Rapid synthesis of internal peptidyl α-ketoamides by on resin oxidation for the construction of rhomboid protease inhibitors.

Rhomboid proteases are intramembrane serine proteases, which are involved in a wide variety of biological processes and have been implied in various human diseases. Recently, peptidyl α-ketoamides have been reported as rhomboid inhibitors with high potency and selectivity - owing to their interaction with both the primed and non-primed site of the target protease. However, their synthesis has been performed by solution phase chemistry. Here, we report a solid phase strategy towards ketoamides as rhomboid protease inhibitors, allowing rapid synthesis and optimization. We found that the primed site binding part of inhibitors is crucial for potency.

Correction: Rapid synthesis of internal peptidyl α-ketoamides by on resin oxidation for the construction of rhomboid protease inhibitors.

[This corrects the article DOI: 10.1039/D0RA10614C.].

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

Cyclic 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 ß-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.

Synthesis and Application of Activity-Based Probes for Proteases.

The detection, visualization, and identification of active proteases can be facilitated by activity-based probes, which covalently bind to a catalytic residue of the target protease. The synthesis of activity-based probes can be challenging. We here outline a simple protocol for probe synthesis based on standard solid phase peptide synthesis followed by capping of the N-terminus with a reactive electrophile as a warhead. The applicability of the probes is illustrated by labeling cysteine proteases in cell and tissue lysates with Western blotting or fluorescence scanning as a readout.

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.