C O M M U N I C A T I O N S
Table 2. Summary of QS Modulation by C1-Alkylated DPD
To lend credence to the use of these compounds in in vivo settings,
we have examined the effects of DPD and the corresponding
C1-substituted DPD analogues against a mouse leukemic monocyte
macrophage cell line (RAW 264.7) using an XTT based in vitro
toxicology assay kit (Sigma). The panel of compounds, including DPD
itself, were found to be nontoxic toward mammalian cells as cells
retained at least 90% viability in the presence of 50 µM compound
(Supporting Information, Table S1). These results, coupled with the
differential activity of these compounds in the two reporter assays,
make these analogues candidates for the study and inhibition of AI-
2-based QS in vivo.
Analogues
IC50 in
S. typhimurium assay (µM)a
fold-activation in
V. harveyi assayb
compound
ethyl-DPD (5b)
propyl-DPD (5c)
butyl-DPD (5d)
hexyl-DPD (5e)
phenyl-DPD (5f)
azidobutyl-DPD (5g)
>50
6.30 ( 0.72
7.69 ( 0.30
6.05 ( 0.93
2.74 ( 0.24
1.81 ( 0.12
7.44 ( 0.77
5.30 ( 0.43
5.04 ( 0.61
24.9 ( 5.4
>50
20.3 ( 1.3
a Assay performed in the presence of 50 µM DPD and varying
concentrations of test compound. b Assay performed in the presence of 1
µM DPD and 25 µM compound. Luminescence was measured after 8 h.
In conclusion, we have shown how a panel of alkyl-substituted
DPD analogues can elicit strikingly different biological effects in
two different species with known AI-2 QS systems: V. harVeyi and
S. typhimurium. This difference in activity could not be predicted
solely on the basis of the crystal structures of the AI-2 signals nor
the receptor proteins but rather was revealed through chemical
synthesis and the exploration of bacterial phenotypes with the use
of a series of structurally related compounds. In sum, these findings
validate, in principle, our approach to the design of DPD-based
analogues for the modulation of AI-2-based QS. Although it remains
to be seen if these compounds will affect other species that respond
to AI-2, this class of compounds nevertheless represents a logical
starting point for the development of broad range modulators of
QS and identification of unknown AI-2 receptor proteins.
Acknowledgment. We gratefully acknowledge Prof. Bonnie
Bassler (Princeton University) for providing us with Met844 and Dr.
Michael Meijler (Ben Gurion University) and Dr. Tobin Dickerson
for insightful conversations. We also thank the Skaggs Institute for
Chemical Biology and Sanofi-Aventis Graduate Fellowship (C.A.L.)
for funding.
of ꢀ-galactosidase activity in S. typhimurium and bioluminescence
production in V. harVeyi. These two assays were selected for biological
evaluation because our analogues were designed from the AI-2 signals
employed by both species as well as because of the rapid readout
provided by each assay.
The screens performed for (ant)agonistic activity in S. typhimurium
were measured via a ꢀ-galactosidase activity assay.15 Assays were
conducted using S. typhimurium strain Met844, a ∆luxS strain with a
lacZ-lsr fusion, and assays were performed in the absence of DPD
(agonist assay) or in the presence of 50 µM DPD (antagonist assay).
The lacZ fusion, which encodes for the biosynthesis of ꢀ-galactosidase
under lsr promoter control allows for the monitoring of AI-2-dependent
lsr activation.16 No agonists were uncovered from the screening, but
in the presence of 50 µM DPD, all compounds were found to act as
antagonists of AI-2-based QS (Table 2). Notably, the propyl-substituted
(5c) and butyl-substituted (5d) analogues were potent inhibitors with
IC50 values 10-fold below the concentration of the natural DPD signal,
placing these two analogues among the most potent inhibitors of QS
relative to the concentration of natural autoinducer.3,17,18 Importantly,
none of these compounds affected the growth of S. typhimurium,
implicating their role in the specific antagonism of AI-2 QS. Also
notable is the activity of the azidobutyl-DPD, which may be used in
the identification of unknown AI-2 receptor proteins using the recently
developed tag-free approach, utilizing click chemistry, to protein
identification.19 Intriguingly, addition or deletion of a single methyl
group between DPD (5a), ethyl-DPD (5b), and propyl-DPD (5c),
completely alters the biological actiVity of these substances ranging
from the natural substrate to no actiVity to a potent antagonist.
To explore an expanded role of these compounds as modulators of
QS, we evaluated their effects on the QS of V. harVeyi in a
bioluminescence assay.20 Modulation of bioluminescence was exam-
ined using V. harVeyi MM32 cells (ATCC BAA-1121, ∆luxS, ∆luxN),
a cell line incapable of producing luminescence either through the acyl-
homoserine lactone pathway or AI-2 pathways in the absence of
exogenous DPD. Although V. harVeyi responds to a borate diester form
of DPD, boric acid was not added during these assays, as the presence
of boric acid itself induces QS activity, rendering V. harVeyi less
sensitive to different concentrations of DPD. Additionally, V. harVeyi
does respond to DPD without the addition of boric acid.12,13,20 Thus,
the test compounds were evaluated for agonist activity, but only the
ethyl-DPD exhibited weak agonistic activity (50-fold less active than
natural DPD, data not shown). However, when the test compounds
were incubated with V. harVeyi and 1 µM DPD to monitor antagonism,
a synergistic effect was observed (Table 2). This synergistic activity
was observed across the entire series of analogues, with ethyl-DPD
(5b) and butyl-DPD (5d) exhibiting greater than 6-fold activation, and
propyl-DPD (5c) and azidobutyl-DPD (5g) exhibiting at least 7-fold
activation over 1 µM DPD. Interestingly, these compounds were
inactive in the absence of DPD, leading to the hypothesis that these
analogues are interacting with the AI-2 receptor protein LuxP in a
manner productive only in the presence of natural DPD.
Supporting Information Available: Experimental procedures, spec-
tral data, and biological protocols. This material is available free of charge
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