A guiding map for inflammation.

Biologists, physicians and immunologists have contributed to the understanding of the cellular participants and biological pathways involved in inflammation. Here, we provide a general guide to the cellular and humoral contributors to inflammation as well as to the pathways that characterize inflammation in specific organs and tissues.

NRF2 activators inhibit influenza A virus replication by interfering with nucleo-cytoplasmic export of viral RNPs in an NRF2-independent manner.

In addition to antioxidative and anti-inflammatory properties, activators of the cytoprotective nuclear factor erythroid-2-like-2 (NRF2) signaling pathway have antiviral effects, but the underlying antiviral mechanisms are incompletely understood. We evaluated the ability of the NRF2 activators 4-octyl itaconate (4OI), bardoxolone methyl (BARD), sulforaphane (SFN), and the inhibitor of exportin-1 (XPO1)-mediated nuclear export selinexor (SEL) to interfere with influenza virus A/Puerto Rico/8/1934 (H1N1) infection of human cells. All compounds reduced viral titers in supernatants from A549 cells and vascular endothelial cells in the order of efficacy SEL>4OI>BARD = SFN, which correlated with their ability to prevent nucleo-cytoplasmic export of viral nucleoprotein and the host cell protein p53. In contrast, intracellular levels of viral HA mRNA and nucleocapsid protein (NP) were unaffected. Knocking down mRNA encoding KEAP1 (the main inhibitor of NRF2) or inactivating the NFE2L2 gene (which encodes NRF2) revealed that physiologic NRF2 signaling restricts IAV replication. However, the antiviral effect of all compounds was NRF2-independent. Instead, XPO1 knock-down greatly reduced viral titers, and incubation of Calu3 cells with an alkynated 4OI probe demonstrated formation of a covalent complex with XPO1. Ligand-target modelling predicted covalent binding of all three NRF2 activators and SEL to the active site of XPO1 involving the critical Cys528. SEL and 4OI manifested the highest binding energies, whereby the 4-octyl tail of 4OI interacted extensively with the hydrophobic groove of XPO1, which binds nuclear export sequences on cargo proteins. Conversely, SEL as well as the three NRF2 activators were predicted to covalently bind the functionally critical Cys151 in KEAP1. Blocking XPO1-mediated nuclear export may, thus, constitute a "noncanonical" mechanism of anti-influenza activity of electrophilic NRF2 activators that can interact with similar cysteine environments at the active sites of XPO1 and KEAP1. Considering the importance of XPO1 function to a variety of pathogenic viruses, compounds that are optimized to inhibit both targets may constitute an important class of broadly active host-directed treatments that embody anti-inflammatory, cytoprotective, and antiviral properties.

The Biological Activities of Polyether Ionophore Antibiotic Routiennocin is Independent of Absolute Stereochemistry.

Carboxylic polyether ionophores (CPIs) are among the most prevalent agricultural antibiotics (notably in the US) and these compounds have been in use for decades. The potential to reposition CPIs beyond veterinary use, e. g. through chemical modifications to enhance their selectivity window, is an exciting challenge and opportunity, considering their general resilience towards resistance development. Given the very large societal impact of these somewhat controversial compounds, it is surprising that many aspects of their mechanisms and activities in cells remain unclear. Here, we report comparative biological activities of the CPI routiennocin and two stereoisomers, including its enantiomer. We used an efficient convergent synthesis strategy to access the compounds and conducted a broad survey of antibacterial activities against planktonic cells and biofilms as well as the compounds' effects on mammalian cells, the latter assessed both via standard cell viability assays and broad morphological profiling. Interestingly, similar bioactivity of the enantiomeric pair was observed across all assays, strongly suggesting that chiral interactions do not play a decisive role in the mode of action. Overall, our findings are consistent with a mechanistic model involving highly dynamic behaviour of CPIs in biological membranes.

Enantioselective Synthesis of α-Quaternary Isochromanes by Oxidative Aminocatalysis and Gold Catalysis.

A novel strategy that combines oxidative aminocatalysis and gold catalysis allows the preparation of chiral α-quaternary isochromanes, a motif that is prevalent in natural products and synthetic bioactive compounds. In the first step, α-branched aldehydes and propargylic alcohols are transformed into α-quaternary ethers with excellent optical purities (>90 % ee) via oxidative umpolung with DDQ and an amino acid-derived primary amine catalyst. Subsequent gold(I)-catalyzed intramolecular hydroarylation affords the isochromane products with retention of the quaternary stereocenter. A second approach explores the use of allylic alcohols as reaction partners for the oxidative coupling to furnish α-quaternary ethers with generally lower enantiopurities. Stereoretentive cyclization to isochromane products is achieved via intramolecular Friedel-Crafts type alkylation with allylic acetates as a reactive handle. A number of synthetic elaborations and a biological study on these α-quaternary isochromanes highlight the potential applicability of the presented method.

