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Novel peptide 33mers have been designed by incorporating beta-conformation stabilizing residues from the beta-sheet domains of alpha-chemokines and functionally important residues from the beta-sheet domain of human neutrophil bactericidal protein (B/PI). B/PI is known for its ability to kill bacteria and to neutralize the action of bacterial endotoxin (lipopolysaccharide, LPS) which can induce septic shock leading to eventual death. Here, the goal was to make short linear peptides which demonstrate good beta-sheet folding and maintain bioactivity as in native B/PI. A library of 24 peptide 33mers (betapep-1 to betapep-24) were synthesized with various amino acid substitutions. CD and NMR data acquired in aqueous solution indicate that betapep peptides form beta-sheet structure to varying degrees and self-associate as dimers and tetramers like the alpha-chemokines. Bactericidal activity toward Gram-negative Pseudomonas aeruginosa was tested, and betapep-19 was found to be only about 5-fold less potent (62% kill at 1.2 x 10(-7) M) than native B/PI (80% kill at 2.9 x 10(-8) M). At LPS neutralization, betapep-2 and -23 were found to be most active (66-78% effective at 1.2 x 10(-6) M), being only about 50-100-fold less active than B/PI (50% at 1.5 x 10(-8) M). In terms of structure-activity relations, beta-sheet structural stability correlates with the capacity to neutralize LPS, but not with bactericidal activity. Although a net positive charge is necessary for activity, it is not sufficient for optimal activity. Hydrophobic residues tend to influence activities indirectly by affecting structural stability. Furthermore, results show that sequentially and spatially related residues from the beta-sheet domain of native B/PI can be designed into short linear peptides which show good beta-sheet folding and retain much of the native activity. This research contributes to the development of solutions to the problem of multiple drug-resistant, opportunistic microorganisms like P. aeruginosa and of agents effective at neutralizing bacterial endotoxin.

Human neutrophil bactericidal protein (B/PI) is known for its ability to kill bacteria and to neutralize the action of endotoxin. Short linear peptides derived from residues 80-109 have been synthesized and their bactericidal and endotoxin neutralizing activities have been assayed. A series of 'walk-through' decapeptides, overlapping 3 to 4 residues, indicates that endotoxin neutralizing and partial bactericidal activities can be localized within the N- and C-terminal portions, respectively, of the 80-109 sequence. Bactericidal activity toward Pseudomonas aeruginosa was localized in central peptides of the walk-through series and greatest in peptide 90-99. By using longer peptides, residues 86-104 and 82-108, both bactericidal and endotoxin neutralizing activities are significantly enhanced. Bactericidal activity of peptide 82-108 is now only 6-fold less than that of parent B/PI and 9-fold more potent than peptide 86-104. The 82-108 peptide was 7-fold more active at endotoxin neutralization than 86-104 but showed less enhanced activity, being approx. 470-times less active than B/PI. Cyclized 82-108 peptide retained bactericidal activity but did not improve in capacity to neutralize endotoxin.