A 2-aminobenzimidazole that inhibits and disperses gram-positive biofilms through a zinc-dependent mechanism

Article abstract of DOI:10.1021/ja9024676

(Chemical presented) A number of 2-aminobenzimidazole derivatives were synthesized and screened for their ability to inhibit and disperse bacterial biofilms. From these compounds, a lead 2-aminobenzimidazole was identified that both inhibited and disperse

Full text of DOI:10.1021/ja9024676

Published on Web 07/02/2009
A 2-Aminobenzimidazole That Inhibits and Disperses Gram-Positive Biofilms
through a Zinc-Dependent Mechanism
Steven A. Rogers, Robert W. Huigens III, and Christian Melander*
Department of Chemistry, North Carolina State UniVersity, Raleigh, North Carolina 27695
Received March 27, 2009; E-mail: Christian_Melander@ncsu.edu
Scheme 1. Synthesis of the 2-Aminobenzimidazole Librarya
Bacterial biofilms, which are defined as surface-attached com-
munities of bacteria encased in an extracellular matrix of biomol-
ecules, represent a significant hurdle for infectious disease control.1
Within the biofilm state, bacteria are upward of 1000-fold more
resistant to antibiotics and are inherently resistant to host immune
responses.2 The National Institutes of Health estimate that 75% of
all infections are biofilm-based and that biofilms drive many
hospital-acquired infections (such as those resulting from implanted
medical devices).3
Because of the prominence of biofilms in the biomedical
community, there has been a significant effort to identify molecules
that target bacteria in the biofilm state.4 To define additional motifs
with antibiofilm properties, we became interested in assessing the
activity of 2-aminobenzimidazole (2-ABI) derivatives. The decision
to study the 2-ABI scaffold was based upon previous studies in
our group that analyzed the antibiofilm properties of a number of
small molecules based upon the natural product bromoageliferin
(Figure 1).5-7 One of the first derivatives studied was TAGE,6 a
bicyclic 2-aminoimidazole (2-AI) that represents the core archi-
tecture of bromoageliferin. The 2-ABI scaffold is a readily
accessible, aromatized analogue of TAGE, which we hypothesized
would provide unique and/or improved antibiofilm properties in
a Reaction conditions: (a) cyclic anhydride, CH2Cl2; (b) HCl, H2O, THF;
(c) cyclic anhydride, toluene, 110 °C.
comparison with 2-AIs.
development in three Gram-positive bacterial strains that are
prominent in human medicine. The three strains chosen were
MRSA, vancomycin-resistant Enterococcus faecium (VRE), and
Staphylococcus epidermidis. Again, all of the 2-ABI derivatives
were initially screened at 100 µM. Each compound was able to
inhibit biofilm development of at least two of the bacterial strains.
A dose-response study was then initiated to determine both the
IC50 and EC50 values of each compound toward the bacterial strains
(Table 1). Here, IC50 is defined as the concentration of compound
that inhibits biofilm development by 50%, while EC50 is defined
as the concentration of compound that disperses 50% of a preformed
biofilm. Of these compounds, 2-ABI derivative 3 had the best
activity profile, with IC50 and EC50 values of 890 nM and 2.9 µM
(MRSA), 1.4 µM and 75 µM (VRE), and 570 nM and 7.3 µM (S.
epidermidis), respectively. Growth-curve and colony-count analyses
demonstrated that each compound was nonmicrobicidal at the IC50
value.
Next, the mechanistic basis of how 3 was able to inhibit and
disperse bacterial biofilms was investigated. Iron levels are known
to effect Gram-positive biofilm development9 and were deemed a
plausible driver of the observed antibiofilm activity. To examine
Fe(II)-related antibiofilm behavior, a dose-dependent study was
performed against each Gram-positive bacteria in which the ability
of 3 to inhibit biofilm development was measured under increasing
Fe(II) concentration. It was noted that the activity of 3 was not
affected by increasing the Fe(II) concentration, thus indicating iron
homeostasis is not involved in 2-ABI antibiofilm activity.
Next, Zn(II) homeostasis was examined. Zn(II) has been
implicated in the pathogenesis of Gram-positive bacterial infections
Figure 1
To explore this hypothesis, a preliminary library of 2-ABI
analogues was synthesized for antibiofilm evaluation (Scheme 1).
An isomeric mixture of tri-Boc-protected 5-amino-2-ABI (1)8 was
acylated with an array of cyclic anhydrides, and the products were
subsequently deprotected to generate an initial set of isomerically
pure 2-ABI derivatives. A related set of 2-ABI derivatives was also
synthesized in which reaction of 1 with cyclic anhydrides under
refluxing toluene followed by Boc deprotection delivered the target
2-ABI-imides.
Each compound was screened at 100 µM for its ability to inhibit
the formation of Pseudomonas aeruginosa PAO1 and multidrug-
resistant Acinetobacter baumannii (MDRAB) biofilms. As opposed
to the 2-AI class of antibiofilm agents, none of the 2-ABI derivatives
were able to inhibit formation of biofilms of either γ-proteobacteria.
Next, each compound was screened for its ability to inhibit biofilm
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9868 J. AM. CHEM. SOC. 2009, 131, 9868–9869
10.1021/ja9024676 CCC: $40.75  2009 American Chemical Society

