Species selective diazirine positioning in tag-free photoactive quorum sensing probes

Article abstract of DOI:10.1039/c3cc43092h

The synthesis and comparison of activities of 'tag-free' probes with diazirines at various positions are described. Remarkable differences in their effects on P. aeruginosa and on human bronchial epithelial cells were observed, supporting the efforts to i

Full text of DOI:10.1039/c3cc43092h

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Species selective diazirine positioning in tag-free
photoactive quorum sensing probes†
Luba Dubinsky,a Antonia Delago,a Neri Amara,a Pnina Krief,a Josep Rayo,a
Tsaffrir Zor,b Vladimir V. Kravchenkoc and Michael M. Meijler*a
Cite this: Chem. Commun., 2013,
49, 5826
Received 26th April 2013,
Accepted 8th May 2013
DOI: 10.1039/c3cc43092h
www.rsc.org/chemcomm
The synthesis and comparison of activities of ‘tag-free’ probes with
diazirines at various positions are described. Remarkable differ-
ences in their effects on P. aeruginosa and on human bronchial
epithelial cells were observed, supporting the efforts to isolate and
identify receptors for N-acyl homoserine lactones.
Fig. 1 Structures of 3-oxo-C12-HSL (C12, 1) and diazirine alkynyl probes 2, 3 and 4.
Quorum sensing (QS) is a mechanism that enables organisms to
regulate gene expression in response to changes in cell population for P. aeruginosa virulence genes.4 While QS plays an important role
density.1 The main purpose of QS is to coordinate the behavior of in the pathogenicity of P. aeruginosa and contributes to its virulence
single cells, in order for them to function as one multicellular and resistance to host responses, many molecular details regarding
organism. In bacteria a variety of physiological processes are its regulation and timing are still underexplored, including for
regulated by QS. Some established phenotypes are the induction instance the recognition of C12 by more than one regulator.5
of bioluminescence, virulence factor expression, biofilm formation
In the past decades several lines of research have revealed an
and motility. Common to all these processes is that biological exchange of QS related signals between bacteria and higher orga-
function is attained only upon simultaneous molecular recogni- nisms. Prokaryotes and eukaryotes have co-existed for millions of
tion by a large number of individuals.2
years and recent studies suggest the evolution of intricate mechan-
Bacterial QS occurs through secretion and recognition of small isms of interaction that are based on chemical signaling events.1,2
diffusible molecules, called autoinducers (AIs). Upon reaching a One example of such an interaction can be found in Candida
certain threshold concentration these AIs diffuse back into the cell albicans, a pathogenic yeast. C. albicans is part of the natural
where they bind their respective receptors, setting in motion cascades microfauna found in humans. However, in immune compromised
of gene transcription and protein expression. In Gram negative individuals it can cause serious infections and recent studies have
bacteria the main AIs are N-acyl homoserine lactones (AHLs). Our shown that the pathogenicity of Candida is modulated by C12.6
research focuses on a representative member of the AHL family, Since P. aeruginosa and C. albicans are often found in the same
N-(3-oxododecanoyl) homoserine lactone (3-oxo-C12-HSL, C12, Fig. 1, 1), tissues or organs, it is reasonable to assume that some form of
the primary QS signal of Pseudomonas aeruginosa. This microorgan- cross talk has evolved between these two organisms.
ism is ubiquitous and highly adaptable, and can be found in soil, in
Kolter and co-workers have described an effect of C12 on the
water, in the skin fauna, but also in most man-made environments, morphology of C. albicans, which indicates an adaptive defense
where it can take advantage of pre-existing weaknesses in the host’s mechanism of the fungus.6 Moreover, yeast growth is reduced by
immune system.3 The primary receptor for C12 in P. aeruginosa is the bacterial phenazines, such as pyocyanin. In turn, farnesol, the
transcriptional activator LasR, which is considered a global regulator designated QS signal of Candida, leads to a decrease in pyocyanin.
Therefore, secreted factors from both species have mutually antago-
nistic effects on each other’s virulence and eventually on survival.6
a Department of Chemistry and the National Institute for Biotechnology in the Negev,
Interaction of bacterial QS signals with mammalian cells to date has
mainly focused on C12-mediated effects on inflammatory and innate
immune responses. However, the observed effects have been shown
to vary, probably depending on the concentration of C12. Multiple
studies,7–20 conducted using a wide range of C12 concentrations,
showed a variety of pro- and anti-inflammatory responses in several
different types of cells, such as fibroblasts,7–11 endothelial8 and
Ben-Gurion University of the Negev, Be’er-Sheva 84105, Israel.
