Tài liệu Báo cáo Y học: Antimicrobial dendrimeric peptides pot

Antimicrobial dendrimeric peptides
James P. Tam, Yi-An Lu and Jin-Long Yang
Vanderbilt University, Department of Microbiology and Immunology, MCN A5119, Nashville, TN, USA
Dendrimeric peptides selective for microbial surfaces have
been developed t o a chieve broad a ntimicrobial activity and
low hemolytic activity to human erythrocytes. The dendri-
meric core is an asymmetric lysine branching tethered with
two to eight copies of a tetrapeptide (R4) or an octapeptide
(R8). T he R4 tetrapeptide (RLYR) contains a putative
microbial surface r ecognition B HHB motif ( B ¼ basic,
H ¼ hydrophobic amino acid) found in protegrins a nd
tachyplesins whereas the octapeptide R8 (RLYRKVYG)
consists of an R4 and a degenerated R4 repeat. Antimicro-
bial assays against 10 organisms in high- and low-salt con-
ditions showed that the R4 and R8 monomers a s well as their
divalent dendrimers contain no to low activity. In contrast,
the tetra- and octavalent R 4 a nd R8 dendrimers are broadly
active under either c onditions, exhibiting relatively similar
potency with minimal inhibition concentrations < 1 l
M
against both bacteria and fungi. Based o n their size and
charge similarities, the potency and activity spectrum of t he
tetravalent R4 dendrimer are comparable to protegrins and
tachyplesins, a family of potent a ntimicrobials containing
17–19 residues. Compared with a series of linearly repeating
R4 peptides, the R4 dendrimers show comparable anti-
microbial potency, but ar e m ore aqueous soluble, more
stable to proteolysis, less toxic t o human cells and m ore
easily synthesized chemically. These results suggest repeating
peptides that cluster the charge and hydrophobic residues
may represent a primitive form of microbial pattern-recog-

nition. Incorporating s uch knowledge in a dendrimeric
design therefore presents an attractive approach for devel-
oping novel peptide antibiotics.
Keywords: dendrimeric peptide; p eptide antibiotics.
Cationic antimicrobial peptides constitute an important
component of the innate immunity against microbial
infections [1–6]. Recently there is renewed interest in
developing novel approaches for designing peptide-based
antibiotics m anifested by k illing mechanisms t hat are less
likely than conventional antibiotics to develop multidrug
resistance [7–12]. Design elements desirable for therapeutics
include activity under physiological conditions (100–
150 m
M
or high-salt c on ditions), low t oxicity and proteo-
lytic stability. Guided by these c onsiderations, we and others
have designed antimicrobial peptides with unusual struc-
tural a rchitectures using rigid sc affoldings such as cyclic
peptides highly constrained with a cystine-knot motif on
two or three b strands [10–12] to cluster hydrophobic and
charge regions that produce amphipathic structures impor-
tant for antimicrobial activity. Furthermore, these
constraints confer metabolic stability, and impart mem-
branolytic selectivity that minimizes toxicity.
Another approach for designing antimicrobial peptides is
based o n their mechanisms of action. An example would
exploit mechanisms of recognizing conserved motifs on
microbial surfaces that are not found in higher eukaryotes.
Janeway & Medzhitov [13] have recently classified a family
of proteins and receptors specific for pathogen associated

molecular patterns (PAMPs) essential for innate and
adaptive responses. P athogen-associated motifs include
various microbial cell-wall components such as lipopoly-
saccharide ( LPS), peptidioglycans, teichoic acids, mannans,
N-formyl peptides, and lipidated peptides [14,15]. Some
well-studied motif-recognizing proteins i nclude LPS -bind-
ing p rotein, soluble and mem brane-anchored CD14 and
Toll-like LPS receptors as well as mannose-binding protein
and the receptors for mannans and manoproteins [16–18].
Cationic antimicrobial peptides may have also evolved t o
recognize PAMPs on microbial s urfaces. T hey o ften possess
a broad spectrum of a ntimicrobial activities against bacte-
ria, fungi or viruses through mechanisms that generally
involve the disruption of microbial envelopes. In general, at
their effective killing doses, most antimicrobial peptides are
nontoxic to host cells, s uggesting pattern-recognition selec-
tivity under evolutionary pressure. Although more t han 200
antimicrobial peptides with various types of structures are
known, they can b e classified into two broad categories
based on their primary sequences: those that contain
repeating sequences ranging from two to 14 amino a cids
and those that are nonrepeating [19,20]. Found in these two
types of peptides a re basic amino acids useful f or electro-
Correspondence to J. P. Tam, Vanderbilt University, Department of
Microbiology and Immunology, A-5119 MCN, 1161 21st A venue
South, Nashville, TN 37232-2363, USA. Fax: + 1 615 343 1467,
Tel.: + 1 615 343 1465, E-mail:
Abbreviations:CHCA,a-cyano-4-hydroxycinnamic acid; DCC,
N,N-dicyclohexylcarbodiimide; DCM, dichloromethane; DIC,
N,N-diisopropylcarbodiimide; DIEA, N,N-diisopropylethylamine;

DMF, dimethylformamide; EC
50
, peptide concentration causing 50%
hemolysis; Fmoc, 9-fluorenylmethyloxycarbonyl; Fmoc-DPA,
p-(R,S)-a-[1-(9H-fluoren-9-yl)methoxyformamide]-2,4-dimethoxy-
benzylphenoxyacetic acid; HOBt, N-hydroxybenzotriazole; LPS,
lipoplysaccharide; MBHA resin, methylbenzhydrylamine resin; M IC,
minimal inhibition concentration; PAMPs, pathogen associated
molecular patterns; PG-1, protegrin-1; R
t
, retention time; RTD-1,
rhesus theta defensin; TP-1, tachyplesin-1; TCEP, tris(carboxyethyl)
phosphine; TSB, trypticase soy broth; SPPS, solid-phase peptide
synthesis.
(Received 2 October 2001, revised 2 December 2001, accepted 5
December 2001)
Eur. J. Biochem. 269, 923–932 (2002) Ó FEBS 2002
static interactions with microbial membranes. Other amino
acids useful for structural and hydrophobic roles have also
been observed including Pro, Phe, and Trp [21,22]. A
structural feature commonly found in antimicrobial pep-
tides, whether t hey contain repeating or nonrepeating
sequences, is their ability to cluster charge and hydrophobic
amino a cids as amphipathic molecules to interact with the
negatively charged lipidic microbial surfaces. We reasoned
that this amphipathic structure might function partly for
pattern recognition. Thus, we have explored an approach to
exploit the polyvalency of a dendrimer to tether an array of
short Ôpattern-recognition Õ peptides frequently found in
b-stranded peptide antibiotics t o enhance interactions with

microbial lipid surfaces. A s s hort peptides < 12 amino acids
without conformational constraints are not likely to fold into
a stable structure that provide strong antimicrobial actions
under physiological conditions containing 100 m
M
NaCl, we
also incorporated a dendrimeric design with a lipid-like
backbone that facilitates interaction with microbial surfaces.
Dendrimeric peptides or peptide dendrimers are biopoly-
mers of unusual a rchitectures. First evolved in the 1980s
[23,24], they contain a multivalent core that tethers an array
of bran ching peptides. An early e xample i s the multiple
antigen peptides (MAPs) introduced by our laboratory as
immunogens for producing site-specific polyclonal and
monoclonal antibodies. The dendrimeric core of a MAP
(Fig. 1) consists of a d ivalent L ys core whose a and e amines
double geometrically with each branching generation.
Although a single lysine core with two amino moieties has
been extensively used to design divalent-branched bioac tive
peptides [25–27], a MAP-like dendrimeric peptide contain-
ing the Lys
2
Lys (K
2
K) as a dendrimeric core is better suited
to serve t he purpose o f providing with short peptides
polyvalency for our dendrimeric design.
Little is known a bout the structures of dendrimeric
peptides with the K
2

