Compared with a series of linearly repeating R4 peptides, the R4 dendrimers show comparable anti-microbial potency, but are more aqueous soluble, more stable to proteolysis, less toxic t
Trang 1Antimicrobial 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 to achieve broad antimicrobial 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) The R4 tetrapeptide (RLYR) contains a putative
microbial surface recognition BHHB motif (B ¼ basic,
H ¼ hydrophobic amino acid) found in protegrins and
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 as well as their
divalent dendrimers contain no to low activity In contrast,
the tetra- and octavalent R4 and R8 dendrimers are broadly
active under either conditions, exhibiting relatively similar
potency with minimal inhibition concentrations < 1 lM
against both bacteria and fungi Based on their size and charge similarities, the potency and activity spectrum of the tetravalent R4 dendrimer are comparable to protegrins and tachyplesins, a family of potent antimicrobials containing 17–19 residues Compared with a series of linearly repeating R4 peptides, the R4 dendrimers show comparable anti-microbial potency, but are more aqueous soluble, more stable to proteolysis, less toxic to human cells and more 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 such knowledge in a dendrimeric design therefore presents an attractive approach for devel-oping novel peptide antibiotics
Keywords: dendrimeric peptide; peptide 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 manifested by killing mechanisms that are less
likely than conventional antibiotics to develop multidrug
resistance [7–12] Design elements desirable for therapeutics
include activity under physiological conditions (100–
150 mMor high-salt conditions), low toxicity and
proteo-lytic stability Guided by these considerations, we and others
have designed antimicrobial peptides with unusual
struc-tural architectures using rigid scaffoldings 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 on 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 Pathogen-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 include LPS-bind-ing protein, soluble and membrane-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 to recognize PAMPs on microbial surfaces They often possess
a broad spectrum of antimicrobial 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, suggesting pattern-recognition selec-tivity under evolutionary pressure Although more than 200 antimicrobial peptides with various types of structures are known, they can be classified into two broad categories based on their primary sequences: those that contain repeating sequences ranging from two to 14 amino acids and those that are nonrepeating [19,20] Found in these two types of peptides are basic amino acids useful for
electro-Correspondence to J P Tam, Vanderbilt University, Department of
Microbiology and Immunology, A-5119 MCN, 1161 21st Avenue
South, Nashville, TN 37232-2363, USA Fax: + 1 615 343 1467,
Tel.: + 1 615 343 1465, E-mail: james.tam@mcmail.vanderbilt.edu
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; MIC,
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)
Trang 2static 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 they contain repeating or nonrepeating
sequences, is their ability to cluster charge and hydrophobic
amino acids 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-recognitionÕ peptides frequently found in
b-stranded peptide antibiotics to enhance interactions with
microbial lipid surfaces As short 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 mMNaCl, 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 architectures First evolved in the 1980s
[23,24], they contain a multivalent core that tethers an array
of branching peptides An early example is 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 divalent Lys 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 bioactive
peptides [25–27], a MAP-like dendrimeric peptide
contain-ing the Lys2Lys (K2K) as a dendrimeric core is better suited
to serve the purpose of providing with short peptides
polyvalency for our dendrimeric design
Little is known about the structures of dendrimeric
peptides with the K2K cores The three-lysine K2K core is
asymmetric because each lysine contains a short a and a
long e amino arm that results in unequally spaced amines
with four branches, two 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 end-to-end distance of 21 atoms separating the two e-amines of the K2K core, most of which are methylene 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 K2K core with tethered short a peptides can achieve a barrel-like structure mimicking those of helical peptides, which is essential for membranolytic activity In addition, judicious selection of a short a peptide with cationic charged and hydrophobic residues appropriate for Ôpattern recognitionÕ may enhance the hypothetical bioactive structures through electrostatic and hydrophobic interactions with the nega-tively charged microbial surfaces
Tachyplesins [28] are potent broad-spectrum antibiotics containing four degenerated repeats of a tetrapeptide with a HBCH motif (H, hydrophobic; B, basic and C, Cys) These cystine-stabilized antiparallel peptides have side chains arranged in an up-and-down topology One face of this topology contains a consensus BHHB motif Similar BHHB motifs can be found in protegrins [29] and RTD-1 (rhesus monkey theta defensin) [30] Based on this topological motif, our prototypic pattern-recognition peptides consist of
a tetrapeptide R4, RLYR, and a degenerated double-repeating octapeptide R8, RLYR-KVYG (Fig 2) Here, we describe the design and properties of dendrimeric peptides employing the tetravalent and octavalent dendrimeric cores tethered with cationic peptides, a R4 tetrapeptide or a R8 octapeptide For comparison, the R4 or R8 peptides are then tethered to three different cores consisting of a single lysine (K), a three-lysine (K2K) or a heptalysine [(K2K)2K] 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 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 three different font style (A) two
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 with a and e
branche bearing peptides Arg-Leu-Tyr-Arg.
