Next Generation of Fluorometric Protease Assays: 7-Nitrobenz-2-oxa-1,3-diazol-4-yl-amides (NBD-Amides) as Class-Spanning Protease Substrates.

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 colorimetric p-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.

Investigation of the Compatibility between Warheads and Peptidomimetic Sequences of Protease Inhibitors-A Comprehensive Reactivity and Selectivity Study.

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.

Structural Modifications of Covalent Cathepsin S Inhibitors: Impact on Affinity, Selectivity, and Permeability.

Cathepsin S (catS) is a member of the cysteine protease family with limited tissue distribution, which is predominantly found in antigen-presenting cells. Due to overexpression and overactivity of catS in numerous cancers, inhibition of catS is supposed to improve the antitumor response. Here, we explore the potential of small-molecule catS inhibitors emphasizing their in vitro pharmacodynamics and pharmacokinetics. Membrane permeability of selected inhibitors was measured with a Parallel Artificial Membrane Permeation Assay and correlated to calculated physicochemical parameters and inhibition data. The binding kinetics and inhibition types of potent and selective new inhibitors with unexplored warheads were investigated. Our unique approach involves reversible masking of these potent warheads, allowing for further customization without compromising affinity or selectivity. The most promising inhibitors in this study include covalent aldehyde and ketone derivatives reversibly masked as hydrazones as potential candidates for therapeutic interventions targeting catalytic enzymes and modulating the immune response in cancer.

Dual Strategy to Design New Agents Targeting Schistosoma mansoni: Advancing Phenotypic and SmCB1 Inhibitors for Improved Efficacy.

In this study, we have identified and optimized two lead structures from an in-house screening, with promising results against the parasitic flatworm Schistosoma mansoni and its target protease S. mansoni cathepsin B1 (SmCB1). Our correlation analysis highlighted the significance of physicochemical properties for the compounds' in vitro activities, resulting in a dual approach to optimize the lead structures, regarding both phenotypic effects in S. mansoni newly transformed schistosomula (NTS), adult worms, and SmCB1 inhibition. The optimized compounds from both approaches ("phenotypic" vs "SmCB1" approach) demonstrated improved efficacy against S. mansoni NTS and adult worms, with 2h from the "SmCB1" approach emerging as the most potent compound. 2h displayed nanomolar inhibition of SmCB1 (Ki = 0.050 µM) while maintaining selectivity toward human off-target cathepsins. Additionally, the greatly improved efficacy of compound 2h toward S. mansoni adults (86% dead worms at 10 µM, 68% at 1 µM, 35% at 0.1 µM) demonstrates its potential as a new therapeutic agent for schistosomiasis, underlined by its improved permeability.

Subnanomolar Cathepsin S Inhibitors with High Selectivity: Optimizing Covalent Reversible α-Fluorovinylsulfones and α-Sulfonates as Potential Immunomodulators in Cancer.

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 (Ki =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.

Novel Opportunities for Cathepsin S Inhibitors in Cancer Immunotherapy by Nanocarrier-Mediated Delivery.

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.