K cores. The three-lysine K
2
Kcoreis
asymmetric because each lysine c ontains a short a and a
long e amino arm that results in unequally spaced amines
with four branches, t wo e and two a amines. This
combination of a and e peptide has an e-peptide backbone,
which is torsionally flexible, with five methylenes separating
the amine from the carboxylic acid. In an extended form,
the en d-to-end d istance of 21 atoms separating the two
e-amines of the K
2
K core, most of which are methylen e units
mimicking the lipid chains, can be considered as lipid-like
biopolymers sufficient for transversing a lipid membrane.
Under a lipidic environment, modeling shows that a K
2
K
core with tethered short a p eptides can achieve a barrel-like
structure mimicking those o f helical peptides, which is
essential for membranolytic activity. In addition, judicious
selection of a short a peptide with cationic c harged and
hydrophobic residues appropriate for Ôpattern recognitionÕ
may enhance t he hypothetical bioactive structures through
electrostatic and hydrophobic i nteractions with the n ega-
tively charged microbial surfaces.
Tachyplesins [28] are potent broad-spectrum antibiotics
containing four degenerated repeats of a t etrapeptide with a
HBCH motif (H, hydrophobic; B, basic a nd C, Cys). These
cystine-stabilized antiparallel peptides have s ide chains

arranged in an up-and-down topology. On e face o f this
topology contains a consensus B HHB motif. Similar BHHB
motifs can be found in protegrins [29] and RTD-1 (rhesus
monkey theta defensin) [30]. Based on this topological
motif, our prot otypic pattern-recognition peptides c onsist of
a t etrapeptide R4, RLYR, and a degenerated double-
repeating o ctapeptide R 8, RLYR-KVYG (Fig. 2). Here, we
describe the d esign and properties of dendrimeric pep tides
employing the tetravalent and octavalent dendrimeric cores
tethered with cationic p eptides, a R4 tetrapeptide or a R8
octapeptide. For comparison, the R4 or R 8 peptides a re
then tethe red t o three different cores consisting of a single
lysine (K), a three-lysine (K
2
K) or a heptalysine [(K
2
K)
2
K]
core to give divalent, tetravalent or octavalent dendrimeric
peptides, respectively. The R4 dendrimeric peptides are also
compared with a controlled series of linearly repeating R4
peptides of (RLYR)
n
, n ¼ 1, 2, 4 and 8. Our results show
Fig. 1. Schematic representations of three type of dendrimeric cores with
three generation of lysines shows in t hree different font style. (A) t wo
branched Lys (bold ); (B) four branched (Lys)
2
Lys (regular); (C ) eight

branched [(Lys)
2
Lys]
2
Lys (bold italic); (D) (Lys)
2
Lys core w ith a and e
branche bearing peptides Arg-Leu-Tyr-Arg.
Fig. 2. Topological distributions of BHH B and BHHX motif i n the
antiparallel b sheet s tructured protegrin-1 (PG-1), tachyplesin-1 (TP-1)
and rhesus theta defensin (RTD-1).
924 J. P. Tam et al. (Eur. J. Biochem. 269) Ó FEBS 2002
that the tetravalent and octavalent dendrimeric short
peptides display p roperties desirable in antimicrobials. They
are broadly active with very similar potency against 10 test
organisms, but are also less toxic and more proteolytic
resistant than the corresponding linearly repeating peptides.
MATERIALS AND METHODS
Materials
Solvents, all of HPLC grade, were obtained from VWR
Scientific C o. and used without further purification.
Fmoc amino acid derivatives, N-hydroxybenzotriazole
(HOBt), N,N¢-dicyclohexyl carbodiimide (DCC), and
p-[(R,S)-a-[1-(9H-Fluoren-9-yl)-methoxylformamido]-2,4-
dimethoxylbenzyl]-phenoxyacetic acid (Fmoc-DPA) were
obtained from Chem-impex I nternational Inc. N,N-diiso-
propylethylamine (DIEA) a nd p-cresol w ere purchased
from Aldrich Chemical Co. Trypsin, a-chymotrypsin a nd
a-cyano -4-hydroxycinnamic acid were purchased from
Sigma Chemical C o. Trifluoroacetic acid was obtain ed

from Halocarbon. Tris(carboxyethyl)-phosphine (TCEP)
was obtained from Calbiochem.
Ten organisms obtained from the American Type
Culture Collection (ATCC, Rockville, MD, USA) were
used for antimicrobial assays. Four Gram-negative bacteria
included Escherichia c oli ATCC 25922, Pseudomon as aeru-
ginosa ATCC 27853, Klebsiella oxytoca ATCC 49131, and
Proteus vulgaris ATCC 49132. The three Gram-positive
bacteria were Staphylococcus aureus 29213, Micrococcus
luteus ATCC 49732 and Enterococcus faecalis ATCC 29212.
The three fungi were Candida albicans ATCC 370 92,
Candida kefyr ATCC 37095, and Candida tropicalis ATCC
37097. The s trains were incubated in trypticase soy broth
(TSB) which was p repared in double d istilled water and
autoclaved for sterilization. TSB was purchased from
Becton–Dickinson (Cockeysville, MD, USA).
Peptide syntheses and purification
Solid-phase peptide synthesis on an automated s ynthesizer
(CS Bio Co. 536) was performed using Fmoc-tBu chemistry
and a sin gle coupling protocol with B OP/DIEA [31] in
DMF. Linear peptides were synthesized on F moc-DPA-
resin a nd dendrimeric peptides wer e synthesized on d ifferent
branching core matrix Fmoc-DPA-NH-resins. A nalytical
RP-HPLC was conducted on a Shimadzu L G-6 A system
with an C
18
Vydac column ( 4.6 · 250 mm). A linear
gradient of 10–90% buffer B ran for 30 min at 1 mLÆmin
)1
with detection at 225 nm. Eluen t A contained 0.04% TFA/

