Chemical Tools for the Study of Intramembrane Proteases

Intramembrane proteases (IMPs) reside inside lipid bilayers and perform peptide hydrolysis in transmembrane or juxtamembrane regions of their substrates. Many IMPs are involved in crucial regulatory pathways and human diseases, including Alzheimer's disease, Parkinson's disease, and diabetes. In the past, chemical tools have been instrumental in the study of soluble proteases, enabling biochemical and biomedical research in complex environments such as tissue lysates or living cells. However, IMPs place special challenges on probe design and applications, and progress has been much slower than for soluble proteases. In this review, we will give an overview of the available chemical tools for IMPs, including activity-based probes, affinity-based probes, and synthetic substrates. We will discuss how these have been used to increase our structural and functional understanding of this fascinating group of enzymes, and how they might be applied to address future questions and challenges.status: publishe

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

Short Peptides with Uncleavable Peptide Bond Mimetics as Photoactivatable Caspase-3 Inhibitors

Chemical probes that covalently interact with proteases have found increasing use for the study of protease function and localization. The design and synthesis of such probes is still a bottleneck, as the strategies to target different families are highly diverse. We set out to design and synthesize chemical probes based on protease substrate specificity with inclusion of an uncleavable peptide bond mimic and a photocrosslinker for covalent modification of the protease target. With caspase-3 as a model target protease, we designed reduced amide and triazolo peptides as substrate mimetics, whose sequences can be conveniently constructed by modified solid phase peptide synthesis. We found that these probes inhibited the caspase-3 activity, but did not form a covalent bond. It turned out that the reduced amide mimics, upon irradiation with a benzophenone as photosensitizer, are oxidized and form low concentrations of peptide aldehydes, which then act as inhibitors of caspase-3. This type of photoactivation may be utilized in future photopharmacology experiments to form protease inhibitors at a precise time and location.status: publishe

Short Peptides with Uncleavable Peptide Bond Mimetics as Photoactivatable Caspase-3 Inhibitors

Chemical probes that covalently interact with proteases have found increasing use for the study of protease function and localization. The design and synthesis of such probes is still a bottleneck, as the strategies to target different families are highly diverse. We set out to design and synthesize chemical probes based on protease substrate specificity with inclusion of an uncleavable peptide bond mimic and a photocrosslinker for covalent modification of the protease target. With caspase-3 as a model target protease, we designed reduced amide and triazolo peptides as substrate mimetics, whose sequences can be conveniently constructed by modified solid phase peptide synthesis. We found that these probes inhibited the caspase-3 activity, but did not form a covalent bond. It turned out that the reduced amide mimics, upon irradiation with a benzophenone as photosensitizer, are oxidized and form low concentrations of peptide aldehydes, which then act as inhibitors of caspase-3. This type of photoactivation may be utilized in future photopharmacology experiments to form protease inhibitors at a precise time and location

Influence of CLP structure on antitumor potential

Cyclic lipodepsipeptides (CLPs) are generally produced by bacteria such as Pseudomonas and Bacillus as secondary metabolites. These amphiphilic compounds consist of an oligopeptide chain where a fatty acid moiety is linked to the N-terminus and cyclization occurs via ester-bond formation that involves the C-terminus. Although they have this quite characteristic molecular blueprint, substantial structural diversity can be observed between and within the existing groups. This is mainly as a 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. Besides this high diversity in chemical features, CLPs also exhibit a broad range of diverse biological functions including antimicrobial, antifungal and antiviral activity. More recently, for some CLPs including viscosin, antitumoral activity has been described as well. Despite their potential in clinical and agricultural context, there is not much known about their mechanism of action – for Pseudomonas CLPs in particular – and the corresponding structure-activity relation of CLPs. 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[1][2] and an NMR and modelling methodology was established allowing a detailed understanding of CLP structure and conformation.[3][4] In this presentation, both expertise areas are combined in order to explore how differences in structure and composition of various CLPs may lead to variations in their antitumoral activity. References [1] M. De Vleeschouwer, D. Sinnaeve, J. Van den Begin, T. Coenye, J. C. Martins, A. Madder; 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] 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, 331–339. [4] 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