Consequences of isostructural main-chain modifications for the design of antimicrobial foldamers: Helical mimics of host-defense peptides based on a heterogeneous amide/urea backbone

Article abstract of DOI:10.1002/anie.200905591

"Chemical Equation Presented" Fraternal twins: Oligoureas and Y-peptides are isosteric, quasi-isostructural helical foldamers endowed with distinct blomolecular recognition properties. Combination of the two backbones to generate urea/amide hybrids (see picture) was found to give more potent yet less cytotoxic antimicrobial helical foldamers.

Full text of DOI:10.1002/anie.200905591

Angewandte
Chemie
DOI: 10.1002/anie.200905591
Helical Foldamers
Consequences of Isostructural Main-Chain Modifications for the
Design of Antimicrobial Foldamers: Helical Mimics of Host-Defense
Peptides Based on a Heterogeneous Amide/Urea Backbone**
Paul Claudon, Aude Violette, Karen Lamour, Marion Decossas, Sylvie Fournel,
Bꢀatrice Heurtault, Julien Godet, Yves Mꢀly, Brigitte Jamart-Grꢀgoire, Marie-Christine Averlant-
Petit, Jean-Paul Briand, Guy Duportail, Henri Monteil, and Gilles Guichard*
4
The design of unnatural oligomers with predictable folding
patterns (i.e. foldamers) and function has attracted consid-
a urea moiety for the CH -CO-NH units in the g -peptide
2
backbone is an interesting case of quasi-isostructural replace-
[1]
[3]
[3]
erable attention over the last ten years. Applications in
biology include the development of antimicrobial agents, cell-
penetrating agents, and inhibitors of protein–protein inter-
ment. High-resolution structural studies in solution and in
[
4]
the solid state have shown that the oligourea backbone
adopts a remarkably stable helical fold reminiscent of the 14-
helix described for the cognate g-peptides (Figure 1).
[
1a,2]
[5,6]
actions.
Non-natural oligoamides built from a-amino acid
residues (e.g. peptoids) and higher homologues (e.g. b-, g-,
and d-peptides) are the epitomes of peptidomimetic foldam-
[
1,2]
ers.
A substantial increase in the number of backbones
with folding propensity came from the exploration of the b-
and g-peptide families (i.e. by introduction of isosteric or
[1d]
isoelectronic backbone modifications). The substitution of
[
*] P. Claudon, Dr. A. Violette, K. Lamour, Dr. M. Decossas,
Prof. S. Fournel, Dr. J.-P. Briand, Dr. G. Guichard
[
+]
CNRS, Institut de Biologie Molꢀculaire et Cellulaire
Laboratoire d’Immunologie et Chimie Thꢀrapeutiques
15 rue Renꢀ Descartes, F-67000 Strasbourg (France)
E-mail: g.guichard@iecb.u-bordeaux.fr
Dr. B. Heurtault
Laboratoire de chimie enzymatique et vectorisation
Universitꢀ Strasbourg 1, Institut Gilbert Laustriat CNRS - UMR
Figure 1. Superimposition of helical structures formed by
oligoureas (green) and g -peptides (gray).
[4]
4
[5]
7
7
175, Facultꢀ de Pharmacie
4 route du Rhin, BP 60024, 67401 Illkirch Cedex (France)
[
6]
How such quasi-isostructural backbones compare in
molecular recognition is presently unknown. We reported
previously that oligoureas designed to mimic globally amphi-
philic a-helical host-defense peptides display broad antibac-
terial activity with selectivity for prokaryotic versus mamma-
Prof. B. Jamart-Grꢀgoire, Dr. M.-C. Averlant-Petit
LCPM, UMR 7568 CNRS, Nancy-Universitꢀ
1
rue Grandville BP20451, Nancy (France)
J. Godet, Prof. Y. Mꢀly, Dr. G. Duportail
Laboratoire de Biophotonique et Pharmacologie
CNRS UMR 7213, Facultꢀ de Pharmacie, Universitꢀ de Strasbourg
BP 60024, 67401 Illkirch Cedex (France)
[7]
lian red blood cell membranes. To better understand
structure–property relationships and to gain insight into the
mechanisms of membrane disruption, we have now under-
taken detailed comparative studies of oligoureas, their g-
peptide counterparts, and various mixed amide/urea conge-
ners (1–8) bearing identical side chains (Scheme 1).