H
2
O; eluant B contained 0.04% trifluoroacetic acid/60%
CH
3
CN/H
2
O. Preparative RP-HPLC was performe d on a
Waters 600 system with C
18
Vydac column ( 20 · 250 mm).
MALDI-MS was measured on a Pe rS eptive Biosystems
Voyager instrument. Samples were d issolved in 1 lLofa
1:2mixtureofH
2
O/CH
3
CN. Measurements were m ade in
a linear m odel, with a-cyano-4-hydroxycinnamic acid a s the
matrix.
Preparation of Fmoc-DPA-NH-resin [32]
The amine resin (2 g, 0 .22 mmol) was first swollen and
washed with DCM. Fmoc-DPA (539 mg, 1 mmol) in DMF
(40 mL) was added, shaken for 5 min ( not drained), a nd
followed by D CC (206 mg, 1 mmol) and HOBt (157 mg,
1 mmol) to anchor the F moc-DPA handle onto the resin
support. After 24 h, the resultant Fmoc-DPA-resin was
drained, was hed sequen tially with DMF, DCM, MeOH
and dried in vacuo. Substitution of the functionalized resin
was 0.1 mmol Æg

)1
(Fig. 3).
Preparation of branching lysyl core resin
Syntheses of d i- tetr a- and octa-branching cores required
one, t wo and three coupling cycles, respectively, using a four
molar excess of Fmoc-Lys (Fmoc) via BOP/DIEA in DMF
on the Fmoc-DPA-NH-resin. The Fmoc group was
removed b y treatment with 20% piperidine/DMF. Each
coupling cycle doubled the branching level of lysyl core and
after three cycles, the octa-branching Fmoc-Lys
4
-Lys
2
-Lys-
DPA-NH-resin was achieved (Fig. 3).
Synthesis of DmR4 and DmR8
These peptides were synthesized on different branching
lysyl c ore resin according to what has been described
above. Th e HPLC retention time and m ass data showed
in Table 1.
Fig. 3. Synthetic scheme for preparing different
branched (two, four, and eight) core. Lo w
substituted a mine resin 1 coupled with DP A 2
by DCC/HOBt in DMF to f ormFmoc -DPA-
resin 3. After deprotection, 3 was coupled to
Fmoc-Lys(Fmoc)-OH throughBop/DIEA to
form two branching core resin 4. Repeating
one or two rounds of c ouplings with Fmoc-
Lys(Fmoc)-OH fo rmed f our b ranchin g c ore 5
or eight branching core 6.

Ó FEBS 2002 Antimicrobial dendrimeric peptides (Eur. J. Biochem. 269) 925
Antimicrobial and hemolytic assays
A sensitive and reproducible two-stage radial d iffusion
antimicrobial assay of Lehrer et al .[33]wasemployed.
Antimicrobial activities were expressed in units
(0.1 mm ¼ 1 U ), and the MICs were determ ined from
the x-intercepts of the dose–response curves. Hemolytic
activity was determined u sing fresh human er ythrocytes.
Peptide concentrations causing 50% hemolysis (EC
50
)were
derived from the dose–response curve [34]. The membrano-
lytic selectivity index is expressed as EC
50
/MIC.
Proteolytic stability
Dendrimeric o r linear peptides i n various concentrations
were dissolved in NaCl/P
i
buffer at pH 7.4 and aliquoted
into microtubes. Trypsin was m ixed with peptides in the
ratio of 1 : 100 (enzyme/peptide, w/w). The enzymatic
degradation was carried out at 37 °C and stopped b y
adding an appropriate enzyme inhibitor such as Typ e II-S
trysin inhibitor i nto the samples. The r esidual a ntimicro-
bial activity of each sample was determined by a two -
stage r adial diffusion assay using E. coli as previously
described [33]. The d iameter o f t he clear z one of control
(nonenzyme treatment) is d efined as 100% active a nd the
antimicrobial activity of samples is expressed as percent-

age o f c on trol.
RESULTS
Design and synthesis
The tetrapeptide R4 and the o ctapeptide R8 were derived
from the topological motifs of the cystine-stabilized
b-stranded a ntimicrobial peptid es, PG ( protegrins), TP
(tachyplesins) and RTD-1 (rhesus monkey t heta defensin).
These n aturally occurring peptides consisting of 17–19
residues containing two antiparallel b stran ds stabilized by
two or three cross-strand disulfides that rigidify an up-and-
down side-chain arrangement with to p a nd bottom faces
[35–37]. The arbitrarily assigned bottom hydrophobic faces
of protegrins, tachyplesins and RTD-1 are packed with two
or three disulfide bonds, while a spatially arranged BHHB
motif ( B ¼ basic a nd H ¼ hydrophobic amino acids)
can be found on their top faces. These include tetrapeptides
KWFR and RVYR in tachyplesin-1, RLYR in protegrin-1
and RITR in RTD-1 (Fig. 2 ). For convenience, we selected
RLYR of protegrin-1 a s the consensus BHHB sequence for
designing the R4 tetrapeptide. The octapeptide R 8, Arg-
Leu-Tyr-Arg-Lys-Val-Tyr-Gly, was designed to contain a
degenerated BHHB double-repeat with alternating clusters
of charge regions and hydrophobic a mino acids. The double
charge motif of Arg-Arg is found spatially or contiguously
in TP-1, PG-1 and RTD-1 w hile the degenerate tetrapeptide
repeat (KVYG) containing a single b ase and three hydro-
phobic amino acid sequences are also f ound as retro-
sequences in TP-1 (RYIG), and in PG-1 (RVVF).
Two dendrimeric and a linearly repeating peptide series
ranging f rom four to 32 amino acids were prepared

(Table 1). The dendrimeric series contained R 4 and R8
peptides tethered on an asymmetric core as dendrimeric
peptide D
m
R4 and D
m
R8 with branch numbers (m) of t wo,
four, and eight, respectively. Two dendrimeric peptide series
were compared with a series c ontaining linearly repeating
R4 peptides (R4)
n
with n ¼ 1, 2, 4 and 8.
All p eptides were prepared chemically by a stepwise solid-
phase method [38] purified by RP-HPLC a nd characterized
by mass spectrometry. An advantage of a dendrimeric over
a linear peptide of comparable molecular size is that they
require far fewer steps in their chemical synthesis. As
peptide dendrimers a re prepared by a controlled polymer-
ization in w hich multip le peptides copes grow simultane-
ously on the branching cores, the number of assembling
steps in their syntheses is sharply reduced when comparing
to linear p eptides o f similar lengths (Fig. 2). Thus, the R8
dendrimeric peptides required only eight cycles (120 steps)
by a solid-phase method for assembly on the lysyl cores to
afford th ree D
m
R8 dendrimers containing 16, 32 a nd 64
amino-acid residues, respectively. Three dendrimeric R4
peptides required only four cycles. In contrast, the similarly
sized 32-residue (R4)

