Cathepsin S (CatS) is a secreted cysteine protease that cleaves certain extracellular matrix proteins, regulates antigen presentation in antigen-presenting cells (APC), and promotes M2-type macrophage and dendritic cell polarization. CatS is overexpressed in many solid cancers, and overall, it appears to promote an immune-suppressive and tumor-promoting microenvironment. While most data suggest that CatS inhibition or knockdown promotes anti-cancer immunity, cell-specific inhibition, especially in myeloid cells, appears to be important for therapeutic efficacy. This makes the design of CatS selective inhibitors and their targeting to tumor-associated M2-type macrophages (TAM) and DC an attractive therapeutic strategy compared to the use of non-selective immunosuppressive compounds or untargeted approaches. The selective inhibition of CatS can be achieved through optimized small molecule inhibitors that show good pharmacokinetic profiles and are orally bioavailable. The targeting of these inhibitors to TAM is now more feasible using nanocarriers that are functionalized for a directed delivery. This review discusses the role of CatS in the immunological tumor microenvironment and upcoming possibilities for a nanocarrier-mediated delivery of potent and selective CatS inhibitors to TAM and related APC to promote anti-tumor immunity.

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 cysteine protease cathepsin S (CatS) is overexpressed in many tumors. It is known to be involved in tumor progression as well as antigen processing in antigen‐presenting cells (APC). Recent evidence suggests that silencing CatS improves the anti‐tumor immune response in several cancers. Therefore, CatS is an interesting target to modulate the immune response in these diseases. Here, we present a series of covalent‐reversible CatS inhibitors based on the α‐fluorovinylsulfone and ‐sulfonate warheads. We optimized two lead structures by molecular docking approaches, resulting in 22 final compounds which were evaluated in fluorometric enzyme assays for CatS inhibition and for selectivity towards the off‐targets CatB and CatL. The most potent inhibitor in the series has subnanomolar affinity (K_i=0.08 nM) and more than 100,000‐fold selectivity towards cathepsins B and L. These new reversible and non‐cytotoxic inhibitors could serve as interesting leads to develop new immunomodulators in cancer therapy.

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.