Prof. H. Monteil
Laboratoire de Physiopathologie et d’Antibiologie des Infections
Nosocomiales et Emergentes
Institut de Bactꢀriologie, Facultꢀ de Mꢀdecine
3
rue Koeberlꢀ, 67000 Strasbourg (France)
+
Oligourea 1 was found previously to display both 1) a
strong helix propensity in a lipidic environment, and 2) sig-
nificant antibacterial activity against Gram negative and
Gram positive bacteria. However, further development of 1
was limited by synthetic hurdles. Stepwise elongation of 1 on a
[
] Present address: Institut Europꢀen de Chimie et Biologie
Universitꢀ de Bordeaux—CNRS UMR 5248, CBMN
2
rue R. Escarpit, 33607 Pessac (France)
[
7]
[
**] This research was supported in part by the CNRS, the Rꢀgion Alsace
and ImmuPharma France and by grants from ANR (project no. 05
PRIB 02-903 and NT05-4-42848). Fellowships from ImmuPharma
France, ANRT, and Rꢀgion Alsace (P.C., A.V., K.L.) are gratefully
acknowledged. We thank Daniele Sphener for her help in using the
EM-PACT2 apparatus. Access to the RIO Imaging Facility (Plate-
forme de Microscopie et d’Imagerie Cellulaire Strasbourg Espla-
nade) is gratefully acknowledged.
Rink resin using monomers protected with N-fluorenylme-
[3e,7,8]
thoxycarbonyl (Fmoc) groups
resulted in poor overall
yields. The purity of crude product based on C reverse-phase
1
8
[
9]
HPLC was only 17%. Although chemistry using building
blocks protected with the N-tert-butoxycarbonyl (Boc) group
is more robust, it was found that the urea linkage formed by
anchoring the first residue on a 4-methylbenzhydrylamine
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200905591.
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333

Communications
at the cost of mammalian versus
bacterial cell selectivity. How-
ever, substituting iBu for iPr
side chains in 3 (to give 4) led
only to a modest improvement
À1
(MIC = 128 mgmL
on the
three bacterial strains). In con-
trast, the same modification on
2
gave the most potent antibac-
terial oligourea (5) reported to
date with MIC and MBC values
À1
as low as 8 and 32 mgmL on S.
aureus, respectively.
We next examined the sus-
ceptibility of host cell mem-
branes to oligoureas and g-pep-
tides by monitoring lysis of
sheep red blood cells. Oligourea
À1
2
(HD100 = 256 mgmL ) com-
pared favorably with melittin,
which is strongly toxic towards
red blood cells (HD100
=
À1
4
3
2 mgmL ). g -Peptides 3 and
4
display the highest HD100
values.
With the aim to investigate
further the effects of insertion
4
Scheme 1. Globally amphiphilic oligoureas, g -peptides, and related hybrids 1–8.
4
(
(
MBHA) resin or the less acid-sensitive benzhydrylamine
BHA) resin does not resist the acidic conditions (trifluoro-
acetic acid or 3n HCl in dioxane) required to cleave the Boc
group. To overcome this difficulty, we envisaged the prepa-
ration of oligoureas bearing a stable terminal amide group by
of g -amino acid residues on 2.5-helix propensity and
4
antimicrobial activity, we prepared mixed oligourea/g -pep-
4
tides 6–8 containing two to four g -amino acid residues.
Information about the conformational preferences of oligo-
mers 6–8 was gained by monitoring chemical shift differences
4
1
a
direct attachment of an isosteric g -amino acid on MBHA
(Dd) in the H NMR spectra between diastereotopic CH
resin. This strategy, together with the use of N-Boc-protected
monomers, proved to be very effective, with purities of crude
protons of “diaminoethylene” residues (Figures S4–S6 and
[
9]
Table S1 in the Supporting Information) and by circular
dichroism (CD) spectroscopy (Figure S7 in the Supporting
2
routinely over 60% (Figure S1 in the Supporting Informa-
[
9]
[3d]
[9]
[3]
tion). It is noteworthy that neither 2.5-helix propensity
Information), as described previously. Comparison with
[
9]
(
Figure S2 and S3 in the Supporting Information) nor
oligomer 2 revealed several trends. The insertion of an
antibacterial activity were affected by this point mutation.
