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 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 mM or 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: [email protected]

Abbreviations: CHCA, a-cyano-4-hydroxycinnamic acid; DCC,

N,N-dicyclohexylcarbodiimide; DCM, dichloromethane; DIC,

N,N-diisopropylcarbodiimide; DIEA, N,N-diisopropylethylamine;

DMF, dimethylformamide; EC50, 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; Rt, 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