Natural products and synthetic small molecules can be used to perturb, dissect and manipulate biological processes, thereby providing the basis for drug development. Over the past decades, the ...evolution of molecular biology protocols and microscopy techniques has made it possible to visually detect proteins in living systems with valuable spatiotemporal resolution, in which dynamic topological information has proved to be insightful. By contrast, although small molecules have become essential for biological studies, general methods to track them in cells remain underexplored. In this Review, we discuss how bioorthogonal chemistry, and click chemistry in particular, can be exploited to label and visualize almost any biologically active small molecule in cells and tissues. We review recent developments, highlighting cases in which visualizing small molecules has provided crucial mechanistic insights. This methodology is facile to implement, is versatile and is illuminating.Click chemistry enables efficient chemical labelling of small molecules in cells, providing a powerful method to visualize almost any biologically active compound. This versatile methodology can provide valuable information about the mechanisms of action of small molecules in various biological settings.
CD44 is a transmembrane glycoprotein linked to various biological processes reliant on epigenetic plasticity, which include development, inflammation, immune responses, wound healing and cancer ...progression. Although it is often referred to as a cell surface marker, the functional regulatory roles of CD44 remain elusive. Here we report the discovery that CD44 mediates the endocytosis of iron-bound hyaluronates in tumorigenic cell lines, primary cancer cells and tumours. This glycan-mediated iron endocytosis mechanism is enhanced during epithelial-mesenchymal transitions, in which iron operates as a metal catalyst to demethylate repressive histone marks that govern the expression of mesenchymal genes. CD44 itself is transcriptionally regulated by nuclear iron through a positive feedback loop, which is in contrast to the negative regulation of the transferrin receptor by excess iron. Finally, we show that epigenetic plasticity can be altered by interfering with iron homeostasis using small molecules. This study reveals an alternative iron-uptake mechanism that prevails in the mesenchymal state of cells, which illuminates a central role of iron as a rate-limiting regulator of epigenetic plasticity.
Cancer stem cells (CSCs) represent a subset of cells within tumours that exhibit self-renewal properties and the capacity to seed tumours. CSCs are typically refractory to conventional treatments and ...have been associated to metastasis and relapse. Salinomycin operates as a selective agent against CSCs through mechanisms that remain elusive. Here, we provide evidence that a synthetic derivative of salinomycin, which we named ironomycin (AM5), exhibits a more potent and selective activity against breast CSCs in vitro and in vivo, by accumulating and sequestering iron in lysosomes. In response to the ensuing cytoplasmic depletion of iron, cells triggered the degradation of ferritin in lysosomes, leading to further iron loading in this organelle. Iron-mediated production of reactive oxygen species promoted lysosomal membrane permeabilization, activating a cell death pathway consistent with ferroptosis. These findings reveal the prevalence of iron homeostasis in breast CSCs, pointing towards iron and iron-mediated processes as potential targets against these cells.
Methods for generating molecular diversity provide a route to screen a wider section of chemical space, to discover compounds with useful biological properties. Now, a complexity-to-diversity ...strategy has enabled the discovery of a multi-cyclic structure from a complex natural product that induces ferroptotic cell death in cancer cells.
Persister cancer cells represent rare populations of cells resistant to therapy. Cancer cells can exploit epithelial-mesenchymal plasticity to adopt a drug-tolerant state that does not depend on ...genetic alterations. Small molecules that can interfere with cell plasticity or kill cells in a cell state-dependent manner are highly sought after. Salinomycin has been shown to kill cancer cells in the mesenchymal state by sequestering iron in lysosomes, taking advantage of the iron addiction of this cell state. Here, we report the chemo- and stereoselective synthesis of a series of structurally complex small molecule chimeras of salinomycin derivatives and the iron-reactive dihydroartemisinin. We show that these chimeras accumulate in lysosomes and can react with iron to release bioactive species, thereby inducing ferroptosis in drug-tolerant pancreatic cancer cells and biopsy-derived organoids of pancreatic ductal adenocarcinoma. This work paves the way toward the development of new cancer medicines acting through active ferroptosis.
Enoxacin is a small molecule that stimulates RNA interference (RNAi) and acts as a growth inhibitor selectively in cancer but not in untransformed cells. Here, we used alkenox, a clickable enoxacin ...surrogate, coupled with quantitative mass spectrometry, to identify PIWIL3 as a mechanistic target of enoxacin. PIWIL3 is an Argonaute protein of the PIWI subfamily that is mainly expressed in the germline and that mediates RNAi through piRNAs. Our results suggest that cancer cells re-express PIWIL3 to repress RNAi through miRNAs and thus open a new opportunity for cancer-specific targeting.
