Ferroptosis is a form of nonapoptotic cell death for which key regulators remain unknown. We sought a common mediator for the lethality of 12 ferroptosis-inducing 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.
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•Metabolomic profiling revealed GSH depletion as one mechanism of ferroptosis•Chemoproteomics identified GPX4 as a target for RSL3, a second ferroptosis inducer•GPX4 is a central regulator of ferroptosis, akin to bcl-2 in apoptosis•DLBCLs and renal cell carcinomas are sensitive to ferroptotic cell death
A chemoproteomic screen identifies a glutathione peroxidase as an essential regulator of a nonapoptotic form of cell death known as ferroptosis.
Target-identification and mechanism-of-action studies have important roles in small-molecule probe and drug discovery. Biological and technological advances have resulted in the increasing use of ...cell-based assays to discover new biologically active small molecules. Such studies allow small-molecule action to be tested in a more disease-relevant setting at the outset, but they require follow-up studies to determine the precise protein target or targets responsible for the observed phenotype. Target identification can be approached by direct biochemical methods, genetic interactions or computational inference. In many cases, however, combinations of approaches may be required to fully characterize on-target and off-target effects and to understand mechanisms of small-molecule action.
Synthetic lethality occurs when the inhibition of two genes is lethal while the inhibition of each single gene is not. It can be harnessed to selectively treat cancer by identifying inactive genes in ...a given cancer and targeting their synthetic lethal (SL) partners. We present a data-driven computational pipeline for the genome-wide identification of SL interactions in cancer by analyzing large volumes of cancer genomic data. First, we show that the approach successfully captures known SL partners of tumor suppressors and oncogenes. We then validate SL predictions obtained for the tumor suppressor VHL. Next, we construct a genome-wide network of SL interactions in cancer and demonstrate its value in predicting gene essentiality and clinical prognosis. Finally, we identify synthetic lethality arising from gene overactivation and use it to predict drug efficacy. These results form a computational basis for exploiting synthetic lethality to uncover cancer-specific susceptibilities.
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•Genome-scale data-driven identification of synthetic lethality in cancer•Synthetic lethality networks successfully predict cancer gene essentiality•Synthetic lethality networks predict 15 year survival in breast cancer patients•Synthetic dosage lethality networks predict drug response in cancer
A data-driven computational pipeline named DAISY captures known and novel synthetic lethal (SL) interactions, enabling the utilization of genome-wide cancer SL networks to predict gene essentiality, drug response, and clinical prognosis.
Ferroptosis-an iron-dependent, non-apoptotic cell death process-is involved in various degenerative diseases and represents a targetable susceptibility in certain cancers
. The ...ferroptosis-susceptible cell state can either pre-exist in cells that arise from certain lineages or be acquired during cell-state transitions
. However, precisely how susceptibility to ferroptosis is dynamically regulated remains poorly understood. Here we use genome-wide CRISPR-Cas9 suppressor screens to identify the oxidative organelles peroxisomes as critical contributors to ferroptosis sensitivity in human renal and ovarian carcinoma cells. Using lipidomic profiling we show that peroxisomes contribute to ferroptosis by synthesizing polyunsaturated ether phospholipids (PUFA-ePLs), which act as substrates for lipid peroxidation that, in turn, results in the induction of ferroptosis. Carcinoma cells that are initially sensitive to ferroptosis can switch to a ferroptosis-resistant state in vivo in mice, which is associated with extensive downregulation of PUFA-ePLs. We further find that the pro-ferroptotic role of PUFA-ePLs can be extended beyond neoplastic cells to other cell types, including neurons and cardiomyocytes. Together, our work reveals roles for the peroxisome-ether-phospholipid axis in driving susceptibility to and evasion from ferroptosis, highlights PUFA-ePL as a distinct functional lipid class that is dynamically regulated during cell-state transitions, and suggests multiple regulatory nodes for therapeutic interventions in diseases that involve ferroptosis.
Clear-cell carcinomas (CCCs) are a histological group of highly aggressive malignancies commonly originating in the kidney and ovary. CCCs are distinguished by aberrant lipid and glycogen ...accumulation and are refractory to a broad range of anti-cancer therapies. Here we identify an intrinsic vulnerability to ferroptosis associated with the unique metabolic state in CCCs. This vulnerability transcends lineage and genetic landscape, and can be exploited by inhibiting glutathione peroxidase 4 (GPX4) with small-molecules. Using CRISPR screening and lipidomic profiling, we identify the hypoxia-inducible factor (HIF) pathway as a driver of this vulnerability. In renal CCCs, HIF-2α selectively enriches polyunsaturated lipids, the rate-limiting substrates for lipid peroxidation, by activating the expression of hypoxia-inducible, lipid droplet-associated protein (HILPDA). Our study suggests targeting GPX4 as a therapeutic opportunity in CCCs, and highlights that therapeutic approaches can be identified on the basis of cell states manifested by morphological and metabolic features in hard-to-treat cancers.