An Enantioselective Aminocatalytic Cascade Reaction Affording Bioactive Hexahydroazulene Scaffolds.

A novel cascade reaction initiated by an enantioselective aminocatalysed 1,3-dipolar [6+4] cycloaddition between catalytically generated trienamines and 3-oxidopyridinium betaines is presented. The [6+4] cycloadduct spontaneously undergoes an intramolecular enamine-mediated aldol, hydrolysis, and E1cb sequence, which ultimately affords a chiral hexahydroazulene framework. In this process, three new C-C bonds and three new stereocenters are formed, enabled by a formal unfolding of the pyridine moiety from the dipolar reagent. The hexahydroazulenes are formed with excellent diastereo-, regio- and periselectivity (>20 : 1), up to 96 % ee, and yields up to 52 %. Synthetic elaborations of this scaffold were performed, providing access to a variety of functionalised hydroazulene compounds, of which some were found to display biological activity in U-2OS osteosarcoma cells in cell painting assays.

PSL Chemical Biology Symposia Third Edition: A Branch of Science in its Explosive Phase.

This symposium is the third PSL (Paris Sciences & Lettres) Chemical Biology meeting (2016, 2019, 2023) held at Institut Curie. This initiative originally started at Institut de Chimie des Substances Naturelles (ICSN) in Gif-sur-Yvette (2013, 2014), under the directorship of Professor Max Malacria, with a strong focus on chemistry. It was then continued at the Institut Curie (2015) covering a larger scope, before becoming the official PSL Chemical Biology meeting. This latest edition was postponed twice for the reasons that we know. This has given us the opportunity to invite additional speakers of great standing. This year, Institut Curie hosted around 300 participants, including 220 on site and over 80 online. The pandemic has had, at least, the virtue of promoting online meetings, which we came to realize is not perfect but has its own merits. In particular, it enables those with restricted time and resources to take part in events and meetings, which can now accommodate unlimited participants. We apologize to all those who could not attend in person this time due to space limitation at Institut Curie.

oxSTEF Reagents Are Tunable and Versatile Electrophiles for Selective Disulfide-Rebridging of Native Proteins.

Site-selective disulfide rebridging has emerged as a powerful strategy to modulate the structural and functional properties of proteins. Here, we introduce a novel class of electrophilic reagents, designated oxSTEF, that demonstrate excellent efficiency in disulfide rebridging via double thiol exchange. The oxSTEF reagents are prepared using an efficient synthetic sequence which may be diverted to obtain a range of derivatives allowing for tuning of reactivity or steric bulk. We demonstrate highly selective rebridging of cyclic peptides and native proteins, such as human growth hormone, and the absence of cross-reactivity with other nucleophilic amino acid residues. The oxSTEF conjugates undergo glutathione-mediated disintegration under tumor-relevant glutathione concentrations, which highlights their potential for use in targeted drug delivery. Finally, the α-dicarbonyl motif of the oxSTEF reagents enables "second phase" oxime ligation, which furthermore increases the thiol stability of the conjugates significantly.

A randomized, double-blind, crossover study of the effect of the fluoroquinolone moxifloxacin on glucose levels and insulin sensitivity in young men and women.

AIM: The voltage-gated potassium channel Kv 11.1 is important for repolarizing the membrane potential in excitable cells such as myocytes, pancreatic α- and ß-cells. Moxifloxacin blocks the Kv 11.1 channel and increases the risk of hypoglycaemia in patients with diabetes. We investigated glucose regulation and secretion of glucoregulatory hormones in young people with and without moxifloxacin, a drug known to block the Kv 11.1 channel. MATERIALS AND METHODS: The effect of moxifloxacin (800 mg/day for 4 days) or placebo on glucose regulation was assessed in a randomized, double-blind, crossover study of young men and women (age 20-40 years and body mass index 18.5-27.5 kg/m2 ) without chronic disease, using 6-h oral glucose tolerance tests and continuous glucose monitoring. RESULTS: Thirty-eight participants completed the study. Moxifloxacin prolonged the QTcF interval and increased heart rate. Hypoglycaemia was more frequently observed with moxifloxacin, both during the 8 days of continuous glucose monitoring and during the oral glucose tolerance tests. Hypoglycaemia questionnaire scores were higher after intake of moxifloxacin. Moxifloxacin reduced the early plasma-glucose response (AUC0-30 min ) by 7% (95% CI: -9% to -4%, p < .01), and overall insulin response (AUC0-360 min ) decreased by 18% (95% CI: -24% to -11%, p < .01) and plasma glucagon increased by 17% (95% CI: 4%-33%, p = .03). Insulin sensitivity calculated as the Matsuda index increased by 11%, and MISI, an index of muscle insulin sensitivity, increased by 34%. CONCLUSIONS: In young men and women, moxifloxacin, a drug known to block the Kv 11.1 channel, increased QT interval, decreased glucose levels and was associated with increased muscle insulin sensitivity and more frequent episodes of hypoglycaemia.