Fig 2 Topological distributions of BHHB and BHHX motif in the antiparallel b sheet structured protegrin-1 (PG-1), tachyplesin-1 (TP-1) and rhesus theta defensin (RTD-1).
Trang 3that the tetravalent and octavalent dendrimeric short
peptides display properties 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
M A T E R I A L S A N D M E T H O D S
Materials
Solvents, all of HPLC grade, were obtained from VWR
Scientific Co and used without further purification
Fmoc amino acid derivatives, N-hydroxybenzotriazole
(HOBt), N,N¢-dicyclohexylcarbodiimide (DCC), and
p-[(R,S)-a-[1-(9H-Fluoren-9-yl)-methoxylformamido]-2,4-dimethoxylbenzyl]-phenoxyacetic acid (Fmoc-DPA) were
obtained from Chem-impex International Inc
N,N-diiso-propylethylamine (DIEA) and p-cresol were purchased
from Aldrich Chemical Co Trypsin, a-chymotrypsin and
a-cyano-4-hydroxycinnamic acid were purchased from
Sigma Chemical Co Trifluoroacetic acid was obtained
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 coli ATCC 25922, Pseudomonas
aeru-ginosaATCC 27853, Klebsiella oxytoca ATCC 49131, and
Proteus vulgaris ATCC 49132 The three Gram-positive
bacteria were Staphylococcus aureus 29213, Micrococcus
luteusATCC 49732 and Enterococcus faecalis ATCC 29212
The three fungi were Candida albicans ATCC 37092,
Candida kefyrATCC 37095, and Candida tropicalis ATCC
37097 The strains were incubated in trypticase soy broth
(TSB) which was prepared in double distilled 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 synthesizer
(CS Bio Co 536) was performed using Fmoc-tBu chemistry
and a single coupling protocol with BOP/DIEA [31] in
DMF Linear peptides were synthesized on
Fmoc-DPA-resin and dendrimeric peptides were synthesized on different branching core matrix Fmoc-DPA-NH-resins Analytical RP-HPLC was conducted on a Shimadzu LG-6 A system with an C18 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 Eluent A contained 0.04% TFA/
H2O; eluant B contained 0.04% trifluoroacetic acid/60%
CH3CN/H2O Preparative RP-HPLC was performed on a Waters 600 system with C18Vydac column (20· 250 mm) MALDI-MS was measured on a PerSeptive Biosystems Voyager instrument Samples were dissolved in 1 lL of a
1 : 2 mixture of H2O/CH3CN Measurements were made in
a linear model, with a-cyano-4-hydroxycinnamic acid as 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), and followed by DCC (206 mg, 1 mmol) and HOBt (157 mg,
1 mmol) to anchor the Fmoc-DPA handle onto the resin support After 24 h, the resultant Fmoc-DPA-resin was drained, washed sequentially with DMF, DCM, MeOH and dried in vacuo Substitution of the functionalized resin was 0.1 mmolÆg)1(Fig 3)
Preparation of branching lysyl core resin Syntheses of di- tetra- and octa-branching cores required one, two 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 by treatment with 20% piperidine/DMF Each coupling cycle doubled the branching level of lysyl core and after three cycles, the octa-branching Fmoc-Lys4-Lys2 -Lys-DPA-NH-resin was achieved (Fig 3)
Synthesis of DmR4 and DmR8 These peptides were synthesized on different branching lysyl core resin according to what has been described above The HPLC retention time and mass data showed
in Table 1
Fig 3 Synthetic scheme for preparing different
branched (two, four, and eight) core Low
substituted amine resin 1 coupled with DPA 2
by DCC/HOBt in DMF to
formFmoc-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 couplings with
Fmoc-Lys(Fmoc)-OH formed four branching core 5
or eight branching core 6.
