Bead diffusion assay for discovering antimicrobial cyclic peptides

Article abstract of DOI:10.1039/c0cc04670a

A combinatorial library of 15536 cyclic decapeptide analogues of tyrocidine A and gramicidin S was prepared on photocleavable TentaGel beads. The beads were photolyzed without solvent, and spread onto an agar plate inoculated with bacterial lawn. Clear zo

Full text of DOI:10.1039/c0cc04670a

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Bead diffusion assay for discovering antimicrobial cyclic peptidesw
a
a
b
a
´
Viviana S. Fluxa, Noelie Maillard, Malcolm G. P. Page and Jean-Louis Reymond*
Received 28th October 2010, Accepted 22nd November 2010
DOI: 10.1039/c0cc04670a
A combinatorial library of 15 536 cyclic decapeptide analogues
of tyrocidine A and gramicidin S was prepared on photocleavable
TentaGel beads. The beads were photolyzed without solvent, and
spread onto an agar plate inoculated with bacterial lawn. Clear
zones were formed around beads carrying antimicrobially active
peptides such as E18 c(kVOrnLfThiYOrnLq), which inhibited
growth of B. subtilis and selected MRSA strains.
Antimicrobial peptides (AMP) represent a privileged source of
antimicrobial compounds that might help address antibiotic
resistance.1 Split-and-mix synthesis on solid support provides a
versatile and efficient method to prepare thousands of analogues
of such compounds as one-bead-one-compound (OBOC)
combinatorial libraries.2 High throughput antimicrobial screen-
ing uses test compounds in solution, which requires formats that
deliver soluble products such as positional-scanning libraries or
parallel-synthesis.3 Herein, we report an antimicrobial activity
Fig. 1 Principle of bead diffusion assay. The picture illustrates the
screening of the cyclic peptide library prepared on macrobeads (Fig. 2)
against B. subtilis. MTT thiazolyl blue tetrazolium bromide. See ESIw
for details.
assay for OBOC libraries of cyclic peptides using
a
bead diffusion assay4 applied to amino acid analysis (AAA)
decodable combinatorial libraries.5 The assay identified cyclic
decapeptide E18 in a library of tyrocidine A and gramicidin
S analogues. E18 exhibits good activity against selected strains
of Staphylococcus aureus including methicillin-resistant strains
(MRSA), and only weak hemolytic activity.
solvent to release the peptide from the solid support.
The beads were then spread on the surface of an agar
plate inoculated with E. coli. After incubation for 18 hours
at 37 1C, bacterial growth was revealed by staining with
MTT (Fig. 1).10
A
growth inhibition zone was
clearly visible around the indolicidin beads, but not
around the control beads bearing only the acetylated
photolabile linker, consistent with the antimicrobial activity
of indolicidin.
In the disc-diffusion assay, a bacterial growth inhibition
zone is detected on the underlying agar, whose size indicates
the antimicrobial potency of the compound impregnated in the
disc.6 This principle has been adapted to screen combinatorial
libraries of small molecules by spreading synthesis beads on an
agar plate after dry photolysis of a photolabile linker to allow
diffusion to the surrounding agar.4b,c We have used a similar
assay for peptide dendrimers in an off-bead assay for catalysis
involving diffusion from the beads into silicagel plates.7 In
this case partial photolysis was tuned to retain sufficient
amount of material on the bead to allow decoding by AAA.
Because AAA decoding is compatible with cyclic peptides,8 we
set out to test if a bead diffusion assay could be used to discover
antimicrobial cyclic peptides.
The bead diffusion assay was implemented to screen a
15 536-member combinatorial library of cyclic decapeptide
analogues of gramicidin S and tyrocidine A, a pair of
antimicrobial cyclic peptides of natural origin that share five
amino acid residues in their sequence (Fig. 2).11 The sequence
was anchored to the support by the side chain of a Fmoc–
D-glutamate with the a-carboxyl group protected as allyl ester
to allow selective removal with palladium and on-bead
cyclization as the last synthesis step, a known strategy for the
synthesis of cyclic decapeptides12 and suitable for obtaining
combinatorial libraries of cyclic peptides.8
The phe-Leu dipeptide at position X6–X7 and the valine
residue at X9 were kept constant because they are known to
contribute to the activity of gramicidin S.13 Positions X2–X5,
X8 and X10 were varied combinatorially using five different
amino acids at each position following the TAGSFREE
library design algorithm which allows sequence assignment
by AAA of the beads.5 The resulting 15 536-member library
was synthesized by split-and-mix SPPS on a 1.0 g batch of
TentaGel macrobeads (200–250 mm diameter).