8
peptide of the linearly repeating R4
series required 3 2 c ycles and 480 steps for its synthesis,
which clearly indicates th e synthetic advantage in preparing
the dendrimeric series.
Table 1. S equences, HPLC retention time, mass data and number of monomer, a mino acid and charge of linear and dendrimeric peptides.
Compounds Sequence
a
HPLC (min) MH
+
(calculated) Monomer
Number of
Amino acids
b
Charge
c
A Linear R4
1 R4 RLYR 11.3 605.518 (607.691) 1 4 2
2 (R4)
2
(RLYR)
2
12.8 1193.83 (1194.42) 2 8 4
3 (R4)
4
(RLYR)
4
17.1 2369.66 (2371.81) 4 16 8
4 (R4)
8

(RLYR)
8
23.9 4726.71 (4726.84) 8 32 16
B Dendrimer R4
5D
2
R4 (RLYR)
2
-[K] 13.2 1322.44 (1322.55) 2 8 4
6D
4
R4 (RLYR)
4
-[K
2
K] 14.5 2756.01 (2756.27) 4 16 8
7D
8
R4 (RLYR)
8
-([K
2
K]
2
K) 17.2 5623.44 (5623.71) 8 32 16
C Dendrimer R8
8 R8 RLYRKVYGK 11.8 1182.72 (1181.43) 1 8 3
9D
2
R8 (RLYRKVYG)

2
-[K] 14.8 2220.23 (2218.52) 2 16 6
10 D
4
R8 (RLYRKVYG)
2
-[K
2
K] 15.7 4549.02 (4549.55) 4 32 12
11 D
8
R8 (RLYRKVYG)
2
-([K
2
K]
2
K) 17.3 9214.54 (9211.27) 8 64 24
a
C-terminal amide.
b
Excluding the lysyl core.
c
Charge refers to the dendrimeric peptides and the lysyl core.
926 J. P. Tam et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Antimicrobial activity
The minimum inhibitory concentrations (MICs) of all three
series against four Gra m-negative bacteria, three G ram-
positive bacteria and three fungi were determined by two-
stage radial diffusion assay in both low- and high-salt (with

100 m
M
NaCl) conditions. Assays of antimicrobial activity
under high-salt conditions were intended to simulate
physiological conditions of 100–150 m
M
NaCl.
Dendrimeric RLYRKVYG (R8) octapeptides
Under low-salt conditions, the R8 monomer containing an
additional charged Lys at its C-terminus showed low
activity against Gram-positive and Gram-negative b acteria
as well as m oderate activity against t hree tes t fungi w ith
MICs > 10 l
M
(Table 2). However, their activities against
all t est organisms were completely abrogated under high-
salt condition s. The potency of the dimeric 16-r esidue
peptide D
2
R8 was significantly i mproved, with MICs
<10 l
M
against seven organisms and < 1 l
M
against
E. coli and M. luteus under low-salt condition. D
2
R8 did
not retain its activity under high-salt conditions, except
against M. luteus (MIC, 1.8 l

M
). In contrast, the tetravalent
D
4
R8 was broadly active under both l ow- and high-salt
conditions. With the exception of D
4
R8 against Pr. vulgaris
(MIC, 2 l
M
),theirMICswere<1l
M
under low-salt
conditions. T he tetr avalent D
4
R8, w ith a mean MIC o f
 0.67 l
M
against all test organisms under both low- and
high-salt conditions, displayed activity spectra and potency
comparable to naturally occurring antimicrobial peptides
such as tachyplesins and protegrins.
Dendrimeric RLYR (R4) tetrapeptides
Comparing with the previous R8 series, the dendrimeric R4
series h as t he adv antag es o f m olecular and synthetic
simplicity. Because the R4 peptides contain only the first
repeat of the R8 octapeptides, their molecular sizes and the
chemical steps needed for their syntheses are quantitatively
halved. However the d endrimeric R4 peptides of eight, 16
and 32 amino acids with valences of two, four and eight are

nearly as potent as the corresponding D
m
R8 series.
The R 4 monomer was inactive with MICs > 500 l
M
against 10 test organisms in both low- and high-salt assays
(Table 3). In contrast, the eight-residue divalent D
2
R4
displayed c onsiderable activity with MICs ranging from 1 to
6.1 l
M
and a mean MIC of 3.3 l
M
under low-salt
conditions, averaging > 160-fold improvement over the
R4 tetrapeptide. However, its activity decreased twofold to
10-fold under high-salt conditions. Interestingly, D
2
R4 was
selective against three test fungi with MICs of 1–1.3 l
M
under low-salt conditions. T etra-branching to a 16-residue
D
4
R4 dendrimeric peptide increased potency  fivefold
with MICs 0.39–1 l
M
under low salt-conditions and
0.59–1.9 l

M
under high-salt conditions. Based on the
similarity of lengths, this 16-residue D
4
R4 is sixfold and
40-fold more active than the corresponding divalent
16-residue D
2
R8 of the R8 s eries a nd is comparable to
PG-1 under low- and high-salt conditions, respectively. The
D
8
R4 displayed MICs < 0.79 l
M
against all 10 test
organisms under low-salt conditions and MICs < 1 l
M
against eight organisms under high-salt conditions. Further
branching to t he octavalent 32-residue D
8
R4ledtoonly
small improvements in potency, n early all which was
retained under high-salt conditions.
Linearly repeating RLYR (R4) tetrapeptides
A series o f linearly repeating R4 peptides containing two,
four, and eight copies of R4 peptides also was prepared for
comparisons with the D
m
R4 dendrimers. In general, the
antimicrobial a ctivity of the linear p eptides improved as

molecular size increased. The linear octapeptide (R4)
2
exhibited M ICs ranging widely from 1.2 to 3 9 l
M
higher
activity against Gram-positive t han Gram-negative bacte-
ria, but was less active than the corresponding dendrimeric
D
2
R4 peptide (Table 4). The tetrameric (R4)
4
with four R4
repeats a nd 16 residues showed MICs of 0.5–1.8 l
M
, but
was also less active than t he corresponding D
4
R4 and D
8
R4
Table 2. Antimicrobial activity of dendritic R8 peptides. Experiments w ere pe rformed in radial diffusion assay with underlay gel containing 1%
agarose, 10 m
M
phosphate buffer with (high-salt) or without ( low-salt) 100 m
M
NaCl. Activities against multiple strains are expressed as t he
minimum inhibitory concentration (MIC, l
M
).
Organism