Trang 4Antimicrobial and hemolytic assays
A sensitive and reproducible two-stage radial diffusion
antimicrobial assay of Lehrer et al [33] was employed
Antimicrobial activities were expressed in units
(0.1 mm ¼ 1 U), and the MICs were determined from
the x-intercepts of the dose–response curves Hemolytic
activity was determined using fresh human erythrocytes
Peptide concentrations causing 50% hemolysis (EC50) were
derived from the dose–response curve [34] The
membrano-lytic selectivity index is expressed as EC50/MIC
Proteolytic stability
Dendrimeric or linear peptides in various concentrations
were dissolved in NaCl/Pibuffer at pH 7.4 and aliquoted
into microtubes Trypsin was mixed with peptides in the
ratio of 1 : 100 (enzyme/peptide, w/w) The enzymatic
degradation was carried out at 37°C and stopped by
adding an appropriate enzyme inhibitor such as Type II-S
trysin inhibitor into the samples The residual
antimicro-bial activity of each sample was determined by a
two-stage radial diffusion assay using E coli as previously
described [33] The diameter of the clear zone of control
(nonenzyme treatment) is defined as 100% active and the
antimicrobial activity of samples is expressed as
percent-age of control
R E S U L T S
Design and synthesis
The tetrapeptide R4 and the octapeptide R8 were derived
from the topological motifs of the cystine-stabilized
b-stranded antimicrobial peptides, PG (protegrins), TP
(tachyplesins) and RTD-1 (rhesus monkey theta defensin)
These naturally occurring peptides consisting of 17–19
residues containing two antiparallel b strands stabilized by
two or three cross-strand disulfides that rigidify an
up-and-down side-chain arrangement with top and 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 and 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 as the consensus BHHB sequence for designing the R4 tetrapeptide The octapeptide R8, 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 amino acids The double charge motif of Arg-Arg is found spatially or contiguously
in TP-1, PG-1 and RTD-1 while the degenerate tetrapeptide repeat (KVYG) containing a single base and three hydro-phobic amino acid sequences are also found as retro-sequences in TP-1 (RYIG), and in PG-1 (RVVF)
Two dendrimeric and a linearly repeating peptide series ranging from four to 32 amino acids were prepared (Table 1) The dendrimeric series contained R4 and R8 peptides tethered on an asymmetric core as dendrimeric peptide DmR4 and DmR8 with branch numbers (m) of two, four, and eight, respectively Two dendrimeric peptide series were compared with a series containing linearly repeating R4 peptides (R4)nwith n ¼ 1, 2, 4 and 8
All peptides were prepared chemically by a stepwise solid-phase method [38] purified by RP-HPLC and 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 are prepared by a controlled polymer-ization in which multiple peptides copes grow simultane-ously on the branching cores, the number of assembling steps in their syntheses is sharply reduced when comparing
to linear peptides of 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 three DmR8 dendrimers containing 16, 32 and 64 amino-acid residues, respectively Three dendrimeric R4 peptides required only four cycles In contrast, the similarly sized 32-residue (R4)8peptide of the linearly repeating R4 series required 32 cycles and 480 steps for its synthesis, which clearly indicates the synthetic advantage in preparing the dendrimeric series
Table 1 Sequences, HPLC retention time, mass data and number of monomer, amino acid and charge of linear and dendrimeric peptides.
Number of Amino acids b Charge c
A Linear R4
B Dendrimer R4
C Dendrimer R8
a C-terminal amide b Excluding the lysyl core c Charge refers to the dendrimeric peptides and the lysyl core.
