasreported evidence linking the immunosuppressive nature ofC12 to its ability to selectively disrupt the regulation of NF-kBfunctions in macrophages and other cell types activated byTLR agonists, TNF or whole bacteria.19 Interestingly, despiteinhibition of NF-kB-dependent immune system effectors, exposureof mammalian cells to C12 resulted in activation of the endoplasmicreticulum (ER) stress response, protein kinase p38 pathway andapoptosis.15,19 Notably, however, unlike TLR agonists andother pathogen-associated molecular patterns (PAMPs), C12-mediated activation of mammalian cells occurred throughmechanism(s) distinct from currently known PAMP recognitionreceptor pathways.18,19

In order to better understand the structural significance ofC12 on mammalian cell activation, we20 and others21–24 havebeen actively interested in the chemical synthesis of C12analogues. To date, only a few of these reports have examinedthe effect of conservative structural changes around the C12

Fig. 1 Structures of N-(3-oxododecanoyl)-L-homoserine lactone (C12) and itslactam and thiolactone analogues.

a Departments of Chemistry and Immunology and Microbial Science, The Skaggs

Institute for Chemical Biology and The Worm Institute for Research and Medicine,

The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla,

CA 92037, USA. E-mail: kdjanda@scripps.edu; Fax: +1 858-784-2595;

Tel: +1 858-784-2515b Department of Immunology and Microbial Science, The Scripps Research Institute,

10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

E-mail: vkrav@scripps.edu; Tel: +1 858-784-9170

† Electronic supplementary information (ESI) available: Synthesis and character-ization of AHL analogues and autoinducer activities. See DOI: 10.1039/c3cc38851d

Received 11th December 2012,Accepted 7th January 2013

DOI: 10.1039/c3cc38851d

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1516 Chem. Commun., 2013, 49, 1515--1517 This journal is c The Royal Society of Chemistry 2013

scaffold on leukocyte proliferation21,22 or the activation ofsignaling pathways in mammalian cells,18,20 and a systematicanalysis of the role that hydrolytic stability of the lactone playshas yet to be interrogated. In this report, we describe focusedanalogue synthesis studies and cell-based biochemical analysesto elucidate the functional importance of lactone stability withrespect to C12-mediated activation of the ER stress response,p38 pathway and apoptosis. Counterintuitive to most medicinalchemistry efforts of ligand–receptor interactions, our findingsreveal that hydrolytic instability of the heterocyclic ring iscritical for mammalian cell activation. Based on these studies,a new chemical probe was prepared, which will be useful formammalian cell receptor identification.

In Gram-negative bacteria, AHLs are produced as quorumsensing signaling molecules to promote bacterial multicellularityand virulence.9,25 With respect to mammalian cell immunoregula-tion, previous experiments have demonstrated that AHLscontaining a L-homoserine lactone, 3-oxo substituent and an alkylchain of 10–14 carbons were necessary for activity.18,20–22 Moreover,our studies have indicated the importance of lactone ring opening,as the corresponding lactam derivative was completely inactivein mammalian cell activation experiments18 and a thiolactoneanalogue was >35 times less active than C12 in a mouse splenocyteproliferation assay.21

To better understand the effect of lactone stability onmammalian cell activation by AHLs, C12-N and C12-S wereprepared as previously reported by our laboratory18,26 andothers,21,27,28 respectively. Bone marrow-derived macrophages(BMDMs) were then treated with C12-N or C12-S (50 mM) over aperiod of 2 h, and macrophage responsiveness was analyzed byWestern blot for the cleavage of poly(ADP)-ribose polymerase(PARP) and the phosphorylation of eukaryotic translationinitiation factor 2a (p-eIF2a) and protein kinase p38 (p-p38),biochemical markers indicative of C12-induced apoptosis, theER stress response and the p38 pathway, respectively.18

As Fig. 2 shows, neither analogue induced C12-mediated bio-logical effects, thus, corroborating with previous reports.18,21

Because previous studies by our laboratory showed that thelactone of C12 is readily hydrolyzed upon addition to cells withthe cellular fraction containing B10 times more hydrolyzedcompound than the supernatant,18 we were interested to examineif lactone hydrolytic stability was a determinant in the ability ofC12 to activate mammalian cells. Half-lives of C12, C12-N and

C12-S have been previously reported, and were measured asB18 h, >48 h and 82.3 h, respectively.26,28 Coupling this datawith our biochemical analyses, we hypothesized that hydrolyticinstability of the lactone ring may be an important factor forAHL-mediated activation of mammalian cells. To examine thispossibility, we designed and synthesized bC12, which contains ab-lactone in place of the homoserine lactone and should exhibitgreater hydrolytic lability (Scheme 1).29 The synthesis of bC12began with the activation of acid 1 with DCC followed bynucleophilic displacement of the resultant activated ester withMeldrum’s acid and coupling with methanol to yield 2. The 3-oxosubstituent was then protected with ethylene glycol and themethyl ester was hydrolyzed to the corresponding acid 3. bC12was then obtained following coupling with amino-b-lactone 4 anddeprotection of the 3-position. As anticipated, the measured half-life of bC12 in buffered aqueous solution was B2 h, which isdrastically reduced compared to the natural C12 and analoguesC12-N and C12-S.

