Synthesis and activity of biomimetic biofilm disruptors

Article abstract of DOI:10.1021/ja3120955

Biofilms are often associated with human bacterial infections, and the natural tolerance of biofilms to antibiotics challenges treatment. Compounds with antibiofilm activity could become useful adjuncts to antibiotic therapy. We used norspermidine, a natural trigger for biofilm disassembly in the developmental cycle of Bacillus subtilis, to develop guanidine and biguanide compounds with up to 20-fold increased potency in preventing biofilm formation and breaking down existing biofilms. These compounds also were active against pathogenic Staphylococcus aureus. An integrated approach involving structure-activity relationships, protonation constants, and crystal structure data on a focused synthetic library revealed that precise spacing of positively charged groups and the total charge at physiological pH distinguish potent biofilm inhibitors.

Full text of DOI:10.1021/ja3120955

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Communication
Synthesis and activity of biomimetic biofilm disruptors
Thomas Böttcher, Ilana Kolodkin-Gal, Roberto Kolter, Richard Losick, and Jon Clardy
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/ja3120955 • Publication Date (Web): 13 Feb 2013
Downloaded from http://pubs.acs.org on February 17, 2013
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Synthesis and activity of biomimetic biofilm disruptors.
Thomas Böttcher#, Ilana Kolodkin-Gal§, Roberto Kolter+, Richard Losick§, Jon Clardy#,*
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#Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Ave., Boston,
MA 02115, USA.
+Department of Microbiology and Immunobiology, Harvard Medical School, 77 Ave. Louis Pasteur, Boston, MA 02115,
USA
§Department of Molecular and Cell Biology, Harvard University, 16 Divinity Ave., Cambridge, MA 02138, USA.
*Email: jon_clardy@hms.harvard.edu
Supporting Information Placeholder
disintegration of biofilms, and identifying these molecular
signals could lead to therapeutically useful templates.
ABSTRACT: Biofilms are often associated with human bac-
terial infections, and the natural tolerance of biofilms to anti-
We previously identified D-amino acids as potent biofilm
biotics challenges treatment. Compounds with anti-biofilm
disruptors due to their ability to release the protein component
activity could become useful adjuncts to antibiotic therapy.
of EPS from the bacterial cell wall.9 Recently we identified
We used norspermidine, a natural trigger for biofilm disas-
sembly in the developmental cycle of Bacillus subtilis, to de-
velop guanidine and biguanide compounds with up to 20x
norspermidine as a key disruptor of the polymeric component
of EPS.10 Mixtures of norspermidine with D-amino acids were
highly synergistic, single digit nanomolar, in disrupting
increased potency in preventing biofilm formation and break-
biofilms (Fig. 1).10 Here we report synthetic mimics of
ing down existing biofilms. Compounds also were active
against pathogenic Staphylococcus aureus. Using an integrated
approach involving structure-activity relationships, protona-
norspermidine with increased potency and a structure-based
rationale for their activity.
tion constants and crystal structure data on a focused synthetic
library, revealed that precise spacing of positively charged
groups and total charge at physiological pH distinguished po-
tent biofilm inhibitors.
Most bacteria form biofilms
– multicellular microbial
communities embedded in a self-produced exopolymeric
substance (EPS) largely composed of a protein anchor and
different extracellular polymers. Bacteria within a mature
biofilm community exist in an altered metabolic state and
different physical environment than their free-floating, or
planktonic, relatives. Biofilm bacteria generally tolerate
antibiotic treatment1,2, and antibiotics can induce biofilm
formation.3,4 Consequently, biofilm inhibitors can be applied
to decrease antibiotic tolerance of bacteria.5 Biofilms play a
major role in many bacterial infections.2 In humans, the
antibiotic tolerance of biofilm communities frustrates the
treatment of persistent bacterial infections such as those
associated with cystic fibrosis, endocarditis, joint prostheses,
heart catheters, or replacement heart valves.6,7
Figure 1. Stages in the developmental cycle of biofilm formation
and disruption. Norspermidine both prevented the formation of
new biofilms and collapsed the structure of existing biofilms.
