Effect of truncation on antifungal properties of tetrabranched peptides.

Ocular fungal infections are worldwide health concern, resulting in a need to develop new drugs with greater effectiveness. B4010, a tetrabranched antifungal peptide that permeabilizes cytoplasmic membranes, was developed in Prof. Roger Beuerman’s lab. The aims of this project were to determine the effect of structural truncation on the antifungal properties of B4010 tested against five Candida albicans strains and evaluate their therapeutic potential for topical and systemic fungal infections. The effects of truncation were tested with minimum inhibitory concentration (MIC) and membrane permeability via SYTOX green assay. The therapeutic potential was determined via cell viability in 50% tear fluid and MIC in 5% sera. B4010 and its analogues (except B4010∆1-4) showed strong antifungal properties against all five strains. Further analysis demonstrated that the number of amino acids in the repeats contributes to antifungal potency. To maintain optimum antifungal properties, a minimum net charge of +20 and hydrophobicity of -2.1 kcal/mol is required. However, serum stability and candidacidal activity in tear fluid results suggested that shorter copy lengths may be better than longer analogues. This study has presented an optimization profile of B4010 and its analogues that would enable the development of a new class of therapeutically important antifungal compounds.Bachelor of Science in Biomedical Science

Branched Peptide, B2088, Disrupts the Supramolecular Organization of Lipopolysaccharides and Sensitizes the Gram-negative Bacteria

Dissecting the complexities of branched peptide-lipopolysaccharides (LPS) interactions provide rationale for the development of non-cytotoxic antibiotic adjuvants. Using various biophysical methods, we show that the branched peptide, B2088, binds to lipid A and disrupts the supramolecular organization of LPS. The disruption of outer membrane in an intact bacterium was demonstrated by fluorescence spectroscopy and checkerboard assays, the latter confirming strong to moderate synergism between B2088 and various classes of antibiotics. The potency of synergistic combinations of B2088 and antibiotics was further established by time-kill kinetics, mammalian cell culture infections model and in vivo model of bacterial keratitis. Importantly, B2088 did not show any cytotoxicity to corneal epithelial cells for at least 96 h continuous exposure or hemolytic activity even at 20 mg/ml. Peptide congeners containing norvaline, phenylalanine and tyrosine (instead of valine in B2088) displayed better synergism compared to other substitutions. We propose that high affinity and subsequent disruption of the supramolecular assembly of LPS by the branched peptides are vital for the development of non-cytotoxic antibiotic adjuvants that can enhance the accessibility of conventional antibiotics to the intracellular targets, decrease the antibiotic consumption and holds promise in averting antibiotic resistance.NRF (Natl Research Foundation, S’pore)ASTAR (Agency for Sci., Tech. and Research, S’pore)NMRC (Natl Medical Research Council, S’pore)Published versio

Electrostatic potential map on the adsorption of B4010 and B4010_R1A with bilayer.

<p>(A) POPC/POPE/POPS/Erg bilayer (B) B4010-adsorbed bilayer and (C) B4010_R1A-adsorbed bilayer. The negative and positive surfaces are labeled in red and blue, respectively whereas grey color indicates neutral surface.</p

MIC of synthetic linear and branched peptides against various yeasts and fungi.

a<p>Not determined.</p

Effects of various additives on candidacidal and membrane permeabilizing properties of B4010.

<p>(A) Effect of CCCP and NaN₃ on membrane potential. (B) Effect of various additives on (B) viability and (C) ATP release. (D) Effect of ion-channel inhibitors on membrane potential. The colored arrows indicate the time of addition of additives whereas the black arrows indicate B4010.</p

Interaction of B4010 with model membrane.

<p>(A) Time course of calcein release from SUVs of PC:PE:PS:erg and PC:cholesterol. The peptide:lipid ratio is indicated in the graphs. (B)-(D) Snapshots illustrating the interaction between B4010 and mixed bilayer containing ergosterol. The acyl chains of the aggregated POPC (grey), POPE (cyan), POPS (pink) and ergosterol (orange) are presented in line form. The peptide backbone is shown in ribbon form. (E) Translocation of water molecules (green) from inner leaflet to the outer as a consequence of membrane perturbations caused by B4010.</p

MIC of B4010 against <i>S. cerevisiae</i> mutants carrying altered sterol structure and composition.

a<p>Taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087730#pone.0087730-TeWelcher1" target="_blank">[29]</a>.</p

Schematic representation of the peptides used in this study.

<p>The branched lysine is colored in blue. For the scrambled peptide (Sc_B4010) each copy contains the sequence VRGRVRKR.</p