Synthesis, biophysical and functional studies of two BP100 analogues modified by a hydrophobic chain and a cyclic peptide

Abstract

Antimicrobial peptides (AMPs) work as a primary defense against pathogenic microorganisms. BP100, (KKLFKKILKYL-NH), a rationally designed short, highly cationic AMP, acts against many bacteria, displaying low toxicity to eukaryotic cells. Previously we found that its mechanism of action depends on membrane surface charge and on peptide-to-lipid ratio. Here we present the synthesis of two BP100 analogs: BP100‑alanyl‑hexadecyl‑1‑amine (BP100-Ala-NH-CH) and cyclo(1‑4)‑d‑Cys, Ile, Leu, Cys-BP100 (Cyclo(1‑4)‑cILC-BP100). We examined their binding to large unilamellar vesicles (LUV), conformational and functional properties, and compared with those of BP100. The analogs bound to membranes with higher affinity and a lesser dependence on electrostatic forces than BP100. In the presence of LUV, BP100 and BP100-Ala-NH-CH acquired α-helical conformation, while Cyclo(1‑4)‑cILC-BP100) was partly α-helical and partly β-turn. Taking in conjunction: 1. particle sizes and zeta potential, 2. effects on lipid flip-flop, 3. leakage of LUVs internal contents, and 4. optical microscopy of giant unilamellar vesicles, we concluded that at high concentrations, all three peptides acted by a carpet mechanism, while at low concentrations the peptides acted by disorganizing the lipid bilayer, probably causing membrane thinning. The higher activity and lesser membrane surface charge dependence of the analogs was probably due to their greater hydrophobicity. The MIC values of both analogs towards Gram-positive and Gram-negative bacteria were similar to those of BP100 but both analogues were more hemolytic. Confocal microscopy showed Gram-positive B. subtilis killing with concomitant extensive membrane damage suggestive of lipid clustering, or peptide-lipid aggregation. These results were in agreement with those found in model membranes.

Binding and Flip as Initial Steps for BP-100 Antimicrobial Actions

“…BP100 mode of action on membranes is dependent on peptide/lipid ratio 11,47 . As our studies comprised of low peptide/lipid concentration (1/128), we suggest that at low peptide/lipid concentrations, BP100 causes lipid aggregation on negatively charged membranes, leading to phase boundary defects between negatively charged lipid rafts and the rest of the membrane.…”

Sarconesin: Sarconesiopsis magellanica Blowfly Larval Excretions and Secretions With Antibacterial Properties

“…For Esterase activity, 180 μL were transferred to a 96-well black plate which was added 20 μL of 250 μM 5(6)-Carboxyfluorescein diacetate (CFDA), incubated in dark for 30 min, followed by measurement of fluorescence with excitation/emission wavelengths of 485/535 nm ( Nocker et al, 2011 ; Yang et al, 2017 ). PI microscope slides, were made by depositing drops of melted agarose 1% (w/v); after placing 20 μL of the cells onto solidified agar pad for immobilisation, the dried culture was covered with a glass coverslip and observed under a microscope ( Carretero et al, 2018 ). Microscopy was performed using a Leica TCS SP8 confocal laser scanning microscope, the images were processed with Leica software LAS X.…”

Dissecting the mechanism of action of actinoporins. Role of the N-terminal amphipathic α-helix in membrane binding and pore activity of sticholysins I and II

“…Total bilayer disruption, as in the carpet mechanism of action of bio-active peptides [ 47 ] would also result in lipid mixing between the two leaflets. Indeed, this was found in a study with the multi-functional peptide BP100 and two of its hydrophobic analogues [ 48 ]. Lipid mixing was observed; however, the kinetics of this process was different from that of leakage experiments.…”