Norspermidine appears to disrupt biofilms by targeting the
extracellular component of EPS in Bacillus subtilis, and it
seemed likely that it did so by binding to negatively charged or
possibly neutral groups using Coulombic attraction and
hydrogen bonding as important features.10 We tested a set of
commercially available polyamines. Norspermidine was most
active in inhibiting biofilms for B. subtilis and S. aureus,
followed by norspermine which has an additional aminopropyl
unit in its structure (Figure S1, Table S2). Perhaps
surprisingly, spermidine with one longer aminobutyl residue in
place of an aminopropyl and diethylenetriamine with two
shorter aminoethyl groups were inactive in both species. This
sharp length dependence indicated that matching the NH to
NH distance (4.9 Å) of the (poly)propyleneamine motif of
norspermidine and norspermine to the pitch (4.6-5.3 Å) of
various helical EPS structures determined or modeled for
potential exopolymers (Table S1) formed a key feature. Bind-
ing of these polyamines to negatively charged secondary struc-
tures would neutralize the charge and collapse the aqueous
In response to this challenge, high-throughput assays have
been developed to identify small molecules with the ability to
prevent biofilm formation or disrupt existing biofilms.8 We
recently explored an alternative strategy that exploits the
normal developmental cycle of bacteria. Biofilms form when
planktonic bacteria in the aqueous phase aggregate on a solid
surface or at an air-liquid interface. The biofilm colony grows
both by recruitment and cell division to form a mature colony.
Mature colonies eventually disintegrate, and the dispersed
bacteria resume a planktonic lifestyle (Fig. 1). Bacterially
produced small molecules orchestrate the creation and
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meshwork characteristic of mature biofilms.10 This simple
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compared to norspermidine (Figure 3A, Tables 1 and S2). In
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model – three or four positively charged groups separated by
propyl units – could be tested against biofilm formation of B.
subtilis with synthetic mimics, and guanidines and biguanides
emerged as particularly appealing substitutes for polyamines
because of their potentially increased overall charge at physio-
logical pH values.
addition to preventing biofilm formation the most potent com-
pound was also able to disrupt existing biofilms (Figure 3B)
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Figure 3: Enhanced activity of synthetic compounds 6a, 7a and
11a against B. subtilis. A) Inhibition of biofilm formation com-
pared to corresponding polyamines and B) breakdown of pre-
existing biofilms within 12 h. Norspd: norspermidine; Norspn:
norspermine; PBS: buffer.
Early on it became clear that the counter ion of the amines and
guanidines had a significant effect on activity. For instance,
the free base of norspermidine was 3x more active than the
chloride salt, which in turn was 3x more active than the sulfate
salt in the B. subtilis assay (Table S2). Therefore, we gener-
ated the free bases of selected compounds and compared them
with the corresponding salts. For B. subtilis a free base’s activ-
ity for biofilm inhibition was greater than or equal to the salt,
while for S. aureus there was no clear trend. Solubility prod-
ucts (Ks) showed no correlation with the activity of the com-
pounds, and bioavailablitiy and delivery into the biofilm ma-
trix are probably critical parameters (Table S3). None of the
compounds significantly inhibited bacterial growth at or close
to its corresponding MBIC value, which rules out biofilm in-
hibition being an artifact of reduced viability (Supporting In-
formation). Only compound 7 started to affect growth at con-
centrations above 200 µM in B. subtilis which corresponds to
40x its MBIC.
Figure 2: Library of synthetic guanidinylated or biguanidylated
polyamine analogs. aCounter ion of salt or free base, for
stoichiometry see Supporting Information.
We used three different synthetic strategies to generate a small
library of compounds (Figure 2) with guanidinyl or biguanidyl
groups as chloride or sulfate salts (Scheme 1). Guanidines can
be conveniently prepared from amines with S-
methylisothiourea (Scheme 1),11 and that reagent led to termi-
nal guanidines (1, 2, 4, 5, 7-9, 12) from commercially avail-
able primary amines.
