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Screening of small-molecule libraries is a productive method for identifying both chemical probes of disease-related targets and potential starting points for drug discovery. In this article, we focus on strategies such as diversity-oriented synthesis that aim to explore novel areas of chemical space efficiently by populating small-molecule libraries with compounds containing structural features that are typically under-represented in commercially available screening collections. Drawing from more than a decade's worth of examples, we highlight how the design and synthesis of such libraries have been enabled by modern synthetic chemistry, and we illustrate the impact of the resultant chemical probes and drug leads in a wide range of diseases.

Ferroptosis—an iron-dependent, non-apoptotic cell death process—is involved in various degenerative diseases and represents a targetable susceptibility in certain cancers 1 . The ferroptosis-susceptible cell state can either pre-exist in cells that arise from certain lineages or be acquired during cell-state transitions 2 – 5 . 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. The cellular organelles peroxisomes contribute to the sensitivity of cells to ferroptosis by synthesizing polyunsaturated ether phospholipids, and changes in the abundances of these lipids are associated with altered sensitivity to ferroptosis during cell-state transitions.

mTOR is a central regulator of cellular growth and metabolism. Using metabolic profiling and numerous small-molecule probes, we investigated whether mTOR affects immediate control over cellular metabolism by posttranslational mechanisms. Inhibiting the FKBP12/rapamycin-sensitive subset of mTOR functions in leukemic cells enhanced aerobic glycolysis and decreased uncoupled mitochondrial respiration within 25 min. mTOR is in a complex with the mitochondrial outer-membrane protein Bcl-xl and VDAC1. Bcl-xl, but not VDAC1, is a kinase substrate for mTOR in vitro, and mTOR regulates the association of Bcl-xl with mTOR. Inhibition of mTOR not only enhances aerobic glycolysis, but also induces a state of increased dependence on aerobic glycolysis in leukemic cells, as shown by the synergy between the glycolytic inhibitor 2-deoxyglucose and rapamycin in decreasing cell viability.

Cancer persister cells, which survive cytotoxic treatments, are shown to be sensitive to inhibition of the lipid hydroperoxidase GPX4. Reservoir of resistance During cancer treatment, tumours can become drug-resistant. In addition, so-called persister cells can emerge and form a reservoir from which resistant cancer cells can originate. Persister cells are no longer sensitive to some drugs, but Michael McManus and colleagues now report that they exist in a mesenchymal state in which they are selectively sensitive to the inhibition of the lipid hydroperoxidase GPX4. Targeting GPX4 could therefore represent a new therapeutic avenue to potentially prevent drug resistance. Acquired drug resistance prevents cancer therapies from achieving stable and complete responses 1 . Emerging evidence implicates a key role for non-mutational drug resistance mechanisms underlying the survival of residual cancer ‘persister’ cells 2 , 3 , 4 . The persister cell pool constitutes a reservoir from which drug-resistant tumours may emerge. Targeting persister cells therefore presents a therapeutic opportunity to impede tumour relapse 5 . We previously found that cancer cells in a high mesenchymal therapy-resistant cell state are dependent on the lipid hydroperoxidase GPX4 for survival 6 . Here we show that a similar therapy-resistant cell state underlies the behaviour of persister cells derived from a wide range of cancers and drug treatments. Consequently, we demonstrate that persister cells acquire a dependency on GPX4. Loss of GPX4 function results in selective persister cell ferroptotic death in vitro and prevents tumour relapse in mice. These findings suggest that targeting of GPX4 may represent a therapeutic strategy to prevent acquired drug resistance.

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. Nitrile-oxide electrophiles were identified as covalent inhibitors of GPX4 that exhibit increased selectivity and reduced off-target effects relative to chloroacetamide-based inhibitors.

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. Clear-cell carcinomas are aggressive tumours characterised by high accumulation of lipids and glycogen. Here, the authors report that these cancers have a common vulnerability to GPX4 inhibition-induced ferroptosis and using CRISPR screen and lipodomic profiling, they identify HIF-2α- HILPDA axis promotes ferroptosis via enrichment of PUFA lipids.

Lenalidomide is a drug with clinical efficacy in multiple myeloma and other B cell neoplasms, but its mechanism of action is unknown. Using quantitative proteomics, we found that lenalidomide causes selective ubiquitination and degradation of two lymphoid transcription factors, IKZF1 and IKZF3, by the CRBN-CRL4 ubiquitin ligase. IKZF1 and IKZF3 are essential transcription factors in multiple myeloma. A single amino acid substitution of IKZF3 conferred resistance to lenalidomide-induced degradation and rescued lenalidomide-induced inhibition of cell growth. Similarly, we found that lenalidomide-induced interleukin-2 production in T cells is due to depletion of IKZF1 and IKZF3. These findings reveal a previously unknown mechanism of action for a therapeutic agent: alteration of the activity of an E3 ubiquitin ligase, leading to selective degradation of specific targets.

\"Organic synthesis\" is a compound-creating activity often focused on biologically active small molecules. This special issue of PNAS explores innovations and trends in the field that are enabling the synthesis of new types of small-molecule probes and drugs. This perspective article frames the research described in the special issue but also explores how these modern capabilities can both foster a new and more extensive view of basic research in the academy and promote the linkage of life-science research to the discovery of novel types of small-molecule therapeutics [Schreiber SL (2009) Chem Bio Chem 10:26-29]. This new view of basic research aims to bridge the chasm between basic scientific discoveries in life sciences and new drugs that treat the root cause of human disease--recently referred to as the \"valley of death\" for drug discovery. This perspective article describes new roles that modern organic chemistry will need to play in overcoming this challenge.

Despite remarkable success of immune checkpoint inhibitors, the majority of cancer patients have yet to receive durable benefits. Here, in order to investigate the metabolic alterations in response to immune checkpoint blockade, we comprehensively profile serum metabolites in advanced melanoma and renal cell carcinoma patients treated with nivolumab, an antibody against programmed cell death protein 1 (PD1). We identify serum kynurenine/tryptophan ratio increases as an adaptive resistance mechanism associated with worse overall survival. This advocates for patient stratification and metabolic monitoring in immunotherapy clinical trials including those combining PD1 blockade with indoleamine 2,3-dioxygenase/tryptophan 2,3-dioxygenase   (IDO/TDO) inhibitors. Immune-checkpoint inhibition therapy has achieved success in a subset of patients. Here the authors profiled about 200 relevant metabolites in patient serum samples from three independent immunotherapy trials and found the serum kynurenine/tryptophan ratio increases to be associated with worse overall survival.