Mechanism of interaction of optimized Limulus-derived cyclic peptides with endotoxins: thermodynamical, biophysical and microbiological analysis

International audience; On the basis of formerly investigated peptides corresponding to the endotoxin-binding domain from the Limulus anti-lipopolysaccharide factor (LALF), a protein from L. polyphemus, we have designed and synthesized peptides of different lengths with the aim to get potential therapeutic agents against the septic shock syndrom. For an understanding of the mechanisms of action, we performed a profound physico-chemical and biophysical analysis of the interaction of rough mutant lipopolysaccharide (LPS) with these peptides by applying Fourier-transform infrared spectroscopy (FTIR), small-angle X-ray scattering (SAXS), calorimetric techniques (differential scanning calorimetry DSC and isothermal titration calorimetry ITC), and freeze-fracture transmission electron microscopy (FFTEM). Also, the action of the peptides on bacteria of different origin in microbial assays was investigated. Using FTIR and DSC, our data indicated a strong fluidization of the lipid A acyl chains due to peptide binding, with a decrease of the endothermic melting enthalpy change of the acyl chains down to a complete disappearance in the 1:0.5 to 1:2 [LPS]:[peptide] molar ratio range. Via ITC, it was deduced that the binding is a clearly exothermic process which runs into saturation also at a [LPS]:[peptide] = 1:0.5 to 1:2 molar ratio. The data obtained with SAXS indicated a drastic change of the aggregate structures of LPS into a multilamellar stack, which was visualized in electron micrographs as hundreds of lamellar layers. This can be directly correlated with the inhibition of the LPS-induced production of tumor-necrosis-factor-α in human mononuclear cells, but not with the action of the peptides on bacteria.