Scheme 1
In B. subtilis the most active compounds were 6, 7, 8, 10, and
11 (as salt and base, Tables 1 and S2) with MBICs between 2
µM (7b) and 30 µM (10, 11a). Active compounds exhibited
the proposed binding motif10 of three to four amino or gua-
nidine groups formally separated by propyl chains (5-7, 10,
11, Tables 1 and S2). Additionally, the shorter compound (9)
with only a single propyl chain displayed activity only at 100
µM. The activity pattern for S. aureus was slightly different as
the minimal motif required for activity was two guanidine or
one amino and one guanidine groups separated by a propyl
chain (4-11). Compounds with ethyl chains instead of propyl
(1-3, 12) were inactive (•1 mM) for B. subtilis and only
weakly active (750 µM) or inactive for S. aureus. Biguanide
itself was inactive for both species (Tables 1 and S2).
Alternatively, cyanamide (or the alkylated form of its car-
bodiimide tautomer), which reacts with secondary amines
(Scheme 1),12 was used to prepare triguanidinylated com-
pounds (3, 6) and alkylated guanidines (3, 10). Finally, a
biguanide (11) was synthesized from m-phenylenediamine and
dicyandiamide (Scheme 1) according to Cohn.13 While aro-
matic amines are known to readily react with dicyandiamide,13
our attempts to extend the reaction to primary aliphatic amines
were unsuccessful.
Table 1. Activity of selected compounds
MBIC (µM) at pH 7.4
Compound
B. subtilis
S. aureus
Compounds were tested with inhibiting biofilm formation in
B. subtilis, the model organism that led to norspermidine10 and
Staphylococcus aureus, as a related pathogenic species with
high clinical relevance. The minimum biofilm inhibitory con-
centrations (MBICs) of all compounds are given in Table S2.
Some of the compounds exhibited remarkable activity for the
inhibition of biofilms with 5x to 20x increased activity with B.
subtilis and more than 8x increased activity with S. aureus
>1000
500
50
4
5
75
400
>1000 (500a)
a
b
a
b
375±125
10a
6
10
50
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low protonation constant of 3.9 that results in one uncharged
5
2
55±15
250
7
a
1
2
3
4
5
6
7
amino group – (101) – in the wide pH range of 5-8 (Figure
4A). Although guanidine groups in the related structure 2 sig-
nificantly increase the third pKa value to 6.3, the central amino
group remains unprotonated (101) at physiological pH values,
which was confirmed by X-ray structure analysis (Supporting
Figure S17).
b
100
30
30
7±3
500
9
10
11
20±10
300
a
750±250
b
aincomplete inhibition
8
9
The most active inhibitors of biofilm formation of S. aureus
were 4, 5a, 6b, 7a, and 10 with MBICs in the range of 10-75
µM. The activity of the best biofilm inhibitors in this initial
library is comparable to the lower range of what has been re-
ported in the literature for biofilm inhibitory compounds that
do not adversely affect bacterial growth.8
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Table 2. pKa values of selected compounds
pKa values
Compound
1
2
3
4
Norspermidine
11.1±0.1
10.6±0.3
11.1±0.1
11.6±0.1
13.6±0.1
13.5±0.0
13.7±0.1
13.4±0.1
13.8±0.2
13.5±0.1
13.6±0.0
13.6±0.1
12.1±0.1
9.4±0.2
10.5±0.3
9.8±0.1
7.1±0.0
8.7±0.2
7.8±0.1
3.9±0.1
-
-
Norspermine
6.7±0.1
Spermidine
-
DETa
9.0±0.3
-
12.6±0.2
12.8±0.2
13.0±0.3
12.0±0.3
13.2±0.3
12.0±0.3
12.2±0.1
9.4±0.1
-
1
2
6.3±0.0
-
-
-
4
Figure 4: Protonation and charge states. A) Average protonation
of polyamines and corresponding di- or triguanidines as a func-
tion of pH. B) Classical, incorrect representation of protonated
biguanides, C) correct protonation state for 11. D) Cropped
biguanide moiety from crystal structure of 11.
