Apoferritin encapsulation of cysteine protease inhibitors for cathepsin L inhibition in cancer cells

Cysteine proteases play a key role in tumorigenesis causing protein degradation and promoting invasive tumour growth. Cathepsin L is overexpressed in cancer cells and could provide a specific target for delivery of anticancer agents. We encapsulated novel dipeptidyl nitrile based cysteine protease inhibitors (Neq0551, Neq0554 and Neq0568) into biocompatible apoferritin (AFt) protein nanocages to achieve specific delivery to tumours and pH-induced drug release. AFt-encapsulated Neq0554 demonstrated $3-fold enhanced in vitro activity (GI 50 ¼ 79 mM) compared to naked agent against MiaPaCa-2 pancreatic carcinoma cells. Selectivity for cancer cells was confirmed by comparing their activity to non-tumourigenic human fibroblasts (GI 50 > 200 mM). Transferrin receptor (TfR-1) expression, detected only in lysates prepared from carcinoma cells, may contribute to the cancer-selectivity. The G 1 cell cycle arrest caused by AFt-Neq0554 resulting in cytostasis was corroborated by clonogenic assays. Superior and more persistent inhibition of cathepsin L up to 80% was achieved with AFt-encapsulated agent in HCT-116 cells following 6 h exposure to 50 mM agent. The selective anticancer activity of AFt-encapsulated cysteine protease inhibitor Neq0554 reported here warrants further preclinical in vivo evaluation

Synthesis and matched molecular pair analysis of covalent reversible inhibitors of the cysteine protease CPB

Cysteine protease B (CPB) can be targeted by reversible covalent inhibitors that could serve as antileishmanial compounds. Here, sixteen dipeptidyl nitrile derivatives were synthesized, tested against CPB, and analyzed using matched molecular pairs to determine the effects of stereochemistry and p-phenyl substitution on enzyme inhibition. The compound (S)-2-(((S)-1-(4-bromophenyl)-2,2,2-trifluoroethyl)amino)-N-(1-cyanocyclopropyl)-3-phenylpropanamide (5) was the most potent CPB inhibitor (pKi = 6.82), which was also selective for human cathepsin B (pKi < 5). The inversion of the stereochemistry from S to R was more detrimental to potency when placed at the P2 position than at P3. The p-Br derivatives were more potent than the p-CH3 and p-OCH3 derivatives, probably due to intermolecular interactions with the S3 subsite

N-Sulfonyl Dipeptide Nitriles as Inhibitors of Human Cathepsin S: In silico Design, Synthesis and Biochemical Characterization

A library of cathepsin S inhibitors of the dipeptide nitrile chemotype, bearing a bioisosteric sulfonamide moiety, was synthesized. Kinetic investigations were performed at four human cysteine proteases, i.e. cathepsins S, B, K and L. Compound 12 with a terminal 3-biphenyl sulfonamide substituent was the most potent (Ki = 4.02 nM; selectivity ratio cathepsin S/K = 5.8; S/L = 67) and 24 with a 4′-fluoro-4-biphenyl sulfonamide substituent the most selective cathepsin S inhibitor (Ki = 35.5 nM; selectivity ratio cathepsin S/K = 57; S/L = 31). In silico design and biochemical evaluation emphasized the impact of the sulfonamide linkage on selectivity and a possible switch of P2 and P3 substituents with respect to the occupation of the corresponding binding sites of cathepsin S

Crystal structure of Leishmania mexicana cysteine protease B in complex with a high-affinity azadipeptide nitrile inhibitor

Leishmania mexicana is an obligate intracellular protozoan parasite that causes the cutaneous form of leishmaniasis affecting South America and Mexico. The cysteine protease LmCPB is essential for the virulence of the parasite and therefore, it is an appealing target for antiparasitic therapy. A library of nitrile-based cysteine protease inhibitors was screened against LmCPB to develop a treatment of cutaneous leishmaniasis. Several compounds are sufficiently high-affinity LmCPB inhibitors to serve both as starting points for drug discovery projects and as probes for target validation. A 1.4 Å X ray crystal structure, the first to be reported for LmCPB, was determined for the complex of this enzyme covalently bound to an azadipeptide nitrile ligand. Mapping the structure-activity relationships for LmCPB inhibition revealed superadditive effects for two pairs of structural transformations. Therefore, this work advances our understanding of azadipeptidyl and dipeptidyl nitrile structure-activity relationships for LmCPB structure-based inhibitor design. We also tested the same series of inhibitors on related cysteine proteases cathepsin L and Trypanosoma cruzi cruzain. The modulation of these mammalian and protozoan proteases represents a new framework for targeting papain-like cysteine proteases

Synthesis and structure-activity relationship of nitrile-based cruzain inhibitors incorporating a trifluoroethylamine-based P2 amide replacement

The structure-activity relationship for nitrile-based cruzain inhibitors incorporating a P2 amide replacement based on trifluoroethylamine was explored by deconstruction of a published series of inhibitors. It was demonstrated that the P3 biphenyl substituent present in the published inhibitor structures could be truncated to phenyl with only a small loss of affinity. The effects of inverting the configuration of the P2 amide replacement and linking a benzyl substituent at P1 were observed to be strongly nonadditive. We show that plotting affinity against molecular size provides a means to visualize both the molecular size efficiency of structural transformations and the nonadditivity in the structure-activity relationship. We also show how the relationship between affinity and lipophilicity, measured by high-performance liquid chromatography with an immobilized artificial membrane stationary phase, may be used to normalize affinity with respect to lipophilicity