Trang 5Antimicrobial activity
The minimum inhibitory concentrations (MICs) of all three
series against four negative bacteria, three
Gram-positive bacteria and three fungi were determined by
two-stage radial diffusion assay in both low- and high-salt (with
100 mMNaCl) conditions Assays of antimicrobial activity
under high-salt conditions were intended to simulate
physiological conditions of 100–150 mMNaCl
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 bacteria
as well as moderate activity against three test fungi with
MICs > 10 lM(Table 2) However, their activities against
all test organisms were completely abrogated under
high-salt conditions The potency of the dimeric 16-residue
peptide D2R8 was significantly improved, with MICs
< 10 lM against seven organisms and < 1 lM against
E coliand M luteus under low-salt condition D2R8 did
not retain its activity under high-salt conditions, except
against M luteus (MIC, 1.8 lM) In contrast, the tetravalent
D4R8 was broadly active under both low- and high-salt
conditions With the exception of D4R8 against Pr vulgaris
(MIC, 2 lM), their MICs were < 1 lM under low-salt
conditions The tetravalent D4R8, with a mean MIC of
0.67 lMagainst 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 has the advantages of molecular 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 dendrimeric R4 peptides of eight, 16 and 32 amino acids with valences of two, four and eight are nearly as potent as the corresponding DmR8 series The R4 monomer was inactive with MICs > 500 lM against 10 test organisms in both low- and high-salt assays (Table 3) In contrast, the eight-residue divalent D2R4 displayed considerable activity with MICs ranging from 1 to 6.1 lM and a mean MIC of 3.3 lM 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, D2R4 was selective against three test fungi with MICs of 1–1.3 lM under low-salt conditions Tetra-branching to a 16-residue
D4R4 dendrimeric peptide increased potency fivefold with MICs 0.39–1 lM under low salt-conditions and 0.59–1.9 lM under high-salt conditions Based on the similarity of lengths, this 16-residue D4R4 is sixfold and 40-fold more active than the corresponding divalent 16-residue D2R8 of the R8 series and is comparable to PG-1 under low- and high-salt conditions, respectively The
D8R4 displayed MICs < 0.79 lM against all 10 test organisms under low-salt conditions and MICs < 1 lM against eight organisms under high-salt conditions Further branching to the octavalent 32-residue D8R4 led to only small improvements in potency, nearly all which was retained under high-salt conditions
Linearly repeating RLYR (R4) tetrapeptides
A series of linearly repeating R4 peptides containing two, four, and eight copies of R4 peptides also was prepared for comparisons with the DmR4 dendrimers In general, the antimicrobial activity of the linear peptides improved as molecular size increased The linear octapeptide (R4)2 exhibited MICs ranging widely from 1.2 to 39 lMhigher activity against Gram-positive than Gram-negative bacte-ria, but was less active than the corresponding dendrimeric
D2R4 peptide (Table 4) The tetrameric (R4)4with four R4 repeats and 16 residues showed MICs of 0.5–1.8 lM, but was also less active than the corresponding D4R4 and D8R4
Table 2 Antimicrobial activity of dendritic R8 peptides Experiments were performed 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 the minimum inhibitory concentration (MIC, l M ).
Organism
MIC (l M )
Gram-negative
Gram-positive
Fungi
Trang 6dendrimers Comparing with the DmR4 series, linear R4
peptides were generally less active against many tested
organisms The activity profile of octameric (R8)8could 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 the dendrimeric or
linear R4 and R8 peptide series expressing EC50 values
ranging > 500-fold The toxicity of linear peptides against
erythrocytes was generally higher than the dendrimeric R4
peptides The monomeric R4 and R8, peptides which were
ineffective as antimicrobials, were also nonhemolytic with
EC50> 3900 lM The hemolytic toxicity of the linear R4
peptides increased 2.8- and 15-fold from R4 to the dimer
and tetramer, respectively In contrast, the hemolytic
activity of the dendrimeric R4 peptides increased only
2.3- and 3.3-fold from R4 to D2R4 and D4R4 peptides, respectively Interestingly, the hemolytic activity of the dendrimeric D8R4 (EC50 1514 lM) was similar to D4R4 (1510 lM) Based on the molecular size, the toxicity of (R4)2 peptide on human erythrocytes was twofold higher than
D2R4, while the (R4)4peptide was about fourfold higher than the corresponding D4R4 A significant difference in the effects on erythrocytes morphology of was also 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 not found with in the corresponding dendrimeric R8 peptides Although the EC50
of the linear peptide (R4)8 could not be determined accurately because of its poor solubility in phosphate buffers, this peptide rapidly and quantitatively induced erythrocytes aggregations
Proteolytic stability The proteolytic stability of dendrimeric peptides to trypsin and chymotrypsin was determined using peptide and enzyme in ratio of 100 : 1 (w/w) at 37°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 )
L-salt H-salt L-salt H-salt Gram-negative
Gram-positive
Fungi
Table 3 Antimicrobial activity of R4 dendrimer peptides Experiment were performed in radial diffusion assay as described for Table 2.