In a preliminary experiment, the known antimicrobial
peptide indolicidin (ILPWKWPWWPWRR)9 was prepared
by solid-phase peptide synthesis on photocleavable tentagel
resin, which was then deprotected by acidic treatment,
washed with water, and photolyzed in the absence of
a Department of Chemistry and Biochemistry, University of Berne,
Freiestrasse 3, CH-3012 Berne, Switzerland.
E-mail: jean-louis.reymond@ioc.unibe.ch;
Fax: +41 31 631 80 57; Tel: +41 31 631 43 25
b Basilea Pharmaceutica International Ltd., Switzerland
w Electronic supplementary information (ESI) available: Details of
library design, synthesis, and screening and synthesis of peptides. See
DOI: 10.1039/c0cc04670a
The library was screened against E. coli and B. subtilis as
model Gram-negative and Gram-positive bacteria, respectively,
using 50 mg (approx. 11 000 beads) portions of the library per
c
1434 Chem. Commun., 2011, 47, 1434–1436
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Fig. 3 Frequency of occurrence of cationic and hydrophobic residues
in screening hits against E. coli (red bars) and B. subtilis (blue bars),
compared to control beads (white bars) and the overall statistical
distribution of the library (black bars). Cationic residues: Lys, D-Lys,
Orn, Arg. Hydrophobic residues: Ile, Tyr, Pro, Val, Leu, Phe, D-Phe,
Thi, Thz.
(Table 1). All positive hits showed growth inhibition of
B. subtilis and to a lesser extent of E. coli, while the two
negative hit control peptides were inactive, thus confirming the
off-bead agar-plate screening assay. The stronger activity
against B. subtilis probably reflects the greater sensitivity of
Gram-positive bacteria to membrane disrupting compounds.
The five most active products were further tested against
eighteen other bacterial strains from eight types of bacteria,
including several MRSA (Table 2). Significant activities were
observed with cyclic peptides E4 and E18, which both
contained b-(2-thienyl)-L-alanine. The compounds were also
tested for resistance development with B. subtilis by daily MIC
determination and further growth in the presence of
compound at half the MIC. No increase of the MIC values
was observed on any of the compounds, showing
that resistance did not easily appear against these peptides.
For the most active cyclic peptide E18 exchange of the
b-(2-thienyl)-L-alanine residue against the isosteric phenyl-
alanine (Thi - Phe) to give E18a reduced activity, highlighting
the critical role of this non-natural amino acid residue. Activity
was also impaired in the acyclic analogue E18b, showing that
cyclization was essential for activity. While the antimicrobial
activity of E18 was comparable to those reported on similar
Fig.
2
Synthesis of the combinatorial library. Conditions: (a)
hydroxyethyl photolinker, PyBop, iPrNEt2, NMP (1-methyl-2-
pyrrolidinone), 2 h; (b) MSNT (1-(2-mesitylenesulfonyl)-3-nitro-1H-
1,2,4-triazole), Fmoc–D-Glu-1-allyl ester, 1-methylimidazole, CH2Cl2,
4 h, then piperidine 20% in NMP; (c) split-and-mix synthesis with
building blocks X2–X10; (d) PhSiH3, Pd(PPh3)4, CH2Cl2; (e) piperidine
20% in NMP, then PyBoP, iPrNEt2, NMP, 3 h, then HATU, iPrNEt2,
NMP, 15 h. (f) (i) TFA/TIS/EDT/H2O (92 : 5 : 2 : 1), (ii) wash
with CH2Cl2, NMP, MeOH and H2O, dry, (iii) UV 365 nm, 4 hours.
Thz = L-thiazolidine-carboxylic acid, Thi = b-(2-thienyl)-L-alanine.
Orn = L-ornithine. Hyp = 4-hydroxy-L-proline, upper case letters for
natural L-amino acids, lower case letter for D-amino acids.
strains of the parent peptides gramicidin S (1.5–64 mg mlÀ1 15
)
assay. For each agar plate tested, 2–5 beads surrounded by an
inhibition zone were detected and picked for sequence
determination by amino acid analysis. A series of non-
inhibitory beads were also picked as negative controls. The
E. coli screen generally gave small halos around the beads
(18 active beads, Tables S1 and S2, ESIw). The sequences were
diverse but showed a higher proportion of two or three cationic
residues and six hydrophobic residues compared to the control
beads or statistical distribution (Fig. 3). The B. subtilis screen
produced readily detectable halos around active beads (13 active
beads, Table S3, ESIw). In this case there was a similar
enrichment of dicationic sequences but most frequently only
five hydrophobic residues.