MIC (l
M
)
R8 D
2
(R8) D
4
(R8) D
8
(R8)
LH LHLHLH
Gram-negative
E. coli 28.4 > 500 0.8 29.8 0.2 2.0 0.3 0.3
P. aeruginosa 46.2 > 500 6.3 40.4 0.6 0.6 0.5 0.5
P. vulgaris 32.0 > 500 18.4 117 2.0 5.0 0.7 0.5
K. oxytoca 99.8 > 500 1.4 29.6 0.5 2.0 0.4 0.5
Gram-positive
S. aureus 72.4 > 500 2.1 118 0.7 2.1 0.3 0.4
M. luteus 28.4 > 500 0.8 1.8 0.2 0.5 0.2 0.4
E. faecalis 10.3 > 500 5.0 17.2 0.7 0.8 0.3 0.4
Fungi
C. albicans 19.2 > 500 1.9 14.4 0.6 1.7 0.3 0.4
C. kefyr 16.2 > 500 3.1 38.2 0.7 1.0 0.4 0.4
C. tropicalis 10.1 > 500 5.0 28.8 0.5 0.5 0.4 0.4
Ó FEBS 2002 Antimicrobial dendrimeric peptides (Eur. J. Biochem. 269) 927
dendrimers. Comparing with the D
m
R4 series, linear R4
peptides were generally less act ive against many tested
organisms. The activity profile of octameric (R8)

8
could not
be determined accurately because it precipitated in phos-
phate buffers, suggesting aggregation under both low- and
high-salt conditions.
Hemolytic activity
Table 5 shows the hemolytic activities of t he dendrimeric or
linear R4 a nd R8 pep tide s eries expressing E C
50
values
ranging > 500-fold. The toxicity of linear peptides against
erythrocytes was g enerally higher than the dendrimeric R4
peptides. The monomeric R4 and R8, peptides which were
ineffective as a ntimicrobials, were also nonhemolytic with
EC
50
>3900l
M
. The hemolytic toxicity of the linear R4
peptides increased 2 .8- and 15-fold f rom R4 to t he dimer
and tetramer, respectively. In contrast, the hemolytic
activity of the dendrimeric R4 peptides increased only
2.3-and3.3-foldfromR4toD
2
R4 and D
4
R4 peptides,
respectively. Int erestingly, the hemolytic activit y of the
dendrimeric D
8

R4 (EC
50
1514 l
M
) was similar to D
4
R4
(1510 l
M
). Based on the molecular size, the t oxicity o f ( R4)
2
peptide on human erythrocytes was t wofold higher than
D
2
R4, w hile the ( R4)
4
peptide was about fourfold higher
than the corresponding D
4
R4. A significant d ifference in the
effects on e rythrocytes m orphology o f was als o observed
between the linear and dendrimeric peptides when the
peptides and cells were incubated at 37 °C. The higher
ordered linear R8 peptides (R4)
4
and (R4)
8
caused cell
rufflings and aggregations which were n ot found with in the
corresponding dendrimeric R8 peptides. Although t he EC

50
of the linear peptide (R 4)
8
could n ot be determined
accurately because o f its poor solubility in phosphate
buffers, this peptide rapidly and quantitatively induced
erythrocytes aggregations.
Proteolytic stability
The proteolytic stability of d endrimeric peptides to trypsin
and chymotrypsin was determined using peptide and
enzyme in ratio of 100 : 1 (w/w) at 3 7 °C. Enzyme-treated
Table 4. Antimicrobial activity of linear R4 peptides. Experiment were
performed in radial diffusion assay as described for Table 2.
Organism
MIC (l
M
)
(R4)
2
(R4)
4
L-salt H-salt L-salt H-salt
Gram-negative
E. coli 39.0 33.6 1.0 0.7
P. aeruginosa 4.1 8.7 0.5 1.0
P. vulgaris 10.2 18.4 1.8 1.2
K. oxytoca 14.1 13.1 0.9 0.9
Gram-positive
S. aureus 8.1 17.5 1.8 1.1
M. luteus 1.3 12.8 0.6 0.6

E. faecalis 1.2 5.2 0.9 1.4
Fungi
C. albicans 9.0 17.4 1.2 1.9
C. kefyr 1.7 5.5 1.1 2.0
C. tropicalis 1.4 3.2 0.8 1.2
Table 3. Antimicrobial activity of R4 dendrimer peptides. Experiment were performed in radial d iffusion assay as described for Table 2.
MIC (l
M
)
R
4
D
2
(R4) D
4
(R4) D
8
(R4)
Organism L- salt H-salt L- salt H-salt L- salt H-salt L- salt H-salt
Gram-negative
E. coli > 500 > 500 6.1 10.2 0.6 0.7 0.5 0.7
P. aeruginosa > 500 > 500 3.6 18.5 0.5 1.2 0.3 0.9
P. vulgaris > 500 > 500 3.3 28.0 1.0 1.9 0.8 1.3
K. oxytoca > 500 > 500 3.8 39.0 0.4 0.9 0.4 0.8
Gram-positive
S. aureus > 500 > 500 4.5 10.2 0.8 0.6 0.5 0.6
M. luteus > 500 > 500 3.9 12.0 0.5 0.7 0.5 0.7
E. faecalis > 500 > 500 4.7 10.2 0.8 1.8 0.8 1.3
Fungi
C. albicans > 500 > 500 1.0 6.4 0.8 0.8 0.7 0.9

C. kefyr > 500 > 500 1.2 1.5 0.9 1.3 0.7 0.6
C. tropicalis > 500 > 500 1.3 1.8 0.7 0.8 0.7 0.6
Table 5 . H emo lytic activity of R4 and R8 linear and dendritic p eptid es.
Hemolytic activity of peptides is expressed as EC
50
,whichisthepep-
tide concentration producing 50% of human erythrocytes lysis.
Peptide EC
50
(l
M
)
Linear RLYR 5200
(RLYR)
2
1950
(RLYR)
4
338
(RLYR)
8
48
Dendritic R4 (RLYR)
2
K 3700
(RLYR)
4
K
2
K 1510

(RLYR)
8
(K
2
K)
2
K 1514
Dendritic R8 RLYRKVYG 3950
(RLYRKVYG)
2
K 1420
(RLYRKVYG)
4
K
2
K 610
(RLYRKVYG)
8
(K
2
K)
2
K 112
Tachyplesin 108
928 J. P. Tam et al. (Eur. J. Biochem. 269) Ó FEBS 2002
samples collected at different time p oints were tested for
their residual antimicrobial activity against indicator
bacterium E. coli (Fig. 4). When the linear (R4)
4
peptide

was treated with trypsin, its antibacterial activity decreased
rapidly to 25–30% of the untreated control after 2–5 min
and remained at about 20% of control after 24 h. In
contrast, t he ac tivity of the trypsin-treated D
4
R4 peptide
dendrimer was > 9 0% and 80% of control after 2 and
24 h, respectively. Similar results were observed for
the dendrimeric R8 peptides (Fig. 5). Trypsin r apidly
inactivated > 80% of the antibacterial activity of the linear
R8 peptides, but only  30% of D
2
R8 and D
4
R8 activity
after 2 h. To determine whether these results derived from a
loss of trypsin activity during t he assay, D
2
R8 was retreated
with trypsin a t 4 - a nd 8- h i ntervals a fter the first trypsin
treatment. Results showed that eac h treatment d ecrease d
the antibacterial activity of D
2
R8 about 10–20%, while the
antimicrobial activity of D
4
R8 decreased 1 5% after 8 h
trypsin re-treatment. These findings suggest that the
dendrimeric peptides were more resistant to proteolytic
degradation.