E-mail: meijler@bgu.ac.il; Tel: +972-8-6472751
b Department of Biochemistry and Molecular Biology, Life Sciences Institute,
Tel-Aviv University, Tel-Aviv 69978, Israel
c Department of Immunology and Microbial Sciences, The Scripps Research Institute,
10550 N Torrey Pines Road, La Jolla, CA 92037, USA
† Electronic supplementary information (ESI) available: Synthetic procedures,
spectral characterization and analytical protocols. See DOI: 10.1039/c3cc43092h
c
5826 Chem. Commun., 2013, 49, 5826--5828
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epithelial7,9 cells, keratinocytes,12 T cells,13–16 macrophages11 and
Probe 4 was synthesized in a similar fashion (Scheme S3, ESI†).
neutrophils.17 In addition, we have recently discovered that C12 Homologation of THP-protected hydroxycaproic acid 9 with mono
amplifies LPS-stimulated IL-10 expression in macrophages.21
tert-butyl malonate resulted in 10, which was reacted with propargyl
While a lot of new information has been uncovered in recent years bromide to afford 11. Deprotection followed by Jones oxidation and
with regard to the effects of C12 on eukaryotes,22,23 so far no bona fide TFA mediated dealkylation yielded the desired 6-oxodec-9-ynoic
receptor has been found. Several candidate proteins have been acid, 14. The final coupling to prepare probe 4 was performed in
identified using a range of methods, but in none of these studies good yield, following the previously described procedure for probe 3.
was C12 shown to actually bind the protein, let alone bind it in a
In order to analyze binding of the probes to the native
specific manner with high affinity.24–27 One candidate for a eukaryotic Pseudomonas receptor, LasR, we overexpressed the ligand binding
receptor for C12 is the peroxisome proliferator activated receptor domain (LBD) of LasR in E. coli, in the presence of probes 2–4,
gamma (PPARg), a transcription factor that participates in the regula- followed by UV irradiation of the cells (at 365 nm), resulting in a
tion of fatty acid storage and glucose metabolism in mammalian cells. covalent bond between the probes and their receptors. The cells
It has been demonstrated that C12 modulates the transcriptional were then lysed, and LasR-LBD was purified by affinity chromato-
activity of PPARg,24–26 potentiating the expression of pro-inflammatory graphy, after which the protein was irradiated again for 10 min and
response genes. Therefore, additional proteins distinct from PPARg directly analysed by LC/MS (Fig. 2). The additional UV irradiation
are likely to be involved in C12-mediated immunosuppressive improved the labeling, indicating that part of the diazirine probe
¨
responses. In a different study, Vikstrom and co-workers showed undergoes slow activation and that the probes in general have high
that C12 affects the migration of human intestinal epithelial Caco-2 affinity for LasR, remaining in its binding pocket throughout
cells.27 They moreover showed an interaction between the IQ-motif- purification. Cells that were incubated with C12 alone (20 mM)
containing GTPase-activating protein IQGAP1 and a C12-biotin probe, yielded a single LasR-LBD peak (22 427 Da (calc. 22 430 Da), Fig. 3A).
as well as other fluorescently tagged probes. However, no specific An additional peak with a mass difference of 291 Da appeared upon
binding studies with C12 were performed, and given the small size addition of probe 3 (20 mM), ESI†, Fig. S1A, corresponding to the
and hydrophobic nature of AHLs, it is expected that even the smallest expected addition of 291 Da upon reaction with the activated
modification might substantially affect the interaction of C12 with its diazirine probe; probe 4 yielded a similar result (+289 Da). In both
target protein(s).16,22 Therefore, conventional methods of modifying cases the additional peak was absent when C12 (20 mM) was added
C12 with a bulky tag or conjugating it to an affinity matrix28 may cause to the reaction (ESI†, Fig. S1A and B), demonstrating the specificity
a significant loss of affinity and prevent detection by its receptor.
