SUMMARY Ferroptosis is a form of nonapoptotic cell death for which key regulators remain unknown. We sought a common mediator for the lethality of 12 ferroptosisinducing small molecules. We used targeted metabolomic profiling to discover that depletion of glutathione causes inactivation of glutathione peroxidases (GPXs) in response to one class of compounds and a chemoproteomics strategy to discover that GPX4 is directly inhibited by a second class of compounds. GPX4 overexpression and knockdown modulated the lethality of 12 ferroptosis inducers, but not of 11 compounds with other lethal mechanisms. In addition, two representative ferroptosis inducers prevented tumor growth in xenograft mouse tumor models. Sensitivity profiling in 177 cancer cell lines revealed that diffuse large B cell lymphomas and renal cell carcinomas are particularly susceptible to GPX4-regulated ferroptosis. Thus, GPX4 is an essential regulator of ferroptotic cancer cell death.

The phosphoinositide 3-kinase (PI3K)/mechanistic target of rapamycin (mTOR) pathway is frequently overactivated in cancer, and drives cell growth, proliferation, survival, and metastasis. Here, we report a structure–activity relationship study, which led to the discovery of a drug-like adenosine 5′-triphosphate-site PI3K/mTOR kinase inhibitor: (S)-4-(difluoromethyl)-5-(4-(3-methylmorpholino)-6-morpholino-1,3,5-triazin-2-yl)­pyridin-2-amine (PQR530, compound 6), which qualifies as a clinical candidate due to its potency and specificity for PI3K and mTOR kinases, and its pharmacokinetic properties, including brain penetration. Compound 6 showed excellent selectivity over a wide panel of kinases and an excellent selectivity against unrelated receptor enzymes and ion channels. Moreover, compound 6 prevented cell growth in a cancer cell line panel. The preclinical in vivo characterization of compound 6 in an OVCAR-3 xenograft model demonstrated good oral bioavailability, excellent brain penetration, and efficacy. Initial toxicity studies in rats and dogs qualify 6 for further development as a therapeutic agent in oncology.

Covalent protein kinase inhibitors exploit currently noncatalytic cysteines in the adenosine 5′-triphosphate (ATP)-binding site via electrophiles directly appended to a reversible-inhibitor scaffold. Here, we delineate a path to target solvent-exposed cysteines at a distance >10 Å from an ATP-site-directed core module and produce potent covalent phosphoinositide 3-kinase α (PI3Kα) inhibitors. First, reactive warheads are used to reach out to Cys862 on PI3Kα, and second, enones are replaced with druglike warheads while linkers are optimized. The systematic investigation of intrinsic warhead reactivity ( k chem ), rate of covalent bond formation and proximity ( k inact and reaction space volume V r ), and integration of structure data, kinetic and structural modeling, led to the guided identification of high-quality, covalent chemical probes. A novel stochastic approach provided direct access to the calculation of overall reaction rates as a function of k chem , k inact , K i , and V r , which was validated with compounds with varied linker lengths. X-ray crystallography, protein mass spectrometry (MS), and NanoBRET assays confirmed covalent bond formation of the acrylamide warhead and Cys862. In rat liver microsomes, compounds 19 and 22 outperformed the rapidly metabolized CNX-1351, the only known PI3Kα irreversible inhibitor. Washout experiments in cancer cell lines with mutated, constitutively activated PI3Kα showed a long-lasting inhibition of PI3Kα. In SKOV3 cells, compounds 19 and 22 revealed PI3Kβ-dependent signaling, which was sensitive to TGX221. Compounds 19 and 22 thus qualify as specific chemical probes to explore PI3Kα-selective signaling branches. The proposed approach is generally suited to develop covalent tools targeting distal, unexplored Cys residues in biologically active enzymes.

The mechanistic target of rapamycin (mTOR) pathway is hyperactivated in cancer and neurological disorders. Rapalogs and mTOR kinase inhibitors (TORKi) have recently been applied to alleviate epileptic seizures in tuberous sclerosis complex (TSC). Herein, we describe a pharmacophore exploration to identify a highly potent, selective, brain penetrant TORKi. An extensive investigation of the morpholine ring engaging the mTOR solvent exposed region led to the discovery of PQR626 (8). 8 displayed excellent brain penetration and was well-tolerated in mice. In mice with a conditionally inactivated Tsc1 gene in glia, 8 significantly reduced the loss of Tsc1-induced mortality at 50 mg/kg p.o. twice a day. 8 overcomes the metabolic liabilities of PQR620 (52), the first-in-class brain penetrant TORKi showing efficacy in a TSC mouse model. The improved stability in human hepatocytes, excellent brain penetration, and efficacy in Tsc1 GFAPCKO mice qualify 8 as a potential therapeutic candidate for the treatment of neurological disorders.

Glaucoma and age-related macular degeneration are ocular diseases targeted clinically by anecortave acetate (AA). AA and its deacetylated metabolite, anecortave desacetate (AdesA), are intraocular pressure (IOP)-lowering and angiostatic cortisenes devoid of glucocorticoid activity but with an unknown mechanism of action. We used a methotrexate-anchored yeast three-hybrid (Y3H) technology to search for binding targets for AA in human trabecular meshwork (TM) cells, the target cell type that controls IOP, a major risk factor in glaucoma. Y3H hits were filtered by competitive Y3H screens and coimmunoprecipitation experiments and verified by surface plasmon resonance analysis to yield a single target, phosphodiesterase 6-delta (PDE6D). PDE6D is a prenyl-binding protein with additional function outside the PDE6 phototransduction system. Overexpression of PDE6D in mouse eyes caused elevated IOP, and this elevation was reversed by topical ocular application of either AA or AdesA. The identification of PDE6D as the molecular binding partner of AA provides insight into the role of this drug candidate in treating glaucoma.