Covalent peptidomimetic protease inhibitors have gained a lot of attention in drug development in recent years. They are designed to covalently bind the catalytically active amino acids through electrophilic groups called warheads. Covalent inhibition has an advantage in terms of pharmacodynamic properties but can also bear toxicity risks due to non-selective off-target protein binding. Therefore, the right combination of a reactive warhead with a well-suited peptidomimetic sequence is of great importance. Herein, the selectivities of well-known warheads combined with peptidomimetic sequences suited for five different proteases were investigated, highlighting the impact of both structure parts (warhead and peptidomimetic sequence) for affinity and selectivity. Molecular docking gave insights into the predicted binding modes of the inhibitors inside the binding pockets of the different enzymes. Moreover, the warheads were investigated by NMR and LC-MS reactivity assays against serine/threonine and cysteine nucleophile models, as well as by quantum mechanics simulations.

The aim of this study was to investigate the transition from non-covalent reversible over covalent reversible to covalent irreversible inhibition of cysteine proteases by making delicate structural changes to the warhead scaffold. To this end, dipeptidic rhodesain inhibitors with different N-terminal electrophilic arenes as warheads relying on the S_NAr mechanism were synthesized and investigated. Strong structure–activity relationships of the inhibition potency, the degree of covalency, and the reversibility of binding on the arene substitution pattern were found. The studies were complemented and substantiated by molecular docking and quantum-mechanical calculations of model systems. Furthermore, the improvement in the membrane permeability of peptide esters in comparison to their corresponding carboxylic acids was exemplified.

Trypsin-like serine proteases are involved in many important physiological processes like blood coagulation and remodeling of the extracellular matrix. On the other hand, they are also associated with pathological conditions. The urokinase-pwlasminogen activator (uPA), which is involved in tissue remodeling, can increase the metastatic behavior of various cancer types when overexpressed and dysregulated. Another member of this protease class that received attention during the SARS-CoV 2 pandemic is TMPRSS2. It is a transmembrane serine protease, which enables cell entry of the coronavirus by processing its spike protein. A variety of different inhibitors have been published against both proteases. However, the selectivity over other trypsin-like serine proteases remains a major challenge. In the current study, we replaced the arginine moiety at the P1 site of peptidomimetic inhibitors with different bioisosteres. Enzyme inhibition studies revealed that the phenylguanidine moiety in the P1 site led to strong affinity for TMPRSS2, whereas the cyclohexylguanidine derivate potently inhibited uPA. Both inhibitors exhibited high selectivity over other structurally similar and physiologically important proteases.

Fluorometric assays are one of the most frequently used methods in medicinal chemistry. Over the last 50 years, the reporter molecules for the detection of protease activity have evolved from first‐generation colorimetricp‐nitroanilides, through FRET substrates, and 7‐amino‐4‐methyl coumarin (AMC)‐based substrates. The aim of further substrate development is to increase sensitivity and reduce vulnerability to assay interferences. Herein, we describe a new generation of substrates for protease assays based on 7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl‐amides (NBD‐amides). In this study, we synthesized and tested substrates for 10 different proteases from the serine‐, cysteine‐, and metalloprotease classes. Enzyme‐ and substrate‐specific parameters as well as the inhibitory activity of literature‐known inhibitors confirmed their suitability for application in fluorometric assays. Hence, we were able to present NBD‐based alternatives for common protease substrates. In conclusion, these NBD substrates are not only less susceptible to common assay interference, but they are also able to replace FRET‐based substrates with the requirement of a prime site amino acid residue.

The Front Cover presents an experimental weapon changing the outcome of a battle in the respiratory system. The picture shows severe acute respiratory syndrome corona virus (SARS‐CoV) particles and the virus′ own papain‐like protease (PL^pro, PDB‐ID 3E9S) through the window of a cockpit. The essential protease is targeted and “bombarded” with the novel inhibitors shown on the screens. Hitting the protease leads to the destruction of the viruses. The corresponding article describes inhibitors developed for SARS‐CoV PL^pro and examines the transferability of their effectiveness to the close homologue of SARS‐CoV‐2. More information can be found in the Full Paper by Tanja Schirmeister et al.. Cover designed by Hannah and Lea Maus.