To overcome this challenge, we have recently developed an
of the probes for the LasR binding pocket, similar to probe 2.29
In addition, we performed in vivo labeling experiments with LasR-
approach to label and isolate receptors for C12 via a two-step LBD overexpressed in E. coli, using biotin/rhodamine-azide as tags.
tag free photoaffinity purification procedure.29 This approach is Binding of the probes to LasR-LDB was validated using LC/MS. After
based on a powerful methodological approach termed activity cell lysis, by employing the ligand’s alkyne moiety, a bioorthogonal
based protein profiling (ABPP), which was developed by the copper catalyzed azide alkyne cycloaddition (CuAAC) reaction was
groups of Cravatt and Bogyo.30–32
performed, yielding fluorescent or biotinylated proteins. Analysis of
In our previous study we described the design and synthesis of a the lysed proteome (Fig. 3), following SDS-PAGE and western blotting,
diazirine containing C12 mimic with an alkyne as a strategic group for shows fluorescent bands at the expected molecular mass for LasR-
reaction with a fluorescent or biotinylated azide (Fig. 1). The choice of LBD, while for cells that were treated with probes 2–4 in the presence
diazirine as a reactive group was motivated by its small size, effecting of C12 (2-fold excess), the intensity of the bands was much lower,
minimal structural changes to the C12 mimic (allowing maximal again demonstrating the competition for the same binding site. We
receptor recognition) and relatively mild irradiation conditions.33
also evaluated the ability of probes 2–4 to induce bioluminescence in
Here, we describe the synthesis and evaluation of C12 based the QS reporter strain PAO-JP2-luxCDABE (Fig. 4A), a P. aeruginosa
probes with the diazirine moiety at different positions along the alkyl strain that has the complete QS machinery, but cannot synthesize
chain (Fig. 1). The rationale behind this exploration of the potential C12. We found that at low micromolar concentrations probes 2 and 3
impact of the position of the diazirine on biological function is based
on our hypothesis that the microenvironment of the bound probe
strongly affects the probability of successfully targeting true receptors
among the many weaker binders of small hydrophobic ligands.
Both diazirine alkynyl probes 3 and 4 were synthesized based on
procedures that are similar to our published route29 to probe 2 –
with some clear distinctions (see ESI† for details). The synthesis of
probe 3 started from commercially available (5-iodo-1-pentynyl)-
trimethylsilyl-silane (5), to which lithiated 3,4-dihydro-2H-pyran was
added, followed by oxidation with the Jones reagent (Scheme S2,
ESI†). The alkylated product 6 was then treated with liquid ammonia
to afford the diaziridine intermediate, which was oxidized with iodine
to afford the desired diazirine 7. Coupling of 7 with Meldrum’s acid in
Fig. 2 Deconvoluted ESI mass spectra of crude LasR-LBD: (A) overexpressed in
E. coli in the presence of C12 (20 mM). Calculated mass: 22 430 Da. Observed:
the presence of DCC and DMAP, followed by treatment with homo-
serine lactone yielded diazirine alkynyl probe 3.
22 427 Da. (B) Overexpressed in the presence of diazirine probe 4 (20 mM). Calculated
mass increase upon labeling with 4: 291 Da. Observed increase: 289 Da.
c
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This work was supported by the European Research Council
(Starting Grant 240356, MMM), the National Institutes of Health
(AI094348, VVK), and by the US–Israel Binational Science Founda-
tion (Startup Grant 2006287, MMM). We thank R. J. Ulevitch for
valuable insight and support, and M. J. Bottomley and M. G. Surette
for bacterial strains and plasmids.
Notes and references
Fig. 3 Live cell photolabeling of LasR using probes 2–4, followed by lysis and
CuAAC: (A) fluorescent labeling of tagged LasR-LBD with rhodamine azide;
(B) western blot analysis of biotinylated proteins, using streptavidin-HRP.
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Taken together, the functional and binding assays show that while
all three probes are good mimics for C12 in both bacterial and
mammalian systems, significant differences in target binding and/or
function demonstrate that the exact position of the diazirine moiety –
even between adjacent positions along the lipid chain – may be a
crucial factor in photoaffinity labeling and provide information on
the importance of a precise design of such small photoaffinity probes.
We are currently using the three probes to identify putative and
unknown receptors for C12 in different species.
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5828 Chem. Commun., 2013, 49, 5826--5828
This journal is The Royal Society of Chemistry 2013