N-Acylated and d Enantiomer Derivatives of a Nonamer Core Peptide of Lactoferricin B Showing Improved Antimicrobial Activity

Abstract

N-acylated or d enantiomer peptide derivatives based on the sequence RRWQWRMKK in lactoferricin B demonstrated antimicrobial activities greater than those of lactoferricin B against bacteria and fungi. The most potent peptide, conjugated with an 11-carbon-chain acyl group, showed two to eight times lower MIC than lactoferricin B.

Lactoferricin B (LFcin B) is a 25-mer antimicrobial peptide generated by pepsin digestion of bovine lactoferrin (LF) and is more potent than lactoferricin H (LFcin H), a 47-mer peptide similarly derived from human LF (3). LFcin B shows activity against a wide range of microorganisms including bacteria and fungi (2, 15), and its mode of action is microbicidal via membrane perturbation (4, 15). The peptide shows synergistic actions with other antimicrobial agents such as azole antifungal agents (13, 14). Nuclear magnetic resonance studies have revealed that the three-dimensional structure of LFcin B is an antiparallel β-sheet and that hydrophilic and positively charged residues surround the hydrophobic surface, suggesting interactions of this peptide with the biological membrane (9). Although several LFcin analogs with amino acid sequences shorter than LFcin B or LFcin H have been reported, their antimicrobial potency seems to be similar to or weaker than that of the original LFcin (5, 7, 10, 11). Therefore, this study was performed to produce more potent LFcin analogs and demonstrate improved antimicrobial activities displayed by N-acylated or d enantiomer analogs of nonamer peptides based on a core sequence of LFcin B.

Preparation of peptides.

The structures of the LFcin-related peptides and reference antimicrobial peptides used in this study are shown in Table 1. LFcin B was purified from a pepsin hydrolysate of bovine LF as described previously (3). (Ala^8,13,18)-magainin II-NH₂ (6) was purchased from Sigma Chemical Co. (St. Louis, Mo.). Other peptides were chemically synthesized on a 4-(2′,4′-dimethoxyphenyl-9-fluorenylmethoxycarbonyl-aminomethyl) phenoxyacetamido-ethyl resin or preloaded 4-(hydroxymethyl)phenoxymethyl-copoly(styrene-1% divinylbenzene) (HMP) resin (Perkin-Elmer Japan Co. Applied Biosystems Division, Chiba, Japan) by a 9-fluorenylmethoxycarbonyl strategy and solid-phase methodology by means of an automated synthesizer (model 433A; Perkin Elmer). For N-acylation of the peptides, the resin was treated with the desired fatty acid activated in situ as an HOBt/HBTU ester. After removal of the resin and the side-chain-protecting groups, the crude peptides were purified by reverse-phase high-performance liquid chromatography. The structures of the final compounds were confirmed by mass-spectral analysis (LCQ, Finnigan Mat). All peptides including N-acylated ones were soluble in water.

TABLE 1.

Structures of the peptide derivatives used in this study

Peptide name	Structure
LFcin B	FKCRRWQWRMKKLGAPSITCVRRAF
LFcin H(25mer)	TKCFQWQRNMRKVRGPPVSCIKRDS
(4-12)-NH2	RRWQWRMKK-NH2
Acyl-6-(4-12)-NH2	CH3(CH2)4CO-RRWQWRMKK-NH2
Acyl-10-(4-12)-NH2	CH3(CH2)8CO-RRWQWRMKK-NH2
Acyl-11-(4-12)-NH2	CH3(CH2)9CO-RRWQWRMKK-NH2
Acyl-14-(4-12)-NH2	CH3(CH2)12CO-RRWQWRMKK-NH2
Acyl-16-(4-12)-NH2	CH3(CH2)14CO-RRWQWRMKK-NH2
Acyl-18-(4-12)-NH2	CH3(CH2)16CO-RRWQWRMKK-NH2
d-(4-12)-NH2	d-(RRWQWRMKK)-NH2c
Acyl-10-d-(4-12)-NH2	CH3(CH2)8CO-d-(RRWQWRMKK)-NH2c
Ac-RRWWCR-NH2a	CH3CO-RRWWCR-NH2
(Ala8,13,18)-magainin II-NH2b	GIGKFLHAALLFAKAFVAEIMNS-NH2

^aSee reference 8. 

^bSee reference 6. 

^cThese are the d enantiomer derivatives of the peptides. 

Microorganisms tested.

Escherichia coli IID861, Pseudomonas aeruginosa IFO3445, and Staphylococcus aureus JCM2151 (ATCC 6538P) were maintained on plate count agar (Eiken Chemicals Co., Tokyo, Japan) slants. The stock cultures were harvested in 1% Bacto Peptone (Difco Laboratories Co., Detroit, Mich.) and incubated for 7 h at 37°C to obtain inocula. Candida albicans TIMM0144 and Trichophyton mentagrophytes TIMM1189 were grown on Sabouraud glucose agar (1% Bacto Peptone, 2% glucose, 1.5% agar) slants at 27°C before use.

Antimicrobial susceptibility testing.

MICs were determined by the microdilution method using flat-bottom 96-well microplates. As culture media, 1% Bacto Peptone or Mueller-Hinton broth (Difco) was used for bacteria and Sabouraud glucose broth (1% Bacto Peptone, 2% glucose) was used for fungi. Bacterial cells were inoculated into microplates at a final cell density of 10⁶ cells/ml and incubated for 17 h at 37°C. C. albicans cells were harvested from a slant, suspended in the culture medium, inoculated into microplates at 10⁵ cells/ml, and incubated for 17 h at 37°C. Microconidia of T. mentagrophytes were harvested from a slant in saline solution containing 0.85% NaCl and 0.05% Tween 80, suspended in the culture medium, inoculated into microplates at 10⁴ cells/ml, and incubated for 4 days at 27°C. The MIC for triplicate samples was determined.