An atlas of human long non-coding RNAs with accurate 5' ends.

Long non-coding RNAs (lncRNAs) are largely heterogeneous and functionally uncharacterized. Here, using FANTOM5 cap analysis of gene expression (CAGE) data, we integrate multiple transcript collections to generate a comprehensive atlas of 27,919 human lncRNA genes with high-confidence 5' ends and expression profiles across 1,829 samples from the major human primary cell types and tissues. Genomic and epigenomic classification of these lncRNAs reveals that most intergenic lncRNAs originate from enhancers rather than from promoters. Incorporating genetic and expression data, we show that lncRNAs overlapping trait-associated single nucleotide polymorphisms are specifically expressed in cell types relevant to the traits, implicating these lncRNAs in multiple diseases. We further demonstrate that lncRNAs overlapping expression quantitative trait loci (eQTL)-associated single nucleotide polymorphisms of messenger RNAs are co-expressed with the corresponding messenger RNAs, suggesting their potential roles in transcriptional regulation. Combining these findings with conservation data, we identify 19,175 potentially functional lncRNAs in the human genome.

Detrimental Actions of Chlorinated Nucleosides on the Function and Viability of Insulin-Producing Cells.

Neutrophils are innate immune cells that play a key role in pathogen clearance. They contribute to inflammatory diseases, including diabetes, by releasing pro-inflammatory cytokines, reactive oxygen species, and extracellular traps (NETs). NETs contain a DNA backbone and catalytically active myeloperoxidase (MPO), which produces hypochlorous acid (HOCl). Chlorination of the DNA nucleoside 8-chloro-deoxyguanosine has been reported as an early marker of inflammation in diabetes. In this study, we examined the reactivity of different chlorinated nucleosides, including 5-chloro-(deoxy)cytidine (5ClC, 5CldC), 8-chloro-(deoxy)adenosine (8ClA, 8CldA) and 8-chloro-(deoxy)guanosine (8ClG, 8CldG), with the INS-1E ß-cell line. Exposure of INS-1E cells to 5CldC, 8CldA, 8ClA, and 8CldG decreased metabolic activity and intracellular ATP, and, together with 8ClG, induced apoptotic cell death. Exposure to 8ClA, but not the other nucleosides, resulted in sustained endoplasmic reticulum stress, activation of the unfolded protein response, and increased expression of thioredoxin-interacting protein (TXNIP) and heme oxygenase 1 (HO-1). Exposure of INS-1E cells to 5CldC also increased TXNIP and NAD(P)H dehydrogenase quinone 1 (NQO1) expression. In addition, a significant increase in the mRNA expression of NQO1 and GPx4 was seen in INS-1E cells exposed to 8ClG and 8CldA, respectively. However, a significant decrease in intracellular thiols was only observed in INS-1E cells exposed to 8ClG and 8CldG. Finally, a significant decrease in the insulin stimulation index was observed in experiments with all the chlorinated nucleosides, except for 8ClA and 8ClG. Together, these results suggest that increased formation of chlorinated nucleosides during inflammation in diabetes could influence ß-cell function and may contribute to disease progression.

Obesity-induced changes in gene expression in feline adipose and skeletal muscle tissue.

Indoor-confined cats are prone to developing obesity due to a sedentary life and an energy intake exceeding energy requirements. As in humans, feline obesity decreases insulin sensitivity and increases the risk of developing feline diabetes mellitus, but the pathophysiological mechanisms are currently poorly understood. Human obesity-related metabolic alterations seem to relate to changes in the expression of genes involved in glucose metabolism, insulin action and inflammation. The objective of the current study was to investigate changes in the expression of genes relating to obesity, glucose metabolism and inflammation in cats with non-experimentally induced obesity. Biopsies from the sartorius muscle and subcutaneous adipose tissue were obtained from 73 healthy, neutered, indoor-confined domestic shorthaired cats ranging from lean to obese. Quantification of obesity-related gene expression levels relative to glyceraldehyde-3-phosphate dehydrogenase was performed by quantitative real-time polymerase chain reaction. A negative association between obesity and adiponectin expression was observed in the adipose tissue (mean ± SD; normal weight, 27.30 × 10-3 ± 77.14 × 10-3 ; overweight, 2.89 × 10-3 ± 0.38 × 10-3 and obese, 2.93 × 10-3 ± 4.20 × 10-3 , p < 0.05). In muscle, the expression of peroxisome proliferative activated receptor-γ2 and plasminogen activator inhibitor-1 was increased in the obese compared to the normal-weight cats, and resistin was increased in the normal-weight compared to the overweight cats. There were no detectable obesity-related changes in the messenger RNA levels of inflammatory cytokines. In conclusion, a possible obesity-related low-grade inflammation caused by increased expression of key proinflammatory regulators was not observed. This could imply that the development of feline obesity and ensuing insulin resistance may not be based on tissue-derived inflammation, but caused by several determining factors, many of which still need further investigation.