8.4±0.1
12.7±0.5
9.8±0.1
9.2±0.1
-
-
5
-
6
7.6±0.1
7
-
-
-
8
Its lack of activity at pH 7 reflects both structural and charge
liabilities. Not surprisingly, guanidine groups on the scaffold
of norspermidine or norspermine increased all individual pKa
values compared to the corresponding polyamines (Figure 4A)
causing the average degree of protonation to rise, which is in
line with the increased activity of 6 over norspermidine and 7
over norspermine. However, while 5 was active it did not dis-
play increased activity over norspermidine, despite its higher
degree of protonation (Supporting Figure S16). On average,
norspermine carries 3.3 positive charges at pH 7 correspond-
ing to about 30% fully charged (1111) molecules and the rest
triply charged (1110 or 1101) with the protonation micro-state
(1101) with one non-charged secondary amine as the likely
predominant species.15,17,18 The (1101) species does not com-
ply with the triply charged motif (111) and may contribute to
the higher activity of norspermidine over norspermine.
9
10.6±0.1
-
11
adiethylenetriamine
Our results support a model in which the binding of poly-
amine-based inhibitors to the exopolymer depends on the cor-
rect spacing of multiple amino or guanidine groups. The struc-
ture-activity relationship in this library further indicates that
though there is a common motif in both species, the composi-
tion and structure of biofilms of S. aureus and B. subtilis are
different and allow customized inhibition of biofilm formation.
In addition to the structural properties described above, the
charge of the compounds could be an important contributor to
their inhibitory activity.10 To investigate this possibility, we
determined the pKa values of selected compounds at 25 °C at
25 mM. Cumulative association constants were calculated by
HypNMR14,15 (Figures S2-S11) and values for pKa (D2O) were
finally converted to pKa (H2O).16 The pKa values for the com-
pounds are given in Table 2. For comparison, similar pKa val-
ues have been reported before for spermidine with pK1 =
10.90, pK2 = 9.71, and pK3 = 8.25.17
The active compound 11 exists at maximum in a doubly pro-
tonated form due to its high pKa values, and this form is virtu-
ally the only relevant species until pH 8.5 (Supporting Figure
S16). In the literature, the structure and protonation of bigua-
nides is frequently misrepresented as reported by Bharatam et
al.,19 whose computational studies indicated that the central
nitrogen of biguanides is not bonded to a hydrogen both in
neutral and charged states (Figure 4B and C). This N atom is
partially negatively charged while the positive charge is delo-
calized between the terminal nitrogen atoms of a biguanide.
The crystal structure of protonated 11 confirmed these results
(Figure 4D and S19), making 11 analogous to the (1111) motif
of fully charged compound 7 which explains its activity.
Speciation data derived from pKa values were generated using
the program HySS (Supporting Information, Figures S12-S14)
and average charge of the molecules was plotted against pH
(Figure 4). For convenience protonation states will be given by
a digit string with (1) for a protonated site and (0) for a non-
protonated site. In this notation, fully protonated norsper-
midine is (111). Diethylenetriamine (DET) has one extremely
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Author Contributions
1
2
3
4
The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the manu-
script.
5
Funding Sources
6
7
This research was supported by a Leopoldina Research Fellow-
ship (LPDS 2009-45) of the German Academy of Sciences Leo-
poldina (TB), a Human Frontier Science Program LTF fellowship
(IK-G), NIH grant GM086258 (JC), and NERCE-BEID through
5U54 AI057159 (JC), NIH grant GM18568 (RL), the NIAID-
sponsored program on antibiotic resistance P01AI083214 (RL and
RK), a grant from BASF (RL and RK), and NIH grants GM82137
and GM58213 (RK).
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ACKNOWLEDGMENT
We thank Dr. Shao-Liang Zheng for his help with X-ray data
collection and structure determination.
ABBREVIATIONS
EPS exopolymeric substance, MBIC minimal biofilm inhibitory
concentration, PBS phosphate buffered saline.
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Figure 5: Inhibition of biofilm formation (MBICs) is dependent
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ASSOCIATED CONTENT
Supporting Information. Syntheses and characterization, biofilm
inhibition data, pKa measurements, speciation data and crystal
structures. This material is available free of charge via the Inter-
net at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
* Email: jon_clardy@hms.harvard.edu (J. C.)
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