The minimum inhibitory concentrations (MICs) obtained for
Table 1: Antibacterial and haemolytic activities of compounds 2–8
compared to melittin and omiganan.
2
were very similar to those found for melittin, a honeybee
[a]
[
10]
toxin, and omiganan,
a cationic peptide currently in
[
b]
Compound
Bacteria
HD100
phase III clinical trials for the prevention of catheter-related
bloodstream infections (Table 1). Whereas omiganan exhibits
E. coli
P. aeruginosa
S. aureus
(ATCC 25922) (ATCC 27853) (ATCC 25923)
À1
weak bactericidal activity (MBCs > 256 mgmL ), the mini-
MIC
MBC MIC MBC
MIC
MBC
mum bactericidal concentrations (MBCs) measured for 2
were equal to or twice as large as corresponding MICs.
Moreover, 2 was found to be equally potent on methicillin-
sensitive (ATCC 25923) and methicillin-resistant (clinical IBS
[
c]
omiganan
melittin
64 >256 128
>256
256
128
>256 >256
>256
64
128
128
256
32
>256
64
nt
32
64
32
64 128
32 64
32
32
[d]
[d]
2
3
4
5
6
7
8
64
256
>512
>512
64
512
nt
>256 >256 256
128 >256 128
>256
>256
32
64
128
4
5-501) S. aureus strains.
128
8
16
32
128
16
64
32
32 16
128 32
128 64
To determine whether the urea backbone is critical for
4
antibacterial activity, we synthesized 3, the g -peptide ana-
logue of 2. Antibacterial activity of g-peptide 3 was deter-
mined against E. coli, P. aeruginosa, and S. aureus. g-Peptide
128 >256 128
>256
nt
[
a] MIC is the minimum inhibitory concentration and MBC the minimum
3
, although intrinsically less polar than 2, was virtually
À1
bactericidal concentration. MIC, MBC, and haemolysis in mgmL
[
.
À1
inactive (MIC and MBC > 256 mgmL ) on both Gram
b] HD100 values are concentrations required to lyse 100% of the cells
positive and Gram negative bacteria. From studies with
after 48 h. [c] nt=not tested. [d] Identical MIC and MBC values were
obtained on a clinical isolated methicillin-resistant S. aureus strain (IBS
45-501).
[
11]
antibacterial a- and b-peptides, we reasoned that increasing
the hydrophobic content of 3 could increase its potency, albeit
3
34
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Chemie
4
additional g -amino residue at positions P5 and P8 has a local
destabilizing effect on the preceding residues (i.e. residues at
P4 and P7), but it does not seem to alter the overall helical
4
conformation. In contrast, the insertion of two g -amino acid
residue pairs such as in 8 has a more pronounced effect on the
overall structure, with significantly reduced Dd values. CD
spectroscopy experiments show that the positive molar
ellipticity at 203 nm per urea moiety decreases significantly
4
as a result of g -amino acid insertion, which may be
[9,12]
interpreted as 2.5-helix destabilization.
Similarly, antibac-
terial activity was found to decrease with the number of g-
amino acid residues in the backbone. However, octamer 6,
4
with a central g -Val residue, compares favorably with 2 in
term of both bactericidal activity and cytotoxicity against
mammalian cells. This increased selectivity makes mixed
amide/urea oligomer 6 an interesting lead compound for
future studies.
Oligomers 6–8 were also evaluated against three ATCC
isolates of Candida, namely C. krusei ATCC 6258, C. para-
[13]
psilosis ATCC 22019, C. albicans ATCC 90028.
Again,
oligourea 6 was the most active compound in the series, with
À1
MICs of 16–64 mgmL and minimal fungicidal concentra-
tions (MFCs) against all three Candida isolates either equal to
or twice as large as the MICs (Table S2 in the Supporting
[
9]
Information).
Figure 2. Immunoelectron microscopy (biotin, 6 nm) of biot-2 on
E. coli after high-pressure freezing and freeze-substitution. Scale bar
0 nm; inset 10 nm.
Evidence for a direct interaction between oligoureas and
bacterial membranes was gained from immunoelectron
microscopy experiments using postembedding immunogold
labeling experiments. The ultrastructural localization of the
biotinylated analogue of 2 (biot-2) on Gram negative bacteria
5
[
9]
À7
À1
À7
À1
10 molL for 2 and 9 ꢀ 10 molL for 3) calculated by
curve fitting using the binding model given in Equation (13)
in the Supporting Information indicate that g-peptide 3 has a
sixfold lower affinity for the membrane than oligourea 2.
