Cathepsin L (CTSL) is a promising therapeutic target for metabolic disorders. Current pharmacological interventions targeting CTSL have demonstrated potential in reducing body weight gain, serum insulin levels, and improving glucose tolerance. However, the clinical application of CTSL inhibitors remains limited. In this study, we used a combination of artificial intelligence and experimental methods to identify new CTSL inhibitors from natural products. Through a robust deep learning model and molecular docking, we screened 150 molecules from natural products for experimental validation. At a concentration of 100 µM, we found that 36 of them exhibited more than 50 % inhibition of CTSL. Notably, 13 molecules displayed over 90 % inhibition and exhibiting concentration-dependent effects. The molecular dynamics simulation on the two most potent inhibitors, Plumbagin and Beta-Lapachone, demonstrated stable interaction at the CTSL active site. Enzyme kinetics studies have shown that these inhibitors exert an uncompetitive inhibitory effect on CTSL. In conclusion, our research identifies Plumbagin and Beta-Lapachone as potential CTSL inhibitors, offering promising candidates for the treatment of metabolic disorders and illustrating the effectiveness of artificial intelligence in drug discovery.

2024-05-09·Journal of Medicinal Chemistry

Structural Elucidation and Antiviral Activity of Covalent Cathepsin L Inhibitors

Article

作者: Sekirnik, Andreja ; Tsuge, Hideaki ; Usenik, Aleksandra ; Ewert, Wiebke ; Lieske, Julia ; Meents, Alke ; Hinrichs, Winfried ; Dretnik, Klemen ; Lindič, Nataša ; Loboda, Jure ; Turk, Dušan ; Reinke, Patrick Y A ; Turk, Vito ; Karničar, Katarina ; Chapman, Henry N ; Falke, Sven ; Herrmann, Alexander ; Günther, Sebastian ; Ebert, Gregor

Emerging RNA viruses, including SARS-CoV-2, continue to be a major threat. Cell entry of SARS-CoV-2 particles via the endosomal pathway involves cysteine cathepsins. Due to ubiquitous expression, cathepsin L (CatL) is considered a promising drug target in the context of different viral and lysosome-related diseases. We characterized the anti-SARS-CoV-2 activity of a set of carbonyl- and succinyl epoxide-based inhibitors, which were previously identified as inhibitors of cathepsins or related cysteine proteases. Calpain inhibitor XII, MG-101, and CatL inhibitor IV possess antiviral activity in the very low nanomolar EC₅₀ range in Vero E6 cells and inhibit CatL in the picomolar K_i range. We show a relevant off-target effect of CatL inhibition by the coronavirus main protease α-ketoamide inhibitor 13b. Crystal structures of CatL in complex with 14 compounds at resolutions better than 2 Å present a solid basis for structure-guided understanding and optimization of CatL inhibitors toward protease drug development.

2023-02-01·Biomaterials Advances

Inducing cathepsin L expression/production, lysosomal activation, and autophagy of human dental pulp cells by dentin bonding agents, camphorquinone and BisGMA and the related mechanisms

Article

作者: Dhingra, Kunaal ; Chen, Yi-Jane ; Chang, Hsiao-Hua ; Chen, Jen-Hao ; Pan, Yu-Hwa ; Lee, Hui-Na ; Chang, Mei-Chi ; Zhong, Bor-Hao ; Chen, Shyuan-Yow ; Jeng, Jiiang-Huei

Camphorquinone (CQ) and resin monomers are included in dentin bonding agents (DBAs) and composite resin to restore tooth defects due to abrasion, crown fracture, or dental caries. DBAs, CQ, and bisphenol A-glycidyl methacrylate (BisGMA) applications influence the biological activities of the dental pulp. The current investigation aimed to delineate the effect of DBAs, CQ, and BisGMA on cathepsin L production/expression, lysosomal activity, and autophagy induction in human dental pulp cells (HDPCs). HDPCs were exposed to DBAs, CQ, or BisGMA with/without inhibitors for 24 h. Enzyme-linked immunosorbent assay was employed to determine the cathepsin L level in culture medium. The cell layer was utilized to measure cell viability by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl -tetrazolium bromide (MTT) assay. Real-time PCR was used to evaluate the mRNA expression. Western blotting or immunofluorescent staining was used to study protein expression. Lysosomal density was evaluated by lysotracker red staining. We found that DBAs, CQ, and BisGMA stimulated cathepsin L mRNA, protein expression, and production in HDPCs. In addition, CQ and BisGMA induced lysosomal activity, Beclin1, ATG12, LC3B, Bax, and p53 expression in HDPCs, indicating the stimulation of autophagy. Glutathione (GSH) prevented CQ- and BisGMA-induced cytotoxicity. Moreover, E64d, cathepsin L inhibitor (two cathepsin inhibitors), and Pifithrin-α (a p53 inhibitor) showed little preventive effect toward CQ- and BisGMA-induced cytotoxicity. Autophagy inhibitors (NH4Cl, Lys05) mildly enhanced the CQ- and BisGMA-induced cytotoxicity. These results indicate that DBAs stimulated cathepsin L, possibly due to their content of CQ and BisGMA that may induce cathepsin L in HDPCs. CQ and BisGMA stimulated lysosomal activity, autophagy, and apoptosis, possibly via induction of Beclin 1, ATG12, LC-3B, Bax, and p53 expression. In addition, CQ and BisGMA cytotoxicity was related to redox change and autophagy. These events are important role in pulpal changes after the restoration of tooth decay using CQ- and BisGMA-containing DBAs and resin composite.

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