The MICs of LFcin-related peptides for the five microorganisms in peptone-based broth are shown in Table 2. LFcin H(25mer), which has an amino acid sequence homologous to that of LFcin B, was substantially less active than LFcin B against all microorganisms tested. We synthesized several peptide derivatives based on the cationic-amino-acid-rich region RRWQWRMKK (corresponding to positions R4 through K12) of LFcin B and assayed the antimicrobial activity of each. N-acyl derivative peptides having a carbon chain length of six to eleven carbons showed similar or higher activities compared with LFcin B. The most active peptide had an 11-carbon acyl chain and exhibited two- to four-times-lower MIC than LFcin B. d-(4-12)-NH₂, a d enantiomer derivative of (4-12)-NH₂, was also highly active and showed MIC similar to that of Acyl-11-(4-12)-NH₂. N-acylation, however, did not further augment the activity of the d enantiomeric peptide. Ac-RRWWCR-NH₂ was previously identified as the most potent hexameric antimicrobial peptide in synthetic peptide combinatorial libraries (8) and showed sequence similarity to the RRWQWR region in LFcin B. This peptide showed slightly lower activity than LFcin B. (Ala^8,13,18)-magainin II-NH₂ is a peptide consisting of 23 amino acid residues reported to display antimicrobial activity up to two orders of magnitude stronger than that of the original magainin II (6). The activity of this analog was similar to that of Acyl-11-(4-12)-NH₂. In tests using Mueller-Hinton broth, in which the activity of LFcin B, especially against gram-negative bacteria, was decreased, improvement of antimicrobial activities and carbon chain length-dependent activities for N-acylated analogs were clearly indicated (Table 3). Acyl-10-(4-12)-NH₂ and Acyl-11-(4-12)-NH₂ showed four- to eight-times-lower MIC than LFcin B.

TABLE 2.

In vitro antimicrobial activity of LFcin-derived peptides in peptone-based broth

Agent	MIC (μg/ml)
	E. coli	P. aeruginosa	S. aureus	C. albicans	T. mentagrophytes
LFcin B	6	12	12	25	12
LFcin H(25mer)	25	50	25	100	50
(4-12)-NH2	6	25	6	25	6
Acyl-6-(4-12)-NH2	3	25	6	25	6
Acyl-10-(4-12)-NH2	3	6	6	25	6
Acyl-11-(4-12)-NH2	3	6	3	12	6
Acyl-14-(4-12)-NH2	6	12	12	25	12
Acyl-16-(4-12)-NH2	6	12	12	50	12
Acyl-18-(4-12)-NH2	6	25	12	100	12
d-(4-12)-NH2	3	12	3	6	6
Acyl-10-d-(4-12)-NH2	3	6	3	12	6
Ac-RRWWCR-NH2	12	25	12	50	6
(Ala8,13,18)-magainin II-NH2	3	6	3	25	12

TABLE 3.

In vitro antimicrobial activity of LFcin-derived peptides in Mueller-Hinton broth

Agent	MIC (μg/ml)
	E. coli	P. aeruginosa	S. aureus
LFcin B	50	>200	25
LFcin H(25mer)	>200	>200	>200
Acyl-6-(4-12)-NH2	100	>200	100
Acyl-10-(4-12)-NH2	6	50	6
Acyl-11-(4-12)-NH2	12	50	3
Acyl-14-(4-12)-NH2	12	50	12
Acyl-16-(4-12)-NH2	25	50	12
Acyl-18-(4-12)-NH2	50	100	25
d-(4-12)-NH2	50	>200	12
Acyl-10-d-(4-12)-NH2	12	100	6
Ac-RRWWCR-NH2	100	>200	25

N-acylation resulted in an increase in the interaction of LFcin B analogs with microbial membranes and may have caused enhanced antimicrobial activity. Appropriate amino acid substitution is known to increase the activity of antimicrobial peptides (6, 10). The N-acyl derivatization approach using a relatively short peptide, however, can simplify the peptide synthesis process compared to synthesizing larger peptides. Although Ca²⁺ or Mg²⁺ ions (>2 mM) are known to reduce the activity of LFcin B (2), these ions were not likely involved in the increased MIC of the peptide in Mueller-Hinton broth, because the concentrations of Ca²⁺ and Mg²⁺ in Mueller-Hinton broth were low (0.15 and 0.21 mM, respectively) and similar to those in 1% Bacto Peptone. Other constituents may be responsible for the diminished activity of LFcin B in Mueller-Hinton broth, and N-acylated derivatives may overcome such blocking effects of medium ingredients. d enantiomer peptides can survive digestion by proteases secreted by microorganisms. This and previous studies (1, 5, 12) indicate advantages of d enantiomer derivatives in antimicrobial applications.

The hemolysis activity of the 11-mer LFcin B analog is reported to be very low (10), and d-(4-12)-NH₂ did not exert a toxic effect against animal cells at concentrations active for the inhibition of microorganisms in our preliminary evaluation. Oral application is a possible route for the therapeutic uses of the derived compounds, because oral administration of the N-acylated and d enantiomer derivatives in mice resulted in no lethal toxicity at doses as high as 100 mg/kg of body weight (unpublished data).