DISCUSSION
We have demonstrated that dendrimers containing repeat-
ing short tetrapeptides with a BHHB motif modified f rom
naturally occ urring b-stranded antimicrobial peptides func-
tion as potent antimicrobials with membranolytic activity.
Although dendrimeric and linearly repeating peptides differ
in their architectures and t opologies, they may share a
similar ability to form various patterns of hydrophobic and
charge clusters for pattern recognition of microbial surfaces.
Compared with the linearly repeating peptide antimicro-
bials, our results s how that the dendrimeric peptides possess
several desirable attributes. They display potent and broad-
spectrum activity under both low- and high-salt conditions,
enhanced proteolytic stability and dec reased hemolytic
activity. Furthe rmore, they require far fewer chemical steps
for their synthesis than the control s eries of tandemly
repeating peptides.
Although the a ctivity of a ntimicrobial peptides such as
defensins or defensin-like p eptides with M
r
ranging from 3
to 5 kDa is abrogated when tested in high-salt conditions,
this is not true in peptide like p rotegrins, and tachyplesins
with an M
r
of about 2 k Da [1–4]. Thus, these assays would
show whether the dendrimeric peptides ranging from 2 to
5 kDa behave similar t o defensins or protegrins and
tachyplesins and whether molecular s izes have any e ffect
on their activity profiles. F or comparative purposes, Table 6

shows the antimicrobial activity of PG-1 and TP-1 in our
assay system. In general, TP-1 with MICs ranging from 0.2
to 1.3 l
M
is the m ore potent of these two antimicrobial
peptides, d isplaying twofold to threefo ld higher activity
than PG-1 (MICs 0.3–2.8 l
M
)innineofthe10tested
organisms in our assays. I t is also interesting to note that
PG-1 displays higher activity against Gram-positive than
Gram-negative bacteria or fungi under both low- and high-
salt conditions with MICs ranging from 0.3 to 0.8 l
M
.
Correlation of dendrimeric design with antimicrobial
activity
In the current study, th e optimal branching related to
antimicrobial activity and molecular size suitable f or further
development as therapeutics appears to be tetravalent. The
R4 and R8 monomers in t he D
m
R4 and D
m
R8 series are
largely inactive w hereas the dimeric forms do not retain their
Fig. 4. Activity aga inst E. coli of metabolite residures of li near and
dendrimeric R4 by trypsin treatment. The peptides were t reated with
trypsin at 3 7 °C. At devising tim es, the s amples were collected and
trypsin inhibitor was added to samples for stopping the reaction. The

antimicrobial activity of each sample against E. coli was performed in
a two-stage radial diffusion assay. The antimic robial activity of sam-
ples is expressed i n percentage of that of samples w ithout trypsin
treatment.
Fig. 5. Ac tivity against E. coli of metabolite residures of dendrimeric R8
by trypsin treatment. Effect of trypsin on the anitmicrobial activity of
D
2
R8, D
4
R8 and D
8
R8 peptides . Th e ex perimen ts w ere p erformed a s
described in the legend of Fig. 4.
Ó FEBS 2002 Antimicrobial dendrimeric peptides (Eur. J. Biochem. 269) 929
activities under high-salt conditions. In contrast, the tetra-
valent and octavalent (m ¼ four and eight, respectively)
dendrimeric peptides show a broad activity spectrum against
10 test microbes in both low- and high-salt assays. For both
R4 and R8 peptides, tetravalent dendrimers show large
improvements in potency over divalent dendrimers whereas
only small improvements are found from the tetravalent to
octavalent dendrimeric peptides under low-salt conditions.
The higher branching D
8
-dendrimers a nd longer peptide
chain lengths of R8 peptides in retain activity under high-
salt conditions better than the corresponding D
4
-dendrimer

and shorter R4 peptide series. As there are only small
variations in potency (MICs < 1 l
M
) between the tetrava-
lent and octavalent dendrimers, a tetravalent dendrimeric
D
4
R4 design is perhaps more promising for further research
on antimicrobials using other tetrapeptide analogues.
The corresponding controlled series of linear R4 p eptides
exhibits large variations of activity sp ectra and potency that
roughly c orrelate with the decreases i n their lengths. The
linear R4 peptides w ith less than t hree repeats are largely
inactive under high-salt conditions, except against E. coli.
The potency and activity spectra of the D
4
R4 dendrimer
containing four R4 tetrapeptide copies are comparable to
the p rotegrins and tachyplesins of similar lengths. More
significantly, the dendrimeric R4 peptide achieves a
comparable antimicrobial profile without the conformation
constraints found in tachyplesins and protegrins, whose
activities are significant r educed in their unconstrained
forms. Taken together, these results suggest that a dendri-
meric scaffold c ould serve as a template for further analog
studies using a combinatorial approach with short peptides
to improve potency and specificity.
Our previous attempts to exploit t he dendrimeric design
on 33-residue a helical antimicrobial peptide cecropins to
increase potency were disappointing. Tetrameric and

octameric cecropins did not result in e nhanced potency
or specificity. A plausible explanation is that cecropins
form ordered ahelical structures in aqueous environments
[39]. Detailed structural information would thus be
necessary to determine their approximate quaternary
structures by a dendrimeric design. Thus, dendrimeric
antimicrobial peptides based o n s hort p eptides consisting
of four amino acids may h ave to overcome this type of
limitation.
Hemolytic activity and proteolytic stability
The dendrimeric R4 and R8 peptides are essentially
nontoxic to human erythrocytes with EC
50
for h emolysis
ranging from 112 to 3700 l
M
. For example, using the mean
MICs of D
4
R4 and D
4
R8 of 0.7 l
M
and 0.6 l
M
, respect-
ively, for all 10 test organisms, the t herapeutic indices (EC
50
/
MIC) of the two dendrimers will be > 2200. These two

dendrimeric peptides show about 10-fold improvement over
the line ar ( R4)
4
peptide, which has an EC
50
of 338 l
M
and a
therapeutic i ndex of 200. The EC
50
of peptide (R4)
8
cannot
be determined because o f its poor solubility in phosphate
buffers. Peptide (R4)
4
also causes aggregation and mem-
brane r ufflings of erythrocytes at concentrations < 10 l
M
,
which are not observed with the D
4
R4 and D
8
R4 at
concentrations > 1500 l
M
. Together, these results suggest
that the D
m

