Rational Design of Tryptophan‐Rich Antimicrobial Peptides with Enhanced Antimicrobial Activities and Specificities

Abstract

Trp-rich antimicrobial peptides play important roles in the host innate defense mechanism of many plants and animals. A series of short Trp-rich peptides derived from the C-terminal region of Bothrops asper myothoxin II, a Lys49 phospholipase A(2) (PLA(2)), were found to reproduce the antimicrobial activities of their parent molecule. Of these peptides, KKWRWWLKALAKK-designated PEM-2-was found to display improved activity against both Gram-positive and Gram-negative bacteria. To improve the antimicrobial activity of PEM-2 for potential clinical applications further, we determined the solution structure of PEM-2 bound to membrane-mimetic dodecylphosphocholine (DPC) micelles by two-dimensional NMR methods. The DPC micelle-bound structure of PEM-2 adopts an α-helical conformation and the positively charged residues are clustered together to form a hydrophilic patch. The surface electrostatic potential map indicates that two of the three tryptophan residues are packed against the peptide backbone and form a hydrophobic face with Leu7, Ala9, and Leu10. A variety of biophysical and biochemical experiments, including circular dichroism, fluorescence spectroscopy, and microcalorimetry, were used to show that PEM-2 interacted with negatively charged phospholipid vesicles and efficiently induced dye release from these vesicles, suggesting that the antimicrobial activity of PEM-2 could be due to interactions with bacterial membranes. Potent analogues of PEM-2 with enhanced antimicrobial and less pronounced hemolytic activities were designed with the aid of these structural studies.

Rational Design of Engineered Cationic Antimicrobial Peptides Consisting Exclusively of Arginine and Tryptophan, and Their Activity against Multidrug-Resistant Pathogens

“…1) by serial additions of one Arg in the hydrophilic face and one Trp in the hydrophobic face. As previously described, Arg and Trp were chosen because of their membrane perturbation tendencies (43,50,55,56,(58)(59)(60)(61). Amphipathic analysis revealed that the relative hydrophobic moments (Hrel), or the hydrophobic moments relative to a perfect amphipathic peptide, ranged from 0.93 (WR6) to 1.02 (WR16).…”

“…Further studies have shown that polyarginine provides a better membrane-transducing domain than polylysine (58,74). Taken together with earlier studies of the role of Trp as an enhancer of membrane perturbation in the context of a peptide motif (e.g., Trp-rich AMPs indolicidin and tritrpticin) (43,50,54,56), the design of the WR series was a logical next step from the engineering of the LBU series and WLBU2 with the intent to reduce the minimum length required for optimal antimicrobial activity (47,59). Liu et al previously examined a series of Arg-and Trp-based eCAPs for antimicrobial activity, but the peptide sequences were not optimized as idealized amphipathic motifs, and the peptide length was limited to 10 residues (75).…”

A High-Throughput Approach To Identify Compounds That Impair Envelope Integrity in Escherichia coli

“…Further in vitro and in vivo studies are warranted to evaluate a possible clinical application of these findings. Compounds 33 and 79 are both short antimicrobial peptides (13 and 15 residues in length, respectively); compound 79 exhibited 20% hemolysis of washed human red blood cells at 200 g/ml (ϳ80 M), while compound 33 was nonhemolytic to isolated sheep red blood cells on agar plates at a concentration of up to 500 M. Both peptides are expected to act as antimicrobials via a membraneassociated mechanism of action (43,44). The main difference between the two peptides is the tryptophan-rich face (assuming an ␣-helical active conformation) of compound 79, which increases its propensity to associate with the bacterial membrane, thereby disrupting its integrity.…”

Efficacy of a novel antimicrobial peptide against periodontal pathogens in both planktonic and polymicrobial biofilm states

“…The bacterial cytoplasmic membrane was less damaged observed in those treated by penicillin, metronidazole or chlorhexidine, indicating that Nal-P-113 results in higher permeability of the cytoplasmic membrane. We also investigated the membrane disruption capacity of Nal-P-113 by using unilamellar vesicles [45]. The bacterial membrane and mammalian cell membrane were mimicked with POPG alone and POPC/cholesterol (2:1), respectively.…”