We recently described glutathione peroxidase 4 (GPX4) as a promising target for killing therapy-resistant cancer cells via ferroptosis. The onset of therapy resistance by multiple types of treatment ...results in a stable cell state marked by high levels of polyunsaturated lipids and an acquired dependency on GPX4. Unfortunately, all existing inhibitors of GPX4 act covalently via a reactive alkyl chloride moiety that confers poor selectivity and pharmacokinetic properties. Here, we report our discovery that masked nitrile-oxide electrophiles, which have not been explored previously as covalent cellular probes, undergo remarkable chemical transformations in cells and provide an effective strategy for selective targeting of GPX4. The new GPX4-inhibiting compounds we describe exhibit unexpected proteome-wide selectivity and, in some instances, vastly improved physiochemical and pharmacokinetic properties compared to existing chloroacetamide-based GPX4 inhibitors. These features make them superior tool compounds for biological interrogation of ferroptosis and constitute starting points for development of improved inhibitors of GPX4.
Defects in insulin secretion play a central role in the pathogenesis of type 2 diabetes, yet the mechanisms driving beta-cell dysfunction remain poorly understood, and therapies to preserve ...glucose-dependent insulin release are inadequate. We report a luminescent insulin secretion assay that enables large-scale investigations of beta-cell function, created by inserting Gaussia luciferase into the C-peptide portion of proinsulin. Beta-cell lines expressing this construct cosecrete luciferase and insulin in close correlation, under both standard conditions or when stressed by cytokines, fatty acids, or ER toxins. We adapted the reporter for high-throughput assays and performed a 1,600-compound pilot screen, which identified several classes of drugs inhibiting secretion, as well as glucose-potentiated secretagogues that were confirmed to have activity in primary human islets. Requiring 40-fold less time and expense than the traditional ELISA, this assay may accelerate the identification of pathways governing insulin secretion and compounds that safely augment beta-cell function in diabetes.
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•Proinsulin-Gaussia luciferase fusion construct faithfully reports insulin secretion•Luciferase tracks closely with insulin under normal conditions and induced stress•Reporter enables large-scale study of beta-cell function rapidly and at low cost•1,600-compound screen using assay identifies new human glucose-dependent secretagogues
Current assays of beta-cell functions, while useful, have limitations. In this Resource, Burns et al. develop a luminescent reporter of insulin secretion that is 40 times less time-consuming and expensive than ELISA. They perform high-throughput assays of beta-cell function to identify drugs altering human insulin secretion.
The precise control of CRISPR-Cas9 activity is required for a number of genome engineering technologies. Here, we report a generalizable platform that provided the first synthetic small-molecule ...inhibitors of Streptococcus pyogenes Cas9 (SpCas9) that weigh <500 Da and are cell permeable, reversible, and stable under physiological conditions. We developed a suite of high-throughput assays for SpCas9 functions, including a primary screening assay for SpCas9 binding to the protospacer adjacent motif, and used these assays to screen a structurally diverse collection of natural-product-like small molecules to ultimately identify compounds that disrupt the SpCas9-DNA interaction. Using these synthetic anti-CRISPR small molecules, we demonstrated dose and temporal control of SpCas9 and catalytically impaired SpCas9 technologies, including transcription activation, and identified a pharmacophore for SpCas9 inhibition using structure-activity relationships. These studies establish a platform for rapidly identifying synthetic, miniature, cell-permeable, and reversible inhibitors against both SpCas9 and next-generation CRISPR-associated nucleases.
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•Developed high-throughput assays for SpCas9 and performed a small-molecule screen•Identified reversible and cell-permeable inhibitors that disrupt SpCas9-DNA binding•Inhibitors allow dose and temporal control of (non)-nuclease-based SpCas9 systems•Identified a pharmacophore for SpCas9 inhibition using structure-activity studies
A suite of high-throughput assays enables discovery of small-molecule inhibitors of CRISPR-Cas9 that are cell permeable, and non-toxic, providing a chemical means to control SpCas9-based tools.
Computational methods for image-based profiling are under active development, but their success hinges on assays that can capture a wide range of phenotypes. We have developed a multiplex cytological ...profiling assay that "paints the cell" with as many fluorescent markers as possible without compromising our ability to extract rich, quantitative profiles in high throughput. The assay detects seven major cellular components. In a pilot screen of bioactive compounds, the assay detected a range of cellular phenotypes and it clustered compounds with similar annotated protein targets or chemical structure based on cytological profiles. The results demonstrate that the assay captures subtle patterns in the combination of morphological labels, thereby detecting the effects of chemical compounds even though their targets are not stained directly. This image-based assay provides an unbiased approach to characterize compound- and disease-associated cell states to support future probe discovery.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Predicting assay results for compounds virtually using chemical structures and phenotypic profiles has the potential to reduce the time and resources of screens for drug discovery. Here, we evaluate ...the relative strength of three high-throughput data sources-chemical structures, imaging (Cell Painting), and gene-expression profiles (L1000)-to predict compound bioactivity using a historical collection of 16,170 compounds tested in 270 assays for a total of 585,439 readouts. All three data modalities can predict compound activity for 6-10% of assays, and in combination they predict 21% of assays with high accuracy, which is a 2 to 3 times higher success rate than using a single modality alone. In practice, the accuracy of predictors could be lower and still be useful, increasing the assays that can be predicted from 37% with chemical structures alone up to 64% when combined with phenotypic data. Our study shows that unbiased phenotypic profiling can be leveraged to enhance compound bioactivity prediction to accelerate the early stages of the drug-discovery process.
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