C O M M U N I C A T I O N S
Table 1. Biofilm Inhibition and Dispersion Data
IC50 (µM); EC50 (µM)
the antibiofilm properties of the compound. Neither compound was
able to inhibit MRSA, VRE, or S. epidermidis biofilm formation
at 100 µM (the highest concentration tested). Furthermore, no
change was noted in either H NMR spectrum in the presence of
ZnCl2.
compound
MRSA
VRE
S. epidermidis
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
5.9 ( 1.3; 35 ( 2.8
21 ( 2.9; 75 ( 6.7 32 ( 3.8; 44 ( 7.9
0.89 ( 0.01; 2.9 ( 0.4 1.4 ( 0.4; 75 ( 2.1 0.57 ( 0.2; 7.3 ( 0.2
1.5 ( 0.4; 6.6 ( 0.2
16 ( 2.9; 63 ( 6.9
>300; >300
210 ( 35; 280 ( 8.3 1.9 ( 0.4; 19 ( 5.8
63 ( 9.1; 107 ( 11 4.1 ( 2.0; 45 ( 6.1
22 ( 8.3; 88 ( 5.2 1.9 ( 0.9; 13 ( 6.1
0.9 ( 0.3; 94 ( 10 240 ( 20; 260 ( 6.7
69 ( 2.7; 205 ( 15
103 ( 6.3; 250 ( 19 2.4 ( 0.7; >300
73 ( 8.2; 175 ( 8.5
>300; >300
0.9 ( 0.4; 6.3 ( 1.9 6.2 ( 1.2; 30 ( 5.7
7.7 ( 0.8; 150 ( 8.4 180 ( 17; 266 ( 7.3
1.6 ( 0.9; 280 ( 4.8 12 ( 1.0; 137 ( 15
16 ( 4.7; 203 ( 5.5 0.9 ( 0.3; 17 ( 4.0
6.9 ( 2.2; 30 ( 8.5 15 ( 2.7; 70 ( 4.2
1.9 ( 0.5; 230 ( 9.6 170 ( 14; 180 ( 13
69 ( 10; 110 ( 2.9 24 ( 6.3; 81 ( 4.9
7.4 ( 1.7; 31 ( 7.8 1.5 ( 0.5; 33 ( 3.7
2.8 ( 0.8; 36 ( 1.7
17 ( 1.9; 25 ( 2.0
48 ( 4; 101 ( 6
9.6 ( 0.8; 14 ( 2.9
42 ( 2.4; 48 ( 2.1
>300; >300
Figure 3. Control compounds.
In conclusion, a novel inhibitor and disperser of Gram-positive
biofilms that is based upon a 2-ABI scaffold and operates via a
Zn(II)-dependent mechanism has been identified. Preliminary NMR
studies indicated that this compound binds zinc directly. These
2-ABI molecules are unique in that they are some of the most potent
antibiofilm agents identified to date that do not operate through a
microbicidal mechanism. Furthermore, molecules that bind Zn(II)
have recently been shown to be efficacious in animal models of
disease;11,12 therefore, appropriately designed 2-ABI molecules may
provide a basis for remediating biofilm-based infections.
5.7 ( 0.5; 9.5 ( 2.5
and is an important regulator of biofilm formation.10 Furthermore,
generic Zn(II) chelators such as diethylenetriaminepentaacetic acid
(DTPA) are known to inhibit the formation of Staphylococcus spp.
biofilms at midmicromolar concentrations.10 As opposed to the
Fe(II) study, it was noted that Zn(II), in a dose-dependent manner
against each Gram-positive bacteria, suppressed the ability of 3 to
inhibit biofilm development. When supplemented with 200 µM
ZnCl2, 3 was unable to inhibit biofilm formation.
In view of this Zn(II) dependence, the ability of 3 to bind zinc
directly was examined to ascertain whether the biofilm inhibition
was potentially occurring via a Zn(II)-binding mechanism. To
answer this question, an 1H NMR binding experiment was
performed in which the chemical shifts of 3 were measured in the
presence of 0, 0.5, and 1.0 equiv of ZnCl2 (Figure 2). Comparison
of the aromatic peaks clearly indicates peak broadening with
increasing amounts of ZnCl2, indicating that 3 directly binds ZnCl2.
As a control, the same experiment was performed with FeSO4. No
change in the NMR signal of 3 was observed with 0, 0.5, or 1.0
equiv of FeSO4.
Acknowledgment. The authors gratefully acknowledge funding
from the V Foundation and the University of North Carolina
General Administration Competitiveness Research Fund.
Supporting Information Available: Experimental procedures and
characterization data for all new compounds; planktonic growth curves
for MRSA, VRE, and S. epidermidis in the presence and absence of
active 2-ABIs; dose-response curves of 3 in the absence and presence
ZnCl2 and FeSO4; and dose-response curves and NMR binding studies
for 17 and 18. This material is available free of charge via the Internet
at http://pubs.acs.org.
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Finally, two control compounds were tested (17 and 18; Figure
3) to probe whether substructures within 3 were responsible for
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