An ADAM10 Exosite Inhibitor Is Efficacious in an In Vivo Collagen-Induced Arthritis Model.

Rheumatoid arthritis is a systemic autoimmune inflammatory disease that affects millions of people worldwide. There are multiple disease-modifying anti-rheumatic drugs available; however, many patients do not respond to any treatment. A disintegrin and metalloproteinase 10 has been suggested as a potential new target for RA due to its role in the release of multiple pro- and anti-inflammatory factors from cell surfaces. In the present study, we determined the pharmacokinetic parameters and in vivo efficacy of a compound CID3117694 from a novel class of non-zinc-binding inhibitors. Oral bioavailability was demonstrated in the blood and synovial fluid after a 10 mg/kg dose. To test efficacy, we established the collagen-induced arthritis model in mice. CID3117694 was administered orally at 10, 30, and 50 mg/kg/day for 28 days. CID3117694 was able to dose-dependently improve the disease score, decrease RA markers in the blood, and decrease signs of inflammation, hyperplasia, pannus formation, and cartilage erosion in the affected joints compared to the untreated control. Additionally, mice treated with CID 3117694 did not exhibit any clinical signs of distress, suggesting low toxicity. The results of this study suggest that the inhibition of ADAM10 exosite can be a viable therapeutic approach to RA.

Identification of substrate-specific inhibitors of cathepsin K through high-throughput screening.

Cathepsin K (CatK) is a cysteine protease and drug target for skeletal disorders that is known for its potent collagenase and elastase activity. The formation of oligomeric complexes of CatK in the presence of glycosaminoglycans has been associated with its collagenase activity. Inhibitors that disrupt these complexes can selectively block the collagenase activity without interfering with the other regulatory proteolytic activities of the enzyme. Here, we have developed a fluorescence polarization (FP) assay to screen 4761 compounds for substrate-specific ectosteric collagenase inhibitors of CatK. A total of 38 compounds were identified that block the collagenase activity without interfering with the hydrolysis of active site substrates such as the synthetic peptide substrate, benzyloxycarbonyl-Phe-Arg-7-amido-4-methylcoumarin, and gelatin. The identified inhibitors can be divided into two main classes, negatively charged and polyaromatic compounds which suggest the binding to different ectosteric sites. Two of the inhibitors were highly effective in preventing the bone-resorption activity of CatK in osteoclasts. Interestingly, some of the ectosteric inhibitors were capable of differentiating between the collagenase and elastase activity of CatK depending on the ectosteric site utilized by the compound. Owing to their substrate-specific selectivity, ectosteric inhibitors represent a viable alternative to side effect-prone active site-directed inhibitors.

Study on exosite inhibitor of cathepsin K from Herba Ecliptae fractions / 药学学报

Cathepsin K (CTSK) is considered a critical pharmaceutical target in the treatment of osteoporosis. CTSK exerts proteolytic activities against regulatory proteins besides its collagenase function, which may account for some of the adverse reactions when blocked by active site-directed inhibitors. Exosite inhibitors that can discriminate between the therapeutic collagenase and other biological activities of CTSK specifically inhibit the collagenase activity of CTSK without interfering with the other proteolytic activities of the protease. Active recombinant CTSK was expressed in Pichia pastoris, and purified by n-butyl sepharose and SP sepharose column chromatography. Herba Ecliptae is a common traditional Chinese medicine in the treatment of bone diseases. Collagenase assay and benzyloxycarbonyl-Phe-Arg-7-amido-4-methylcoumarin (Z-FR-MCA) substrate assay based on CTSK are applied to verify the exosite inhibitors. n-Butanol extract of Herba Ecliptae are the most active fraction and eclalbasaponin IX isolated from n-butanol fraction is the potential exosite inhibitor of CTSK.

Activity of ADAM17 (a disintegrin and metalloprotease 17) is regulated by its noncatalytic domains and secondary structure of its substrates.

ADAM proteases are implicated in multiple diseases, but no drugs based on ADAM inhibition exist. Most of the ADAM inhibitors developed to date feature zinc-binding moieties that target the active site zinc, which leads to a lack of selectivity and off target toxicity. Targeting secondary substrate binding sites (exosites) can potentially work as an alternative strategy for drug discovery; however, there are only a few reports of potential exosites in ADAM protease structures. In the study presented here, we utilized a series of TNFα-based substrates to probe ADAM10 and 17 interactions with its canonical substrate to identify the structural features that determine ADAM protease substrate specificity. We found that noncatalytic domains of ADAM17 did not directly bind the substrates used in the study but affected the binding nevertheless, most likely because of steric hindrance. Additionally, noncatalytic domains of ADAM17 affected the size/shape of the carbohydrate-binding pocket contained within the catalytic domain of ADAM17. This suggests that noncatalytic domains of ADAM17 play a role in substrate specificity and might help explain differences in substrate repertoires of ADAM17 and its closest homologue, ADAM10. We also addressed the question of which substrate features can affect ADAM protease specificity. We found that all ADAM proteases tested (i.e., ADAM10, 12, and 17) significantly decreased activity when the TNFα-derived sequence was induced into α-helical conformation, suggesting that conformation plays a role in determining ADAM protease substrate specificity. These findings can help in the discovery of ADAM isoform- and substrate-specific inhibitors.