Elena Ilyina

Designed β-sheet-forming peptide 33mers with potent human bactericidal/permeability increasing protein-like bactericidal and endotoxin neutralizing activities

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.

Design of a potent novel endotoxin antagonist

BACKGROUND Bactericidal permeability increasing protein (BPI) binds to and neutralizes lipopolysaccharide (LPS, endotoxin). Small synthetic peptides based on the amino acid sequence of the LPS binding domain of BPI neutralize LPS, albeit inefficiently. Although the LPS binding domain of native BPI possesses a beta-turn secondary structure, this structure is not present in small derivative peptides. The purpose of this study was to determine whether the addition of a beta-turn to a BPI-derived peptide is associated with more potent endotoxin antagonism. METHODS We generated a hybrid peptide (BU3) on the basis of (1) a portion of the LPS binding domain from BPI and (2) amino acids known to initiate a beta-turn. BU3 folds with a beta-turn, and we tested its effects on LPS neutralization and LPS-induced tumor necrosis factor-alpha secretion, comparing it with BPI-derived peptide BG22 that lacks a beta-turn and to an irrelevant peptide (BG16). RESULTS Compared with BG22, BU3 demonstrated enhanced LPS neutralization and inhibition of LPS-induced tumor necrosis factor-alpha secretion in vitro and a similar diminution of endotoxemia and tumor necrosis factor-alpha secretion in a murine model of endotoxemia. CONCLUSIONS These data demonstrate the potential for enhancing the biologic activity of a BPI-derived peptide endotoxin antagonist via manipulation of its conformational structure.

Designing water soluble β-sheet peptides with compact structure

Publisher Summary β-sheet peptide design has proved more complicated primarily because of limited solubility via aggregation in water and to the nature of β-sheet folding that is dictated by long range interactions. The betabellin series and betadoublet peptides, for example, are soluble in water primarily at lower pH values and show non-compact β-sheet conformational properties as monitored by circular dichroism (CD) and nuclear magnetic resonance (NMR). A reason for problems in producing water-soluble, compact β-sheet peptides may lie in the fact that their design usually has been based on a number of structural propensity scales that are derived either statistically from structural databases of known folded proteins or by making single or minimal site-specific changes in a fully folded protein. Even though some β-hairpin and α/β peptides are water-soluble, form β-sheet structures and remain monomeric, larger de novo designed β-sheet- forming peptides, like betadoublet and betabellin, are inherently designed to self-associate through their otherwise solvent-exposed amphipathic hydrophobic surface. Other amphipathic β-sheet forming peptides have been derived from proteins in the α-chemokine family, including platelet factor-4 (PF4), interleukin-8 (IL-8), and growth related protein (Gro-α). The present communication summarizes a novel approach to de novo design of such water-soluble β-sheet sandwich peptides with compact structure. Analysis of the composition, folding, and solubility properties of PF4, IL-8, Gro-α 33mers and betadoublet and betabellin peptides has led to a general recipe for designing water soluble, β-sheet forming peptides.