MIC (l M )
Gram-negative
Gram-positive
Fungi
Table 5 Hemolytic activity of R4 and R8 linear and dendritic peptides Hemolytic activity of peptides is expressed as EC 50 , which is the pep-tide concentration producing 50% of human erythrocytes lysis Peptide EC 50 (l M )
(RLYR) 8 (K 2 K) 2 K 1514
(RLYRKVYG) 4 K 2 K 610 (RLYRKVYG) 8 (K 2 K) 2 K 112
Trang 7samples collected at different time points 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, the activity of the trypsin-treated D4R4 peptide
dendrimer was > 90% and 80% of control after 2 and
24 h, respectively Similar results were observed for
the dendrimeric R8 peptides (Fig 5) Trypsin rapidly
inactivated > 80% of the antibacterial activity of the linear
R8 peptides, but only 30% of D2R8 and D4R8 activity
after 2 h To determine whether these results derived from a
loss of trypsin activity during the assay, D2R8 was retreated
with trypsin at 4- and 8-h intervals after the first trypsin
treatment Results showed that each treatment decreased
the antibacterial activity of D2R8 about 10–20%, while the
antimicrobial activity of D4R8 decreased 15% after 8 h
trypsin re-treatment These findings suggest that the
dendrimeric peptides were more resistant to proteolytic
degradation
D I S C U S S I O N
We have demonstrated that dendrimers containing
repeat-ing short tetrapeptides with a BHHB motif modified from
naturally occurring b-stranded antimicrobial peptides
func-tion as potent antimicrobials with membranolytic activity
Although dendrimeric and linearly repeating peptides differ
in their architectures and topologies, 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 show 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 decreased hemolytic activity Furthermore, they require far fewer chemical steps for their synthesis than the control series of tandemly repeating peptides
Although the activity of antimicrobial peptides such as defensins or defensin-like peptides with Mrranging from 3
to 5 kDa is abrogated when tested in high-salt conditions, this is not true in peptide like protegrins, and tachyplesins with an Mrof about 2 kDa [1–4] Thus, these assays would show whether the dendrimeric peptides ranging from 2 to
5 kDa behave similar to defensins or protegrins and tachyplesins and whether molecular sizes have any effect
on their activity profiles For 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 lM is the more potent of these two antimicrobial peptides, displaying twofold to threefold higher activity than PG-1 (MICs 0.3–2.8 lM) in nine of the 10 tested organisms in our assays It 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 lM
Correlation of dendrimeric design with antimicrobial activity
In the current study, the optimal branching related to antimicrobial activity and molecular size suitable for further development as therapeutics appears to be tetravalent The R4 and R8 monomers in the DmR4 and DmR8 series are largely inactive whereas the dimeric forms do not retain their
Fig 4 Activity against E coli of metabolite residures of linear and
dendrimeric R4 by trypsin treatment The peptides were treated with
trypsin at 37 °C At devising times, the samples 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 antimicrobial activity of
sam-ples is expressed in percentage of that of samsam-ples without trypsin
treatment.
Fig 5 Activity 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 The experiments were performed as described in the legend of Fig 4.
