Angewandte
Chemie
(16) or significant reductions in biofilm inhibitory activity
(Table 1). The most potent biofilm inhibitor identified overall
was the 5,6-dimethyl 2-ABI 21 (IC50 = 4.0 mm), which was
approximately 10-fold more active than the parent compound
7. Amongst the few biofilm inhibitors for which IC50 data has
been reported,[8a,c,e,g] 21 constitutes one of the most active P.
aeruginosa biofilm inhibitors known. Dose-response curves
and images of crystal-violet-stained biofilms in the presence
of 7 and 21 are shown in Figure 2A.
investigate the mechanism by which the 2-ABI scaffold elicits
its biofilm inhibitory and dispersive activity in P. aeruginosa.
Planktonic growth curve analyses (under conditions identical
to biofilm growth) demonstrated that the observed activities
were not a result of a bactericidal mechanism. Melander and
co-workers have shown that 2-ABI derivatives bearing 5-
amido substituents inhibit and disperse biofilms through
a zinc-dependent mechanism, albeit in Gram-positive as
opposed to Gram-negative bacteria (see above).[14] We
screened a wide range of metals, including zinc, in a dose-
dependent manner for mitigating effects on the biofilm
inhibitory activity of 7 in P. aeruginosa, but observed no
change in activity.
As introduced above, the role of QS in biofilm formation
is well documented,[6–9] and therefore we next evaluated the
abilities of 7 and 21 to inhibit QS in P. aeruginosa. For this
purpose we prepared two wild-type P. aeruginosa QS reporter
strains containing the plasmids plasI-LVAgfp and prhlI-
LVAgfp (see the Supporting Information). These strains
report the activity of two intracellular QS receptors in P.
aeruginosa (LasR and RhlR) by the production of green
fluorescent protein (gfp), thereby allowing LasR and RhlR
activities, and thus QS levels, to be quantified by fluorescence.
We observed a significant reduction in both LasR and RhlR
activities in the presence of 7 and 21 at 1–10 ꢀ their IC50 values
for biofilm inhibition (Figure 2C–D). Studies of related 2-
ABI derivatives have shown that this class of molecules is
bacterial cell permeable, thus allowing us to surmise that 7
and 21 could act on the Las and Rhl systems intracellularly.[24]
Further, we utilized a P. aeruginosa strain, which constitu-
tively expresses genomic gfp, to demonstrate that 7 and 21 do
not simply affect global protein synthesis (see the Supporting
Information). Additional experiments are required to eluci-
date the precise targets of 7 and 21 that result in QS
disruption. Nevertheless, these preliminary findings suggest
that biofilm modulation by 2-ABI derivatives could be
occurring, at least in part, through interference with the P.
aeruginosa Las and Rhl QS circuits.
In summary, we have identified 2-ABI derivatives as
potent antibiofilm agents in P. aeruginosa. We uncovered this
compound class through the study of hybrid compounds
derived from the structures of three natural products with
known biofilm and QS inhibitory activities, and the subse-
quent structure–activity analyses of simplified derivatives.
This discovery is significant, as several of these 2-ABI
derivatives are among the most active P. aeruginosa biofilm
modulators to be reported. Moreover, these compounds are
capable of both inhibiting the growth of and dispersing
preformed biofilms. Our results are surprising in light of
previous data on related 2-ABI derivatives which indicated
they were inactive in P. aeruginosa,[14] and support the
continued study of this structurally simple, chemically
robust compound class in Gram-negative bacteria.[25] Lastly,
our studies indicate that the 2-ABIs 7 and 21 are also capable
of QS inhibition in P. aeruginosa, thus suggesting a possible
mechanism for biofilm inhibition. A link between 2-ABI-type
antibiofilm agents and QS, to our knowledge, has been
previously undocumented. Ongoing work in our laboratory is
directed towards the study of additional 2-ABI derivatives, as
Figure 2. Dose-response curves and images of crystal violet biofilm
inhibitory (A), and dispersion (B) assays for 7 and 21 in P. aeruginosa
(PAO1). NA=not available. Dose-response curves for 7 and 21 in
P. aeruginosa PAO1/plasI-LVAgfp (C), and PAO1/pRhI-LVAgfp (D) QS
reporter strains.
Compounds capable of not only inhibiting biofilm growth,
but also dispersing preformed biofilms, are of particular value
for a range of clinical and other applications. We thus tested
the ability of compounds 7 and 21 to disperse 24-hour-old P.
aeruginosa biofilms using the crystal violet staining assay
(Figure 2B). Biofilms were allowed to develop in the absence
of compound for 24 hours, after which nonbiofilm material
was removed by washing with buffer, and fresh media with
compound was added. Biofilm was quantified after an
additional 24 hours, and the amount of the dispersed biofilm
was determined by comparison of the amount of biofilm at
48 hours in the presence of compound versus the amount of
biofilm at 24 hours in the absence of compound. We found
that the 2-ABIs 7 and 21 were capable of strongly dispersing
P. aeruginosa biofilms (ca. 80%), with half-maximal disper-
sion (DC50) values of 84 and 92 mm, respectively (Figure 2B).
Little is known about the actual mechanisms of action of
most small-molecule biofilm inhibitors. As such, we sought to
Angew. Chem. Int. Ed. 2012, 51, 1 – 5
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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