With the bC12 in hand, similar to the above-describedexperiments for the other AHLs, BMDMs were treated withbC12 (50 mM) over a period of 2 h, and cellular extractswere examined by Western blot analysis for C12-mediatedbiomarkers. As Fig. 3a shows, a 15 min treatment with bothC12 and bC12 resulted in comparable activation of PARPcleavage as well as phosphorylation of eIF2a and p38, thusdemonstrating that hydrolytic instability is a critical feature forAHL activity in mammalian immune cells. We also noted thatin contrast to C12, bC12-mediated biological activity rapidlydiminished over time. As this AHL ring analogue contains ab-lactone motif, which could readily modify proteins,29 thismay explain the observed results. Alternatively, it may not beable to reclose due to ring strain and remains in its hydrolyzed(open) form. Interestingly, this structural modification wasnot tolerated by P. aeruginosa, as bC12 was not active as anautoinducer in a P. aeruginosa quorum sensing assay (see ESI†).These results mirror our previously reported findings, whichindicated that the structural requirements for autoinduceractivity and mammalian cell activation are distinct.20

Because many in the field, including our laboratory, areactively interested in uncovering the mammalian cellreceptor(s) of C12 to aid in delineating the mechanisms bywhich AHLs regulate immune cell processes,20,30,31 we wereeager to design an analogue of bC12 for future target identifi-cation studies in immune cells. In particular, as the possibilityof covalent protein modification exists with this AHL derivative,

Fig. 2 Biological activities of C12-N and C12-S in BMDMs. BMDMs were treatedwith 50 mM AHL for the indicated period of time, and cellular extracts wereanalyzed by Western blot for the cleavage of PARP, phosphorylated forms ofeIF2a (p-eIF2a) or p38 (p-p38) and actin as a loading control. C12 was used as apositive control. Scheme 1 Synthesis of bC12.

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we were encouraged to use this scaffold for probe development.bC12-N3 (Fig. 3b, see the ESI† for synthetic details) wasdesigned to utilize the b-lactone functionality as a chemicalwarhead coupled with the installation of an azide moiety as anaffinity tag following click chemistry with a biotinylated orfluorophore-labeled alkyne. The ability of bC12-N3 to activatemammalian cells was then examined, as probe activity iscritical for its use in target identification studies. As Fig. 3ashows, bC12-N3 showed similar activity to the parent bC12and recapitulated C12-mediated biochemical phenotypes withmodified kinetics, again demonstrating that incorporation ofan azido functionality into the AHL tail is a conservativestructural change.20 Target identification studies in immunecells utilizing this AHL probe will be reported in due course.

In conclusion, by completing focused analogue synthesisstudies around the lactone of C12 produced by P. aeruginosa, wehave identified new structural analogues that can be used toexplore the diverse biochemical activities of this bacterialsignaling molecule, which is both an activator of mammaliancells and immunomodulator. Moreover, we have also uncoveredthat hydrolytic instability of the lactone ring is significant for theability of C12 to activate mammalian cells. As our over-archinggoal is to identify the protein target(s) responsible for theparadoxical biological activities of C12, our future studies willfocus on the employment of bC12-N3-based compounds as probemolecules to begin to understand the link between AHL-inducedER stress and immunosuppression. We hope that these futurestudies will help us begin to understand not only how AHLsimpact mammalian cell biology but also how bacterial signalingmolecules are able to impair the human immune system topromote virulence and pathogenicity.

This work was supported by the NIH (Grants AI079503,AI077644 and DE018452 to K.D.J., grant AI085324 to G.F.K.and grant AI094348 to G.F.K. and V.V.K.) and the SkaggsInstitute for Chemical Biology.

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Fig. 3 bC12 AHLs. (a) Biological activities of C12, bC12 and bC12-N3 in BMDMs.BMDMs were treated as previously described and analyzed by Western blot forthe cleavage of PARP, phosphorylated forms of eIF2a (p-eIF2a) or p38 (p-p38)and actin as a loading control. (b) Structure of bC12-N3.

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