Singlet oxygen-mediated one-pot chemoselective peptide–peptide ligation

We here describe a furan oxidation based site-specific chemical ligation approach using unprotected peptide segments. This approach involves two steps: after photooxidation of a furan-containing peptide, ligation is achieved by reaction of the unmasked keto-enal with C- or N-terminal alpha-nucleophilic moieties of the second peptide such as hydrazine or hydrazide to form a pyridazinium or pyrrolidinone linkage respectively

The optimal lipid chain length of a membrane-permeabilizing lipopeptide results from the balance of membrane partitioning and local damage

Pseudodesmin A (PSD) is a cyclic lipodepsipeptide produced by Pseudomonas that kills certain bacteria at MIC1/2 in the single micromolar range, probably by permeabilizing their cellular membranes. Synthetic PSD variants, where the native decanoic (C10) acyl chain is varied in length from C4 to C8 and C12 to C14 carbons, were described to be not or less active against a panel of gram-positive strains, as compared to native PSD-C10. Here, we test the membrane-permeabilizing activity of PSD-C4 through PSD-C14 in terms of calcein release from liposomes, which is characterized in detail by the fluorescence-lifetime based leakage assay. Antagonistic concentrations and their chain length dependence agree well for liposome leakage and antimicrobial activity. The optimal chain length is governed by a balance between membrane partitioning (favoring longer chains) and the local perturbation or "damage" inflicted by a membrane-bound molecule (weakening for longer chains). Local perturbation, in turn, may involve at least two modes of action. Asymmetry stress between outer and inner leaflet builds up as the lipopeptides enter the outer leaflet and when it reaches a system-specific stability threshold, it causes a transient membrane failure that allows for the flip of some molecules from the outer to the inner leaflet. This cracking-in may be accompanied by transient, incomplete leakage from the aqueous cores of the liposomes observed, typically, for some seconds or less. The mismatch of the lipopeptide with the lipid leaflet geometry, expressed for example in terms of a spontaneous curvature, has two effects. First, it affects the threshold for transient leakage as described. Second, it controls the rate of equilibrium leakage proceeding as the lipopeptide has reached sufficient local concentrations in both leaflets to form quasi-toroidal defects or pores. Both modes of action, transient and equilibrium leakage, synergize for intermediate chain lengths such as the native, i.e., for PSD-C10. These mechanisms may also account for the reported chain-length dependent specificities of antibiotic action against the target bacteria.</p

Exploration of CLP antitumor potential

Cyclic lipodepsipeptides (CLPs) are a class of secondary metabolites typically produced by Pseudomonas and Bacillus with interesting biological properties These amphiphilic compounds are composed of an oligopeptide chain, C-terminally cyclized through macrolactonization and N-terminally linked to a lipid tail. They exhibit substantial structural diversity as direct consequence of variation in amino acid composition, including modified proteinogenic, non-proteinogenic and D-amino acids, and the length and degree of saturation of the fatty acid moiety. CLPs receive considerable attention due to their broad range of biological activities including antibiotic, antiviral, antifungal and insecticidal properties. More recently, anticancer activity has been discovered as well. In spite of their potential in clinical and agricultural context, the molecular mechanism of action of most CLPs is yet to be determined, although it is clear that the cell membrane is an interaction partner and possibly the primary target. In order to exploit the promising properties of these compounds, establishment of their structure-activity relationship and a good understanding of their interaction with biological membranes at the molecular level, will be fundamental. Towards this, the site-specific and selective modification of CLPs for generation of molecular probes capable of exposing the mechanism of action represents an essential step forward.Over the last few years, considerable knowledge and expertise have been acquired when it comes to isolation, characterisation and synthesis of CLPs, the latter with emphasis on members of the viscosin group. For instance, a rapid and efficient total chemical synthesis strategy has been developed and optimized which already resulted in the successful synthesis of more than 30 compounds such as viscosin, pseudodesmin, WLIP and a wide variety of analogues thereof.[1–3] In addition, an NMR and modelling methodology was developed allowing a detailed understanding of CLP structure, conformational and self-assembly behaviour.[4–6] The unique combination of both expertise areas enables us to try and grasp the broader picture of CLP antitumor activity and tackle the problem of target identification and elucidation of the molecular events at a cellular level. References [1] M. De Vleeschouwer, J. C. M. and A. M. Davy Sinnaeve, Jos Van den Begin, Tom Coenye, Chem. - A Eur. J. 2014, 79, 1614–1621. [2] M. De Vleeschouwer, J. C. Martins, A. Madder, J. Pept. Sci. 2016, 22, 149–155. [3] M. De Vleeschouwer, D. Sinnaeve, N. Matthijs, T. Coenye, 2017, 640–644. [4] D. Sinnaeve, P. M. S. Hendrickx, J. Van Hemel, E. Peys, B. Kieffer, J. C. Martins, Chem. - A Eur. J. 2009, 15, 12653–12662. [5] N. Geudens, M. De Vleeschouwer, K. Fehér, H. Rokni-Zadeh, M. G. K. Ghequire, A. Madder, R. De Mot, J. C. Martins, D. Sinnaeve, ChemBioChem 2014, 15, 2736–2746. [6] N. Geudens, M. N. Nasir, J.-M. Crowet, J. M. Raaijmakers, K. Fehér, T. Coenye, J. C. Martins, L. Lins, D. Sinnaeve, M. Deleu, Biochim. Biophys. Acta - Biomembr. 2017, 1859, 331–33

Expanding the toolbox : use of biosensing technology to elucidate the mode of action of cyclic lipodepsipeptides

C

Identification of the Molecular Determinants Involved in Antimicrobial Activity of Pseudodesmin A, a Cyclic Lipopeptide From the Viscosin Group

International audienceCyclic lipo(depsi)peptides (CLiPs) from Pseudomonas constitute a class of natural products involved in a broad range of biological functions for their producers. They also display interesting antimicrobial potential including activity against Gram-positive bacteria. Literature has indicated that these compounds can induce membrane permeabilization, possibly through pore-formation, leading to the general view that the cellular membrane constitutes the primary target in their mode of action. In support of this view, we previously demonstrated that the enantiomer of pseudodesmin A, a member of the viscosin group of CLiPs, shows identical activity against a test panel of six Gram-positive bacterial strains. Here, a previously developed total organic synthesis route is used and partly adapted to generate 20 novel pseudodesmin A analogs in an effort to derive links between molecular constitution, structure and activity. From these, the importance of a macrocycle closed by an ester bond as well as a critical length of β-OH fatty acid chain capping the N-terminus is conclusively demonstrated, providing further evidence for the importance of peptide-membrane interactions in the mode of action. Moreover, an alanine scan is used to unearth the contribution of specific amino acid residues to biological activity. Subsequent interpretation in terms of a structural model describing the location and orientation of pseudodesmin A in a membrane environment, allows first insight in the peptide-membrane interactions involved. The biological screening also identified residue positions that appear less sensitive to conservative modifications, allowing the introduction of a non-perturbing tryptophan residue which will pave the way toward biophysical studies using fluorescence spectroscopy