High-grade serous ovarian cancer (HGSOC) remains an unmet medical challenge. Here, we unravel an unanticipated metabolic heterogeneity in HGSOC. By combining proteomic, metabolomic, and bioergenetic ...analyses, we identify two molecular subgroups, low- and high-OXPHOS. While low-OXPHOS exhibit a glycolytic metabolism, high-OXPHOS HGSOCs rely on oxidative phosphorylation, supported by glutamine and fatty acid oxidation, and show chronic oxidative stress. We identify an important role for the PML-PGC-1α axis in the metabolic features of high-OXPHOS HGSOC. In high-OXPHOS tumors, chronic oxidative stress promotes aggregation of PML-nuclear bodies, resulting in activation of the transcriptional co-activator PGC-1α. Active PGC-1α increases synthesis of electron transport chain complexes, thereby promoting mitochondrial respiration. Importantly, high-OXPHOS HGSOCs exhibit increased response to conventional chemotherapies, in which increased oxidative stress, PML, and potentially ferroptosis play key functions. Collectively, our data establish a stress-mediated PML-PGC-1α-dependent mechanism that promotes OXPHOS metabolism and chemosensitivity in ovarian cancer.
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•High-grade serous ovarian cancers display OXPHOS metabolic heterogeneity•High-OXPHOS show high electron transport chain synthesis and respiration rate•Oxidative stress in high-OXPHOS HGSOC activates PML-PGC-1α and ETC transcription•High-OXPHOS HGSOCs show enhanced chemosensitivity through oxidative stress and PML
Gentric et al. identify metabolically heterogeneous OXPHOS subgroups in high-grade serous ovarian cancers (HGSOCs). In high-OXPHOS tumors, chronic oxidative stress promotes aggregation of PML-nuclear bodies that activate PGC-1α, electron transport chain synthesis, and mitochondrial respiration. High-OXPHOS HGSOCs exhibit chemosensitivity, in which increased oxidative stress and PML play key functions.
The success of new therapies hinges on our ability to understand their molecular and cellular mechanisms of action. We modified BET bromodomain inhibitors, an epigenetic-based therapy, to create ...functionally conserved compounds that are amenable to click chemistry and can be used as molecular probes in vitro and in vivo. We used click proteomics and click sequencing to explore the gene regulatory function of BRD4 (bromodomain containing protein 4) and the transcriptional changes induced by BET inhibitors. In our studies of mouse models of acute leukemia, we used high-resolution microscopy and flow cytometry to highlight the heterogeneity of drug activity within tumor cells located in different tissue compartments. We also demonstrate the differential distribution and effects of BET inhibitors in normal and malignant cells in vivo. This study provides a potential framework for the preclinical assessment of a wide range of drugs.
A method to obtain aryl nitriles from the corresponding halides by Pd catalysis, in the absence of any cyanide source, is reported. The reaction of an aryl halide, ethyl nitroacetate, and an olefin ...readily delivers an aromatic nitrile. A variety of aryl iodides/bromides have been converted into the corresponding cyanoarenes in fair to excellent yields. The reaction likely involves the following steps: (a) Pd-catalyzed α-arylation of ethyl nitroacetate; (b) nitrile oxide formation; (c) 3 + 2-cycloaddition with an olefin to provide an isoxazoline; (d) isoxazoline cleavage to benzonitrile formation.
Arf GTPases and their guanine nucleotide exchange factors (ArfGEFs) are major regulators of membrane traffic and organelle structure in cells. They are associated with a variety of diseases and are ...thus attractive therapeutic targets for inhibition by small molecules. Several inhibitors of unrelated chemical structures have been discovered, which have shown their potential in dissecting molecular pathways and blocking disease-related functions. However, their specificity across the ArfGEF family has remained elusive. Importantly, inhibitory responses in the context of membranes, which are critical determinants of Arf and ArfGEF cellular functions, have not been investigated. Here, we compare the efficiency and specificity of four structurally distinct ArfGEF inhibitors, Brefeldin A, SecinH3, M-COPA, and NAV-2729, toward six ArfGEFs (human ARNO, EFA6, BIG1, and BRAG2 and Legionella and Rickettsia RalF). Inhibition was assessed by fluorescence kinetics using pure proteins, and its modulation by membranes was determined with lipidated GTPases in the presence of liposomes. Our analysis shows that despite the intra-ArfGEF family resemblance, each inhibitor has a specific inhibitory profile. Notably, M-COPA is a potent pan-ArfGEF inhibitor, and NAV-2729 inhibits all GEFs, the strongest effects being against BRAG2 and Arf1. Furthermore, the presence of the membrane-binding domain in Legionella RalF reveals a strong inhibitory effect of BFA that is not measured on its GEF domain alone. This study demonstrates the value of family-wide assays with incorporation of membranes, and it should enable accurate dissection of Arf pathways by these inhibitors to best guide their use and development as therapeutic agents.
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