Celastrol induces ferroptosis in activated HSCs to ameliorate hepatic fibrosis via targeting peroxiredoxins and HO-1

Ferroptosis is a form of regulated cell death, characterized by excessive membrane lipid peroxidation in an iron- and ROS-dependent manner. Celastrol, a natural bioactive triterpenoid extracted from Tripterygium wilfordii, shows effective anti-fibrotic and anti-inflammatory activities in multiple hepatic diseases. However, the exact molecular mechanisms of action and the direct protein targets of celastrol in the treatment of liver fibrosis remain largely elusive. Here, we discover that celastrol exerts anti-fibrotic effects via promoting the production of reactive oxygen species (ROS) and inducing ferroptosis in activated hepatic stellate cells (HSCs). By using activity-based protein profiling (ABPP) in combination with bio-orthogonal click chemistry reaction and cellular thermal shift assay (CETSA), we show that celastrol directly binds to peroxiredoxins (PRDXs), including PRDX1, PRDX2, PRDX4 and PRDX6, through the active cysteine sites, and inhibits their anti-oxidant activities. Celastrol also targets to heme oxygenase 1 (HO-1) and upregulates its expression in activated-HSCs. Knockdown of PRDX1, PRDX2, PRDX4, PRDX6 or HO-1 in HSCs, to varying extent, elevated cellular ROS levels and induced ferroptosis. Taken together, our findings reveal the direct protein targets and molecular mechanisms via which celastrol ameliorates hepatic fibrosis, thus supporting the further development of celastrol as a promising therapeutic agent for liver fibrosis.

A targeted covalent inhibitor of p97 with proteome-wide selectivity

A resurging interest in targeted covalent inhibitors (TCIs) focus on compounds capable of irreversibly reacting with nucleophilic amino acids in a druggable target. p97 is an emerging protein target for cancer therapy, viral infections and neurodegenerative diseases. Extensive efforts were devoted to the development of p97 inhibitors. The most promising inhibitor of p97 was in phase 1 clinical trials, but failed due to the off-target-induced toxicity, suggesting the selective inhibitors of p97 are highly needed. We report herein a new type of TCIs (i.e., FL-18) that showed proteome-wide selectivity towards p97. Equipped with a Michael acceptor and a basic imidazole, FL-18 showed potent inhibition towards U87MG tumor cells, and in proteome-wide profiling, selectively modified endogenous p97 as confirmed by in situ fluorescence scanning, label-free quantitative proteomics and functional validations. FL-18 selectively modified cysteine residues located within the D2 ATP site of p97. This covalent labeling of cysteine residue in p97 was verified by LC‒MS/MS-based site-mapping and site-directed mutagenesis. Further structure-activity relationship (SAR) studies with FL-18 analogs were established. Collectively, FL-18 is the first known small-molecule TCI capable of covalent engagement of p97 with proteome-wide selectivity, thus providing a promising scaffold for cancer therapy.

Discovery of a subtype-selective, covalent inhibitor against palmitoylation pocket of TEAD3

The TEA domain (TEAD) family proteins (TEAD1‒4) are essential transcription factors that control cell differentiation and organ size in the Hippo pathway. Although the sequences and structures of TEAD family proteins are highly conserved, each TEAD isoform has unique physiological and pathological functions. Therefore, the development and discovery of subtype selective inhibitors for TEAD protein will provide important chemical probes for the TEAD-related function studies in development and diseases. Here, we identified a novel TEAD1/3 covalent inhibitor (DC-TEADin1072) with biochemical IC

Protein targets of medicinally active molecules based on their original structures and molecular probes / 药学学报

Medicinally active molecules are those that have pharmacological effects. Research on protein targets of these molecules not only clarifies their mechanism of action, but also deepens our understanding of biological systems. Here we review recent advances in protein targets of drugs used in clinical practice or in preclinical research. They have various functions including anti-inflammatory, anti-malarial, anti-tumor and other biological activities. Activity-based protein profiling (ABPP) and cellular thermal shift assay (CETSA) are two useful methods to identify the protein targets of small molecules. ABPP depends on a derivative active molecule probe to pull down the protein targets to reveal the interaction mechanisms between the active molecules and targets. Drug target engagement also can be assessed by means of CETSA based on ligand-induced changes in protein thermal stability. In the CETSA approach, the active molecules do not need to be chemically modified. Combining the CETSA method with quantitative mass spectrometry is an effective approach to study the effect of compounds on the thermal profile of a cellular proteome and identify the protein targets. ABPP and CETSA can be complementary and effectively clarify the protein targets. The study of protein targets will help reveal the mechanism of action of medicinal molecules, reveal toxic mechanisms and aid in the discovery of new medicinal targets to promote the process of drug development.

Progress of methodology for identifying target protein of natural active small molecules / 中国中药杂志

Drug targets are special molecules that can interact with drugs and exert pharmacological functions in human body. The natural active small molecules are the bioactive basis of traditional Chinese medicine, and the mechanism study is a hot topic now, especially for the identification of their target proteins. However, little progress has been made in this field until now. Here, we summarized the recent technologies and methods for the identification of target proteins of natural bioactive small molecules, and introduced the main research methods, principles and successful cases in this field. We also explored the applicability and discussed the advantages and disadvantages among different methods. We hope this review can be used as a reference for the researchers who engaged in natural pharmaceutical chemistry, pharmacology and chemical biology.