Chemoproteomic profiling of targets of berberine and studies on anticancer gold(III) and manganese(III) porphyrins towards intracellular reactive thiols

Abstract

Proteins are involved in almost every biological process in cells and play vital roles in oncogenesis and cancer progression. Elucidation of interactions between protein targets and biologically active small molecules is essential to drug discovery and can lead to the discovery of novel therapeutic target proteins. The natural product berberine can effectively inhibit the proliferation of the HeLa cervical cancer cell line and the HCT 116 colon cancer cell line, while the compound is nontoxic to CCD-19Lu normal lung cells. Berberine was found to exert a significant inhibitory effect on the migration of non-small lung cancer cells (NCI-H1650 cell line). In Chapter 2, the use of quantitative chemical proteomics to identify protein targets of berberine is described. The results show that BBR-probe fished a novel promising protein target, HSP60, and other druggable targets, such as HSP90 and tubulin. High levels of reactive oxygen species (ROS) are typically observed in tumors. To protect cancer cells from oxidative damage, the thiol-dependent thioredoxin (Trx) system is often overexpressed in many types of cancer, making this antioxidant system a promising drug target. Gold(III) 5-(pentafluorophenyl)- 10,15,20-triphenylporphyrin chloride (AuMF) is presented in Chapter 3 as a potential inhibitor of thioredoxin reductase (TrxR). AuMF was found to specifically react with cysteine and reduced glutathione (GSH), by nucleophilic aromatic substitution under physiological conditions, but not with other amino acids. AuMF exhibited a marked inhibition of the activity of purified TrxR, with an IC50 of 225 nM in 30 min. The activity of the thiol-dependent redox system in NCI-H460 non-small lung cancer cells was diminished by AuMF in a dose-dependent manner, with an IC50 of 9.7 M in 2 h. This IC50 value was close to the cytotoxic IC50 value of AuMF against NCI-H460, which was 10.0 M. Therefore, AuMF kills cancer cells by inhibiting the cellular, thiol-dependent redox system. Elevated levels of GSH are usually found in tumors, compared to normal tissues, as a redox adaptation. Additionally, GSH is the factor responsible for drug resistance of some chemotherapeutic agents, such as cisplatin. In contrast to multi-charged manganese(III) porphyrins, studies on mono-cationic Mn(III) porphyrins in biomedicine are sparse. In Chapter 4, the cytotoxicity and anticancer mechanisms of manganese(III) 5-(pentafluorophenyl)-10,15,20-triphenylporphyrin chloride (MnMF) are described. MnMF displays cytotoxicity against many types of cancer cell lines and tumorigenic cells. In addition, MnMF inhibits the migration of NCI-H650 cells. Although MnMF had no effect on angiogenesis, manganese(III) mesoporphyrin IX dimethyl ester (MnMeso) effectively inhibited tube formation by MS1 cells. MnMF treatment resulted in a significant increase in ROS level in NCI-H460 cells. MnMF distinctly, and irreversibly decreased both cellular reduced GSH and total GSH level in NCI-H460 cells thus overcoming the drug resistance caused by GSH. Profiling of MnMF-induced proteome of NCI-H460 cells showed the effect of MnMF on the level of proteins which were mainly involved in cadherin binding, proteostasis, oxidative stress response and glutathione metabolic process. (475 words)