and tyrocidine A (>32 mg mlÀ1),16 its hemolytic activity
(MHC = 2040 mM) was much weaker than that of gramicidin
S (MHC = 30 mM)15b or tyrocidine A (MHC = 8 mM), and
also slightly weaker than analogues E18a (MHC = 390 mM)
and E18b (MHC = 1000 mM).17
In summary, solvent-free photolysis of combinatorial
libraries prepared on photocleavable TentaGel Macrobeads
was used for the rapid identification of antimicrobial
compounds from an AAA-decodable OBOC library of cyclic
decapeptide analogues of tyrocidine A and gramicidin S by a
bead-diffusion assay. The assay identified several sequences
with good antimicrobial activity but low hemolytic effect. The
method should be generally useful to explore the antimicrobial
activities of combinatorial libraries of various compounds and
should greatly facilitate the discovery of new antimicrobial
agents.
Eleven positive hits from the E. coli (E) and B. subtilis (B)
screens featuring one to three cationic residues and four to
seven hydrophobic residues as well as two control inactive (N)
cyclic peptides were prepared by SPPS and their antimicrobial
activity was measured as purified products in solution
This work was supported financially by the University of
Berne and the Swiss National Science Foundation.
c
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Table 1 Antimicrobial activity of synthesized library hits and analogues
Cpd no, sequencea
MS calc./obs.
MIC E. coli
MIC B. subtilis
E1 c(IVTLfPHKThzq)
E3 c(YVHLfThzHfNq)
E4 c(RVRLfThiYThiLq)
E6 c(RVHLfPYOrnThzq)
E8 c(PVTLfThzHKIq)
E9 c(RVOrnLfThiFOrnLq)
E13 c(kVRLfVVfIq)
E14 c(IVOrnLfThzHOrnIq)
E18 c(kVOrnLfThiYOrnLq)
E18a c(kVOrnLfFYOrnLq)
E18b kVOrnLfThiYOrnLqc
B6 c(IVHypLfPVKNq)
B7 c(kVHypLfPYNNq)
B10 c(kVHypLfPHKNq)
N9 c(RVHLfVYfTq)
1179.6/1179.6
1301.6/1301.6
1382.7/1382.7
1269.6/1269.6
1179.6/1179.6
1285.7/1285.4
1230.8/1230.8
1194.7/1194.8
1273.7/1273.8
1267.7/634.6b
1291.7/1291.8
1152.7/1152.6
1217.6/1217.4
1205.7/603.6b
1291.7/646.6b
1237.7/1237.8
>256
128
128
>256
>256
>256
128
64
64
16
>32
>256
128
64
>256
>256
16
32
8
32
64
64
16
32
2
>32
>32
32
32
16
128
128
N29 c(YVHypLfVFOrnIq)
a
E = hits from E. coli assay, B = hits from B. subtilis assay, N = control sequences. See legend of Fig. 2. The mass is for [M + H]+. MIC =
minimal inhibitory concentration in mg mlÀ1
C terminus is amidated –CONH2 and the N terminus is free.
.
This mass corresponds to [(M + 2H)/2]2+. MICs were determined following ref. 14. The
b
c
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Table 2 Extended antimicrobial activity profiles
Bacteriuma
Strain
PB AP
E4
E18
16
16
8
16
8
8
S. aureus
S. aureus
MRSA
ATCC25923 >32
ATCC29213 >32
0.25 >32
2
16 >32
32
16
32
16
887
42080
MRSA
Staphylococcus epidermidis ATCC14990 >32
>32 >32
1
2
2
4
S. epidermidis
J147
ATCC29212 >32
ATCC19434 16
>32
32
Enterococcus faecalis
Enterococcus faecium
E. faecium
>32
>32
>32
>32 >32
>32 >32
32
32
16
Van BE38-10 >32 >32
E. coli
Pseudomonas aeruginosa
ATCC25922
ATCC27853
2
4
8
>32
a
Minimal inhibitory concentration (MIC) values are given in mg mlÀ1
and were determined following standard broth microdilution method
recommended by the Clinical and Laboratory Standards Institute
(M7-A7). The concentration range was 0.06 to 32 mg mlÀ1
Polymyxin (PB) and ampicillin (AP) were used as positive
.
B
controls. The peptides did not show measurable inhibition (MIC >
32 mg mlÀ1) on the following strains: E. faecalis (Van B E80-8), E. coli
(HB101(PAT266)), E. coli (DC2), P. aeruginosa (PAO1(HS)),
P. aeruginosa (18S/H), P. aeruginosa (K799/WT), P. aeruginosa
(K799/61). Peptides E13, E18a, E18b, B7 and B10 were not active in
any of the strains tested (MIC > 32 mg mlÀ1).
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1436 Chem. Commun., 2011, 47, 1434–1436
This journal is The Royal Society of Chemistry 2011