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.

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.

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 IC50 values of 0.61 ± 0.02 and 0.58 ± 0.12 µmol/L against TEAD1 and TEAD3, respectively. Further chemical optimization based on DC-TEAD in 1072 yielded a selective TEAD3 inhibitor DC-TEAD3in03 with the IC50 value of 0.16 ± 0.03 µmol/L, which shows 100-fold selectivity over other TEAD isoforms in activity-based protein profiling (ABPP) assays. In cells, DC-TEAD3in03 showed selective inhibitory effect on TEAD3 in GAL4-TEAD (1-4) reporter assays with the IC50 value of 1.15 µmol/L. When administered to zebrafish juveniles, experiments showed that DC-TEAD3in03 reduced the growth rate of zebrafish caudal fins, indicating the importance of TEAD3 activity in controlling proportional growth of vertebrate appendages.

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

Activity-based protein profiling guided identification of urine proteinase 3 activity in subclinical rejection after renal transplantation.

BACKGROUND: The pathophysiology of subclinical versus clinical rejection remains incompletely understood given their equivalent histological severity but discordant graft function. The goal was to evaluate serine hydrolase enzyme activities to explore if there were any underlying differences in activities during subclinical versus clinical rejection. METHODS: Serine hydrolase activity-based protein profiling (ABPP) was performed on the urines of a case control cohort of patients with biopsy confirmed subclinical or clinical transplant rejection. In-gel analysis and affinity purification with mass spectrometry were used to demonstrate and identify active serine hydrolase activity. An assay for proteinase 3 (PR3/PRTN3) was adapted for the quantitation of activity in urine. RESULTS: In-gel ABPP profiles suggested increased intensity and diversity of serine hydrolase activities in urine from patients undergoing subclinical versus clinical rejection. Serine hydrolases (n = 30) were identified by mass spectrometry in subclinical and clinical rejection patients with 4 non-overlapping candidates between the two groups (i.e. ABHD14B, LTF, PR3/PRTN3 and PRSS12). Western blot and the use of a specific inhibitor confirmed the presence of active PR3/PRTN3 in samples from patients undergoing subclinical rejection. Analysis of samples from normal donors or from several serial post-transplant urines indicated that although PR3/PRTN3 activity may be highly associated with low-grade subclinical inflammation, the enzyme activity was not restricted to this patient group. CONCLUSIONS: There appear to be limited qualitative and quantitative differences in serine hydrolase activity in patients with subclinical versus clinical renal transplant rejection. The majority of enzymes identified were present in samples from both groups implying that in-gel quantitative differences may largely relate to the activity status of shared enzymes. However qualitative compositional differences were also observed indicating differential activities. The PR3/PRTN3 analyses indicate that the activity status of urine in transplant patients is dynamic possibly reflecting changes in the underlying processes in the transplant. These data suggest that differential serine hydrolase pathways may be active in subclinical versus clinical rejection which requires further exploration in larger patient cohorts. Although this study focused on PR3/PRTN3, this does not preclude the possibility that other enzymes may play critical roles in the rejection process.

Monoacylglycerol lipase inhibitors: modulators for lipid metabolism in cancer malignancy, neurological and metabolic disorders.

Monoacylglycerol lipase (MAGL) is a serine hydrolase that plays a crucial role catalysing the hydrolysis of monoglycerides into glycerol and fatty acids. It links the endocannabinoid and eicosanoid systems together by degradation of the abundant endocannabinoid 2-arachidaoylglycerol into arachidonic acid, the precursor of prostaglandins and other inflammatory mediators. MAGL inhibitors have been considered as important agents in many therapeutic fields, including anti-nociceptive, anxiolytic, anti-inflammatory, and even anti-cancer. Currently, ABX-1431, a first-in-class inhibitor of MAGL, is entering clinical phase 2 studies for neurological disorders and other diseases. This review summarizes the diverse (patho)physiological roles of MAGL and will provide an overview on the development of MAGL inhibitors. Although a large number of MAGL inhibitors have been reported, novel inhibitors are still required, particularly reversible ones.