R4 or D
m
R8 peptides are nontoxic to human
erythrocytes at their effective microbe-killing concentra-
tions. However, t he mechanisms that cause t hese differences
are not clear.
D
4
R4 is surprisingly more stable than the (R4)
4
peptide
to proteolysis. The antimicrobial activity of the A rg-rich
(R4)
4
is inactivated by trypsin within 10 min while the
D
4
R4 peptide retains > 80% of its activity after 24 h.
These results suggest that either the dendrimeric structure of
D
4
R4 is more resistant to proteolytic degradation or that it
is a protease inhibitor. Because incubation of D
4
R4 with
trypsin does not inhibit t rypsin activity against the degra-
dation of a chromogenic trypsin substrate, it is likely that
the proteolytic stability o f D
4
R4 is due to its dendrimeric

structure.
Advantages and potential applications
of dendrimeric short peptides
Low m olecu lar mass p ep tide dendrimers have the a dvan-
tage of being less immunogenic than high-molecular-mass
dendrimers. The multivalency of peptide dendrimers
appears to be d esirable in the d esign of m embranolytic
peptides for other biochemical applications. These include
their a bility t o amplify cationic charges and hydrophobic
clusters as the number o f d endrimer branches increases.
Polycationic peptides, whether linear o r branched , are
known to d isplay membrane disruption o r f usion properties
that have been exploited for intracellular peptide, protein
and gene delivery [ 40–44]. Hydrophobic clusters o n a
peptide dendrimer lead to aggregation that may enhance
fusogenic a ctivity. A plausible mechanism is t hat amplifica-
tion by a dendrimeric design increases the effective molarity
of monomeric units and decreases the entrop y of self-
assembly. The end results are t hat t hey may mimic t he
mechanisms of action through w hich high-ordered anti-
microbial peptides exert their membranolytic effects. Thus,
the short dendrimeric peptides may represent a u seful a nd
unusual biopolymer design for effecting various membrano-
lytic activities in lipid environments.
Table 6. Antimicrobial activity of protegrin a nd tachyplesin. Experi-
ment we re performed i n radial diffusion assay as described at Table 2.
Organism
MIC (l
M
)

Protegrin Tachyplesin
L-salt H-salt L-salt H-salt
Gram-negative
E. Coli 0.9 0.8 0.3 0.4
P. aeruginosa 1.2 2.0 0.9 0.5
P. vulgaris 2.4 2.8 0.7 1.0
K. oxytoca 0.7 0.9 0.2 0.5
Gram-positive
S. aureus 0.7 0.6 0.4 0.5
M. luteus 0.3 0.8 1.0 1.1
E. faecalis 0.3 0.7 0.3 0.4
Fungi
C. albicans 1.3 1.0 0.7 0.9
C. kefyr 1.8 1.4 0.9 1.3
C. tropicalis 1.0 1.5 0.5 1.0
930 J. P. Tam et al. (Eur. J. Biochem. 269) Ó FEBS 2002
ACKNOWLEDGEMENT
This work was supported, in p art, by US Public He alth Se rvice NIH
Grants CA36544 and AI46164.
REFERENCES
1. Boman, H.G. (1995) Peptide antibiotics and their role in innate
immunity. Annu.Rev.Immunol.13, 61–92.
2. Nicolas, P. & Mor, A. (1995) Peptide as weapons against micro-
organisms in the chemical d efense system of vertebrates. Annu.
Rev. Microbiol. 49, 277–304.
3. Meister, M., L emaitre, B. & Hoffmann, J.A. (1997) Antimicrobial
peptide defence in Drosophila. Bioassay 19, 1019–1026.
4. Hancock, R.E. (1997) Peptide antibiotics. Lancet. 34 9, 418–422.
5. Ouellette, A.J. & S elsted, M.E. (1996) Paneth cell defensins:
endogenous peptide c ompone nts of intes tinal host defen ce.

FASEB J. 10, 1280–1289.
6. Lehrer, R.I. & G anz, T. (1999) Antimicrobial peptide in
mammalian a nd insect host defence. Curr. Opin. I mmunol. 11,
23–27.
7. S olomon, S., Hu, J., Zhu, Q., Belcourt, D., Bennett, H.P.,
Bateman, A. & Antakly, T . (1991) Corticostatic pe ptides.
J. Steroid B iochem. Mol. B iol. 40, 391–398.
8. Chertov, O., Michiel, D.F., Xu, L., Wang, J.M., Tani, K.,
Murphy, W.J., Longo, D.L., Taub, D.D. & Oppe nheim, J.
(1996) Identification of Defensin-1, Defensin-2, and CAP37/
Azurocidin as T-cell ch emo attractant pr oteins released f rom
interleukin-8-stimulated neutrophils. J. Biol. Chem. 271 , 2935–
2940.
9. Xu, Y ., Tamamura, H., Arakaki, R., N akashima, H., Zhang, X.,
Fujii, N., Uchiyama, T. & Hattori, T. (1999) Marked increase in
anti-HIV activity, as well as inhibitory activity against HIV entry
mediated by CXCR4, linked to enhancement of the binding ability
of tachyplesin analogs to CXCR4. AIDS Res. Human Retroviruses
15, 419–427.
10. T am, J.P., Lu, Y A. & Yang, J L. (2000) Design of salt-insensi-
tive glycine-rich antimicrobial peptides with cyclic tricysti ne
structures. Biochemistry 39, 7159–7169.
11. T am, J.P., Wu, C. & Yang, J L. (2000) Membranolytic selectivity
of cystine-stablizedcyclic protegrins. Eur. J. Biochem. 267, 3289–
3300.
12. T am, J.P., Lu, Y A., Y ang, J L. & Chiu, K W. (1999) A n
unusual structural motif of antimicrobial peptides containing end-
to-end macrocycle and cystine-knot d isulfides. Proc. Natl Acad.
Sci. USA 96, 8913–8918.
13. Medzhitov, R. & Janeway Jr, C. (2000) The toll receptor family

and microbial recognition. Trends Microbiol. 8, 452–456.
14. A derem, A. & Ulevitch, R.J. (2000) Toll-like r ecept ors in the
induction of the innate immune response. Nature 406, 782–787.
15. Muhle, S.A. & Tam, J.P. (2001) Design of Gram-negative selective
antimicrobial peptides. Biochemist ry 40, 5777–5785.
16. S chumann, R.R., Leong, S.R., Flaggs, G.W., Gray, P.W., Samuel,
D.,Wright,S.D.,Mathison,J.C.,Tobias,P.S.&Ulevitch,R.J.
(1990) Structure and function of lipopolysaccharide bind ing pro-
tein. Science 249, 1429–1431.
17. W right, S.D., Ramos, R.A., Tobias, P.S., Ulevitch, R.J. &
Mathison, J.C. ( 1990) CD4, a receptor for complexed o f
lipopolysaccharide (LPS) and L PS binding protein. Science 249,
1431–1433.
18. W illiams, M.J., Rodriguez, A., Kimbrell, D.A. & Eldon, E.D.
(1997) The 18-wheeler mutation r eveals complex a ntibacterial
gene regulation in Drosophila host defence. EMBO J. 16, 6 120–
6130.
19. Frank, R.W., Gennaro, R., Schneider, K. & Prz ybylski, M. (1990)
Amino acid sequences of two proline-rich bactenecins. J. Biol.
Chem. 265, 18871–18874.
20. S hamova, O., Brogden, K .A., Z hao, C., Nguyen, T., Kokryakov,
V.N. & L ehrer, R. (1990) I P urification and p roperties o f p roline-
rich antimicrobial peptides from sheep and goat leukocytes. Inf ect.
Immun. 67, 4106–4111.
21. Zanetti,M.,Sal,G.D.,Storici,P.,Schneider,C.&Romeo,D.
(1993) The cDNA of the n eutrophil antibiotic Bac5 predicts a
pro-sequence homologous to a cysteine proteinase inhibitor that i s
common to other neutrophil a ntibio tics. J. Biol. Chem. 268 ,
522–526.
22. S elsted, M.E., Novotny, M.J., Morris, W.L., Tang, Y Q., Smith,