Trang 8activities under high-salt conditions In contrast, the
tetra-valent and octatetra-valent (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 D8-dendrimers and longer peptide
chain lengths of R8 peptides in retain activity under
high-salt conditions better than the corresponding D4-dendrimer
and shorter R4 peptide series As there are only small
variations in potency (MICs < 1 lM) between the
tetrava-lent and octavatetrava-lent dendrimers, a tetravatetrava-lent dendrimeric
D4R4 design is perhaps more promising for further research
on antimicrobials using other tetrapeptide analogues
The corresponding controlled series of linear R4 peptides
exhibits large variations of activity spectra and potency that
roughly correlate with the decreases in their lengths The
linear R4 peptides with less than three repeats are largely
inactive under high-salt conditions, except against E coli
The potency and activity spectra of the D4R4 dendrimer
containing four R4 tetrapeptide copies are comparable to
the protegrins 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 reduced in their unconstrained
forms Taken together, these results suggest that a
dendri-meric scaffold could 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 the dendrimeric design
on 33-residue a helical antimicrobial peptide cecropins to
increase potency were disappointing Tetrameric and
octameric cecropins did not result in enhanced potency
or specificity A plausible explanation is that cecropins
form ordered a helical 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 on short peptides consisting
of four amino acids may have to overcome this type of limitation
Hemolytic activity and proteolytic stability The dendrimeric R4 and R8 peptides are essentially nontoxic to human erythrocytes with EC50 for hemolysis ranging from 112 to 3700 lM For example, using the mean MICs of D4R4 and D4R8 of 0.7 lMand 0.6 lM, respect-ively, for all 10 test organisms, the therapeutic indices (EC50/ MIC) of the two dendrimers will be > 2200 These two dendrimeric peptides show about 10-fold improvement over the linear (R4)4peptide, which has an EC50of 338 lMand a therapeutic index of 200 The EC50of peptide (R4)8cannot
be determined because of its poor solubility in phosphate buffers Peptide (R4)4 also causes aggregation and mem-brane rufflings of erythrocytes at concentrations < 10 lM, which are not observed with the D4R4 and D8R4 at concentrations > 1500 lM Together, these results suggest that the DmR4 or DmR8 peptides are nontoxic to human erythrocytes at their effective microbe-killing concentra-tions However, the mechanisms that cause these differences are not clear
D4R4 is surprisingly more stable than the (R4)4peptide
to proteolysis The antimicrobial activity of the Arg-rich (R4)4 is inactivated by trypsin within 10 min while the
D4R4 peptide retains > 80% of its activity after 24 h These results suggest that either the dendrimeric structure of
D4R4 is more resistant to proteolytic degradation or that it
is a protease inhibitor Because incubation of D4R4 with trypsin does not inhibit trypsin activity against the degra-dation of a chromogenic trypsin substrate, it is likely that the proteolytic stability of D4R4 is due to its dendrimeric structure
Advantages and potential applications
of dendrimeric short peptides Low molecular mass peptide dendrimers have the advan-tage of being less immunogenic than high-molecular-mass dendrimers The multivalency of peptide dendrimers appears to be desirable in the design of membranolytic peptides for other biochemical applications These include their ability to amplify cationic charges and hydrophobic clusters as the number of dendrimer branches increases Polycationic peptides, whether linear or branched, are known to display membrane disruption or fusion properties that have been exploited for intracellular peptide, protein and gene delivery [40–44] Hydrophobic clusters on a peptide dendrimer lead to aggregation that may enhance fusogenic activity A plausible mechanism is that amplifica-tion by a dendrimeric design increases the effective molarity
of monomeric units and decreases the entropy of self-assembly The end results are that they may mimic the mechanisms of action through which high-ordered anti-microbial peptides exert their membranolytic effects Thus, the short dendrimeric peptides may represent a useful and unusual biopolymer design for effecting various membrano-lyticactivities in lipid environments
Table 6 Antimicrobial activity of protegrin and tachyplesin
Experi-ment were performed in radial diffusion assay as described at Table 2.
Organism
MIC (l M ) Protegrin Tachyplesin L-salt H-salt L-salt H-salt Gram-negative
Gram-positive
Fungi
Trang 9A C K N O W L E D G E M E N T
This work was supported, in part, by US Public Health Service NIH
Grants CA36544 and AI46164.
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