Covalent inhibitors that do not rely on hijacking enzymatic activity have mainly been limited to those targeting cysteine residues. The development of such cysteine-directed covalent inhibitors has greatly profited from the use of competitive residue-specific proteomics to determine their proteome-wide selectivity. Several probes have been developed to monitor other amino acids using this technology, and many more electrophiles exist to modify proteins. Nevertheless, there has been a lack of direct, proteome-wide comparisons of the selectivity of diverse electrophiles. Here we developed an unbiased workflow to analyse electrophile selectivity proteome-wide and used it to directly compare 56 alkyne probes containing diverse reactive groups. In this way, we verified and identified probes to monitor a total of nine different amino acids, as well as the protein amino terminus, across the proteome.

Macromolecular coatings can improve the surface properties of many medical devices by enhancing their wetting behavior, tribological performance, and anti-biofouling properties - and covalent coatings produced from mucin glycoproteins have been shown to be very powerful in all those aspects. However, obtaining highly functional mucin glycoproteins is, at the moment, still a time-consuming process, which renders mucins rather expensive compared to other biomacromolecules. Here, we study a set of commercially available macromolecules that have the potential of substituting mucins in coatings for endotracheal tubes (ETTs). We present an overview of the different properties these macromolecular coatings establish on the ETT surface and whether they withstand storage or sterilization processes. Our study pinpoints several strategies of how to enhance the lubricity of ETTs by applying macromolecular coatings but also demonstrates the limited anti-biofouling abilities of well-established macromolecules such as hyaluronic acid, polyethylene glycol, and dextran. Based on the obtained results, we discuss to what extent those coatings can be considered equivalent alternatives to mucin coatings for applications on medical devices - their applicability does not have to be limited to ETTs, but could be broadened to catheters and endoscopes as well.

Glucosinolates (GSLs) are secondary metabolites produced as part of an herbivore defence system in plants of the order Brassicales. GSLs release isothiocyanates (ITCs) upon activation by the myrosinase. Beyond their herbivore feeding deterrent properties, these ITCs have multiple interesting bioactivities. However, their release is limited by the presence of myrosinase. Here, we report the concept of pseudoglucosinolates (psGSLs) hijacking the natural release mechanism of GSLs for the release of ITCs and adapting it to nitroreductase as the triggering enzymes. We provide proof-of-concept for nitroreductase-responsive psGSLs and demonstrate their potential for peptide labelling, ITC-prodrug approaches and for in vivo applications using the nematode of C. elegans.

With the idea of exploiting metal templated C-S bond forming reactions to achieve modification of cysteines in bacterial proteins, a cyclometalated Au(III) compound was explored in a competitive chemoproteomic approach in S. aureus cell extracts. More than 100 ligandable cysteines were identified, of which more than 50% were not engaged by organic α-chloroacetamides in a previous study, indicating that organometallic compounds expand the ligandable space in bacteria. A selected interaction was validated using an enzyme activity assay, and intact protein mass spectrometry showed cysteine arylation of an unprecedented target. The obtained results demonstrate that this family of organogold compounds has potential for therapeutic protein targeting via selective, covalent modification of cysteine residues in bacteria.

Targeted covalentinhibitors are powerful entities in drug discovery, but their application hasso far mainly been limited to addressing cysteine residues. The development ofcysteine-directed covalent inhibitors has largely profited from determiningtheir proteome-wide selectivity using competitive residue-specific proteomics. Severalprobes have recently been described to monitor other amino acids using thistechnology and many more electrophiles exist to modify proteins. Nevertheless,a direct, proteome‑wide comparison of the selectivity of diverse probes isstill entirely missing. Here, we developed a completely unbiased workflow to analyseelectrophile selectivity proteome‑wide and applied it to directly compare 54 alkyneprobes containing diverse reactive groups. In this way, we verified and newly identifiedprobes to monitor a total of nine different amino acids as well as the N‑terminusproteome‑wide. This selection includes the first probes to globally monitortryptophans, histidines and arginines as well as novel tailored probes formethionines, aspartates and glutamates.