Ferroptosis is triggered by the breakdown of cellular iron-dependent redox homeostasis and the abnormal accumulation of lipid ROS. Cells have evolved defense mechanisms to prevent lipid ROS accumulation and ferroptosis. Using a library of more than 4,000 bioactive compounds, we show that tanshinone fromSalvia miltiorrhiza(Danshen) has very potent inhibitory activity against ferroptosis. Mechanistically, we found that tanshinone functions as a coenzyme for NAD(P)H:quinone oxidoreductase 1 (NQO1), which detoxifies lipid peroxyl radicals and inhibits ferroptosis both in vitro and in vivo. Although NQO1 is recognized as an oxidative stress response gene, it does not appear to have a direct role in ferroptosis inhibition in the absence of tanshinone. Here, we demonstrate a gain of function of NQO1 induced by tanshinone, which is a novel mechanism for ferroptosis inhibition. Using mouse models of acute liver injury and ischemia/reperfusion heart injury, we observed that tanshinone displays protective effects in both the liver and the heart in a manner dependent on NQO1. Our results link the clinical use of tanshinone to its activity in ferroptosis inhibition.

Ferroptosis is a nonapoptotic form of cell death characterized by iron-dependent lipid peroxidation and has been implicated in multiple pathological conditions. Glutathione peroxidase 4 (GPX4) plays an essential role in inhibiting ferroptosis by eliminating lipid peroxide using glutathione (GSH) as a reductant. In this study, we found Ellman's reagent DTNB and a series of disulfide compounds, including disulfiram (DSF), an FDA-approved drug, which protect cells from erastininduced ferroptosis. Mechanistically, DTNB or DSF is conjugated to multiple cysteine residues in GPX4 and disrupts GPX4 interaction with HSC70, an adaptor protein for chaperone mediated autophagy, thus preventing GPX4 degradation induced by erastin. In addition, DSF ameliorates concanavalin A induced acute liver injury by suppressing ferroptosis in a mouse model. Our work reveals a novel regulatory mechanism for GPX4 protein stability control. We also discover disulfide compounds as a new class of ferroptosis inhibitors and suggest therapeutic repurposing of DSF in treating ferroptosis-related diseases.

Hypoxia inducible factor‐1α (HIF‐1α) plays a critical role in cellular adaptation to hypoxia and it is a potential therapeutic target for anti‐cancer drugs. Applying high‐throughput screening, here it is found that HI‐101, a small molecule containing an adamantaniline moiety, effectively reduces HIF‐1α protein expression. With the compound as a hit, a probe (HI‐102) is developed for target identification by affinity‐based protein profiling. The catalytic β subunit of mitochondrial FOF1‐ATP synthase, ATP5B, is identified as the binding protein of HI‐derivatives. Mechanistically, HI‐101 promotes the binding of HIF‐1α mRNA to ATP5B, thus inhibiting HIF‐1α translation and the following transcriptional activity. Further modifications of HI‐101 lead to HI‐104, a compound with good pharmacokinetic properties, exhibiting antitumor activity in MHCC97‐L mice xenograft model, and HI‐105, the most potent compound with an IC50 of 26 nm. The findings provide a new strategy for further developing HIF‐1α inhibitors by translational inhibition through ATP5B.

Hypoxia-inducible factor, also known as HIF, is a transcriptional factor universally found in mammalian cells. HIF-1 is one of the HIF-families and acts as a heterodimer consisting of α and β subunits. It is found to play significant roles in pathologic conditions such as tumor development and metastasis. Here, we first report benzo[d]isoxazole analogues as HIF-1α transcription inhibitors. Thereby, we designed and synthesized 26 benzo[d]isoxazole derivatives and evaluated their inhibitory activities against HIF-1α transcription in HEK293T cells by a dual-luciferase gene reporter assay. Among them, compounds 15 and 31 showed the best efficacy in a cell-based assay with an IC 50 value of 24 nM and have potential antitumor effects for further development.