W. & Cullor, J.S. (1992) Indolici din, a novel bactericidal
tridecapeptide amide from neutrophils. J. Biol. Chem. 267, 4292–
4295.
23. T am, J.P. (1988) Synthetic peptide vaccine design: synthesis and
properties of a h igh-density multiple a ntige nic peptide s ystem.
Proc. Natl Acad. Sci. USA 85, 5409–5403.
24. Posnett, D.N., McGrath, H. & Tam, J.P. (1988) A novel method
for producing anti-peptide antibodies: production of site- specific
antibodies to the T cell antigen receptor b-chain. J. Biol. Chem.
263, 1719–1725.
25. F rancis, M.J., Hastings, G.Z., Brown, F. , McDermed, J.,
Lu, Y A. & Tam, J.P. (1991) I mmunological evaluation of the
multiple antigen peptide (MAP) s ystem using the major immu-
nogenic site o f f oot-and mouth d isease v irus. Immunology 73 ,
249–254.
26. Fassina, G ., Corti, A. & Cassani, G. (1992) Affinity enhancement
of comple mentary p ep tide re cognitio n. Int. J. Pept. Prot. Res. 39,
549–556.
27. Tam, J .P. (1996) Recent advances in multiple antigen p eptides.
J. Immun. Methods 196, 17–32.
28. Nakamura, T., Furanaka, H., Miyata, T., Tokunaga, F., Muta, T.
& Iwanaga, S. (1988) T achyplesin, a class of a ntimicrobial peptide
from the Horseshoe Crab (Tachypleustridentatus). J. Biol. C hem .
263, 16709–16713.
29. Kokryakov, V.N., Harwig, S.S., Pan yutich, E.A., Shevchenko,
A.A., Aleshina, G.M., Shamova, O.V., Korneva, H.A. & Lehrer,
R. (1993) Proteg rins: le ukocyte antimicrobial peptides that com-
bine features of corticostatic defensins and tachyplesins. FEBS
Lett. 327, 231–236.
30. T an g, Y Q., Yu a n, J., O

¨
sapay, G., O
¨
sapay, K., Tran, D., M iller,
C.J., Ouellett e, A .J. & Selsted, M.E. ( 1999) A cyclic antimicro bial
peptide produced in primate leukocytes by the ligation of two
truncated alpha-defensins. Science 286, 498–502.
31. Castro, B., Dormay, J.R., Evin, G . & S elve, C. (1975) Reactifs de
couplage peptidique IV (1)-L¢hexafluorophosphate de benzotria-
zilyl N-oxytrisdimethlamino phosphonium (BOP). Tetra. Lett. 14,
1219–1222.
32. Lu, Y A. & Felix, A.M. (1993) Pegylated peptides I: solid-phase
synthesis of N
a
-pegylated peptides using F moc strategy. Pept. Res.
6, 140–146.
33. L ehrer, R.I., Rosenm an, M., Harwing, S.S.L., Ja ckson, R . &
Eisenhauer, P. (1991) Ultrasensitive assays for endogenous
antimicrobial polypeptides. J. Immunol. Med. 137, 167–173.
34. Fehlbaum, P., Bulet, P ., Machaut, L., Lagueux, M., Broekaert,
W.F., Hetru, C. & Hoffmann, J.A. (1994) Insect immunity, septic
injury of Drosophila indu ces the synth esisof a potent antifungal
peptide with sequence homology to plant antifungal peptides.
J. Biol. C hem. 269, 33159–33163.
35. Kawano, K., Yoneya, T., Miyata, T., Yoshikawa, K., Tokunaga,
T., Terada, Y. & Iwanaga, S. (1990) Anti microbial peptide,
tachyplesin I, isola ted fro m hemoc ytes of the h orse shoecrab
(Tachypleus tridentatus). J. Biol. Chem. 265, 15365–15367.
36. Yonezawa, A., Kuwahara, J., Fujii, N. & Sugiura, Y. (1992)
Binding of tac hyplesin-I to DN A revealed b y footprinting

analysis-significant contribution of secondary structure to DNA-
binding and implication f or biological action. Biochemistry 31 ,
2998–3004.
Ó FEBS 2002 Antimicrobial dendrimeric peptides (Eur. J. Biochem. 269) 931
37. Fahrne r, R.L., Dieckmann, T., Harwig, S.S.L., L ehrer, R.I.,
Eisenberg, D. & Feigon, J. (1996) Solution structure of protegrin-1,
a broad-spectrum antimicrobial peptide from porcine leukocytes.
Chem. Biol. 3, 543–550.
38. Merrifield, R.B. (1986) Solid phase peptide synthesis. Science 57,
1619–1625.
39. Bals,R.,Wang,X.,Zasloff,M.&Wilson,J.M.(1998)Thepeptide
antibiotic LL-37/hCAP-18 is expressed in epithelia of th e human
lung whe re it has broad antimicrobial activity at the airway sur-
face. J. Proc. Natl Acad. Sci. USA 95, 9541–9546.
40. Wadhwa, M.S., Collard, W.T., A dami, R .C. & Rice, K.G. (1997)
Peptide-mediated gene delivery. influence of peptide structure on
gene expression. Bioconj. Chem. 8, 81–88.
41. Pillai, O., Nair, V., Poduri, R. & Panchagnula, R. (1999) Trans-
dermal iontophoresis. Part II: peptide and protein delivery.
Methods Find. Expe. Clin. Pharm. 21, 229–240.
42. Bernkop-Schnurch, A. & Pasta, M. (1998) Intestinal peptide and
protein delivery: novel bioadhesive drug-c arrier matrix shielding
from enzymatic attack. J. Pharm. Sci. 87, 430–434.
43. Murphy, J.E., Uno, T., Hamer, J.D., Cohen, F.E., Dwarki, V. &
Zuckermann, R.N . (1998) A combinatorial approach to th e dis-
covery of efficient c ationic p eptoid reagents fo r g ene d elivery.
Proc. Natl Acad. Sci. USA 95, 1517–1522.
44. Sheldon, K., Liu, D., Ferguson, J. & G ariepy, J. (1995) Loligo-
mers. D esign of de n ovope ptide-based intracellular veh icles. Proc.
Natl Acad. Sci. USA 92, 2056–2060.

932 J. P. Tam et al. (Eur. J. Biochem. 269) Ó FEBS 2002