To assess the extent to which peptides 2 and 3 disrupt
phospholipid membranes, we measured their abilities to
induce efflux of carboxyfluorescein from EYPC/EYPG
(70:30 molar ratio) LUVs. The time course of the leakage
of encapsulated carboxyfluorescein was detected by the
corresponding increase in fluorescence intensity at 518 nm
(Figure 3). Whereas g-peptide 3 was not able to induce
significant efflux of carboxyfluorescein at 4.16 mm even after
prolonged time, very fast and strong carboxyfluorescein
release was observed when LUVs were treated with 2 at the
same concentration. This series of experiments further
confirms the different membrane-disruption properties
exhibited by oligoamide and oligourea backbones.
(
E. coli) reveals the presence of small and well-defined
clusters in the vicinity of the inner and outer bacterial
membranes (Figure 2). These results may suggest that oli-
goureas either alone or as aggregates are able to cross
bacterial membranes.
To determine whether the functional difference between
oligourea and oligoamide backbones results from differential
membrane disruption activities, we have undertaken phys-
icochemical investigations using negatively charged phospho-
4
lipid membranes as model systems. Both oligourea 2 and g -
peptide 3 carry indole side chains at positions 3 and 6 in their
sequence, thus endowing the molecules with intrinsic fluo-
rescence. We first studied the interaction of 2 and 3 with large
unilamellar vesicles (LUV) of egg yolk phosphatidylcholine
EYPC) and egg yolk phosphatidylglycerol (EYPG) in a
0:30 molar ratio by measuring indole emission fluorescence
(
7
and steady-state fluorescence anisotropy upon addition of an
increasing concentration of phospholipids. At a lipid-to-
A number of recent studies have shown that conforma-
tional preorganization is not necessarily a prerequisite to
[
14,15]
oligomer molar ratio R = 20, shifts to lower wavelength
design oligomers with antimicrobial activity.
Thus, it is
i
(
1
blue shift) of the indole emission peak (lmax) of 23 nm and
9 nm were observed for 2 and 3, respectively (Figure S8 in
unlikely that the difference in helix stability between oligoa-
mides and oligoureas alone accounts for the observed
dichotomy in membrane interaction and antibacterial activ-
ities. Instead, helix geometry (side-chain projection) and
backbone polarity are two factors that vary substantially
between the two oligomeric systems and that need to be
considered. Moreover, it is tempting to speculate that
structural features of helical oligoureas revealed by X-ray
[9]
the Supporting Information), thus indicating significant
penetration of the indole moieties into the hydrophobic lipid
bilayer. The restricted mobility of the indole moiety upon
interaction with the membrane was also inferred from an
increase in fluorescence anisotropy. Titrations of 2 and 3 with
EYPC/EYPG LUVs (Figure S9 in the Supporting Informa-
tion) were used to quantify the binding of both oligomers to
[
4]
diffraction studies, that is, conformational adaptability
(helix plasticity) and directional aggregation, may have
[9]
vesicles.
The apparent dissociation constants (1.4 ꢀ
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www.angewandte.org
335

Communications
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of a) 2 and b) 3.
[6] Average (f, q , q , y) backbone torsion angles (8) for oligourea
1 2
4
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+ 80, À170) and (À127, + 66, + 64, À140), respectively.
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[
[
[
some relevance to the unique antimicrobial activities of
oligoureas and related hybrids.
9] See the Supporting Information.
In conclusion, we have documented a case of dichotomy in
the foldamer g-peptide lineage whereby two apparently
isostructural helical backbones (i.e. oligoamide and oli-
gourea) bearing identical side chains appear to strongly
differ in their antibacterial and biomolecular recognition
properties. Our results also point to heterogeneous helical
urea/amide backbones, which may become advantageous in
the development of more potent yet less cytotoxic antimicro-
[
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contain two different, albeit related, chromophores in their
backbone) by using the dichroic maximum at 203 nm.
[
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[
16]
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Received: October 7, 2009
Published online: December 2, 2009
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Keywords: amphiphiles · antimicrobial agents ·
.
12785.
helical structures · peptidomimetics · structure–
activity relationships
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3
36
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Angew. Chem. Int. Ed. 2010, 49, 333 –336