A Lysosome-Targeted Tetrazine for Organelle-Specific Click-to-Release Chemistry in Antigen Presenting Cells.

Bioorthogonal deprotections are readily used to control biological function in a cell-specific manner. To further improve the spatial resolution of these reactions, we here present a lysosome-targeted tetrazine for an organelle-specific deprotection reaction. We show that trans-cyclooctene deprotection with this reagent can be used to control the biological activity of ligands for invariant natural killer T cells in the lysosome to shed light on the processing pathway in antigen presenting cells. We then use the lysosome-targeted tetrazine to show that long peptide antigens used for CD8+ T cell activation do not pass through this organelle, suggesting a role for the earlier endosomal compartments for their processing.

Methyltetrazine as a small live-cell compatible bioorthogonal handle for imaging enzyme activities in situ.

Bioorthogonal chemistry combines well with activity-based protein profiling, as it allows for the introduction of detection tags without significantly influencing the physiochemical and biological functions of the probe. In this work, we introduced methyltetrazinylalanine (MeTz-Ala), a close mimic of phenylalanine, into a dipeptide fluoromethylketone cysteine protease inhibitor. Following covalent and irreversible inhibition, the tetrazine allows vizualisation of the captured cathepsin activity by means of inverse electron demand Diels Alder ligation in cell lysates and live cells, demonstrating that tetrazines can be used as live cell compatible, minimal bioorthogonal tags in activity-based protein profiling.

Facile Synthesis of Aminomethyl Phosphinate Esters as Serine Protease Inhibitors with Primed Site Interaction.

Serine proteases comprise about one-third of all proteases, and defective regulation of serine proteases is involved in numerous diseases. Therefore, serine protease inhibitors are promising drug candidates. Aminomethyl diphenyl phosphonates have been regularly used as scaffolds for covalent serine protease inhibition and the design of activity-based probes. However, they cannot make use of a protease's primed site. Therefore, we developed a facile two-step synthesis toward a set of phenyl phosphinates, which is a related scaffold but can interact with the primed site. We tested their inhibitory activity on five different serine proteases and found that a phenyl group directly attached to the phosphorus atom leads to superior activity compared with phosphonates.

Rapid Solid-Phase Construction of Serine Hydrolase Probes Results in Selective Activity-Based Probes for Acyl Protein Thioesterases-1/2.

Serine hydrolases (SHs) are a large, diverse family of enzymes that play various biomedically important roles. Their study has been substantially advanced by activity-based protein profiling, which makes use of covalent chemical probes for labeling the active site and detection by various methodologies. However, highly selective probes for individual SHs are scarce because probe synthesis usually takes place by time-consuming solution phase chemistry. We here report a general solid-phase synthesis toward SH chemical probes, which will speed up probe library synthesis. It involves the construction of a recognition element ending in a secondary amine followed by capping with different electrophiles. We illustrate the power of this approach by the discovery of selective chemical probes for the depalmitoylating enzymes APT-1/2. Overall, this study reports new methodologies to synthesize SH probes, while providing new reagents to study protein depalmitoylation.

Peptidyl Acyloxymethyl Ketones as Activity-Based Probes for the Main Protease of SARS-CoV-2*.

The global pandemic caused by SARS-CoV-2 calls for the fast development of antiviral drugs against this particular coronavirus. Chemical tools to facilitate inhibitor discovery as well as detection of target engagement by hit or lead compounds from high-throughput screens are therefore in urgent need. We here report novel, selective activity-based probes that enable detection of the SARS-CoV-2 main protease. The probes are based on acyloxymethyl ketone reactive electrophiles combined with a peptide sequence including unnatural amino acids that targets the nonprimed site of the main protease substrate binding cleft. They are the first activity-based probes for the main protease of coronaviruses and display target labeling within a human proteome without background. We expect that these reagents will be useful in the drug-development pipeline, not only for the current SARS-CoV-2, but also for other coronaviruses.

Use of Non-Natural Amino Acids for the Design and Synthesis of a Selective, Cell-Permeable MALT1 Activity-Based Probe.

Constitutive proteolytic activity of MALT1 is associated with highly aggressive B-cell lymphomas. Chemical tools that detect active MALT1 have been reported, but suffer from poor cell permeability and/or cross-reactivity with the cysteine protease cathepsin B. Here, we report that the non-natural amino acid pipecolinic acid in the P2 position of substrates and chemical probes leads to improved selectivity toward MALT1 and results in cell-permeable fluorescent probes.

Recent Advances in Activity-Based Protein Profiling of Proteases.

The activity of proteases is tightly regulated, and dysregulation is linked to a variety of human diseases. For this reason, ABPP is a well-suited method to study protease biology and the design of protease probes has pushed the boundaries of ABPP. The development of highly selective protease probes is still a challenging task. After an introduction, the first section of this chapter discusses several strategies to enable detection of a single active protease species. These range from the usage of non-natural amino acids, combination of probes with antibodies, and engineering of the target proteases. A next section describes the different types of detection tags that facilitate the read-out possibilities including various types of imaging methods and mass spectrometry-based target identification. The power of protease ABPP is illustrated by examples for a selected number of proteases. It is expected that some protease probes that have been evaluated in animal models of human disease will find translation into clinical application in the near future.