A Blunted GPR183/Oxysterol Axis During Dysglycemia Results in Delayed Recruitment of Macrophages to the Lung During Mycobacterium tuberculosis Infection.

BACKGROUND: We previously reported that reduced GPR183 expression in blood from tuberculosis (TB) patients with diabetes is associated with more severe TB. METHODS: To further elucidate the role of GPR183 and its oxysterol ligands in the lung, we studied dysglycemic mice infected with Mycobacterium tuberculosis (Mtb). RESULTS: We found upregulation of the oxysterol-producing enzymes CH25H and CYP7B1 and increased concentrations of 25-hydroxycholesterol upon Mtb infection in the lungs of mice. This was associated with increased expression of GPR183 indicative of oxysterol-mediated recruitment of GPR183-expressing immune cells to the lung. CYP7B1 was predominantly expressed by macrophages in TB granulomas. CYP7B1 expression was significantly blunted in lungs from dysglycemic animals, which coincided with delayed macrophage infiltration. GPR183-deficient mice similarly had reduced macrophage recruitment during early infection. CONCLUSIONS: Taken together, we demonstrate a requirement of the GPR183/oxysterol axis for positioning of macrophages to the site of infection and add an explanation to more severe TB in diabetes patients.

α-Lactam Electrophiles for Covalent Chemical Biology.

Electrophilic groups are one of the key pillars of contemporary chemical biology and medicinal chemistry. For instance, 3-membered N-heterocyclic compounds-such as aziridines, azirines, and oxaziridines-possess unique electronic and structural properties which underlie their potential and applicability as covalent tools. The α-lactams are also members of this group of compounds, however, their utility within the field remains unexplored. Here, we demonstrate an α-lactam reagent (AM2) that is tolerant to aqueous buffers while being reactive towards biologically relevant nucleophiles. Interestingly, carboxylesterases 1 and 2 (CES1/2), both serine hydrolases with key roles in endo- and xenobiotic metabolism, were found as primary covalent targets for AM2 in HepG2 liver cancer cells. All in all, this study constitutes the starting point for the further development and exploration of α-lactam-based electrophilic probes in covalent chemical biology.

Forward Chemical Genetic Screen for Oxygen-Dependent Cytotoxins Uncovers New Covalent Fragments that Target GPX4.

The identification of growth inhibitory compounds with the ability to selectively target the cellular oxygenation state may be of therapeutic interest. Here, a phenotypic screen of a covalent fragment library revealed diverse compounds containing propiolamide warheads with selective toxicity for liver cancer cells in normoxic conditions. Target identification and validation through CETSA and direct pulldown experiments demonstrated that several compounds target glutathione peroxidase 4 (GPX4) and induce ferroptotic cell death. Although being an oxidative cell death mechanism, ferroptosis can be induced also under hypoxic conditions. Prompted by the selective toxicity discovered in the screen, we mapped the oxygen-dependence of several ferroptosis-inducing compounds across three different cell lines. These studies revealed combinations with notable reductions in sensitivity under hypoxic conditions. These observations are mechanistically interesting and may be relevant for the use of ferroptosis-inducers as anti-cancer agents.

Total Synthesis of Natural Products Containing Enamine or Enol Ether Derivatives.

Enamine and enol ethers are nucleophilic functional groups that are well known to most chemists. When enamine or enol ethers are present in natural products, they are nearly exclusively found as derivatives having a direct connection to electron-withdrawing groups for stabilization, and the resulting larger entities, such as enamides or enol acylates, can be further extended or modified in the framework of natural products. The restricted conformational space that is associated with even simple enamine and enol ether derivatives can be a strong determinant of the overall molecular structure, and the more polarized derivatives can endow some natural products with electrophilic properties and thus facilitate covalent interactions with biological targets.In this Account, I describe our efforts (published since 2016) to prepare natural products from several different classes that all feature enamine or enol ether derivatives as key functionalities. Our choice of targets has been guided by a desire to illuminate unknown biological mechanisms associated with the compounds or, alternatively, to improve upon known biological activities that appear to be promising from a biomedical perspective. In the present text, however, the exclusive focus will be on the syntheses.First, I will discuss the basic properties of the functional groups and briefly present a small collection of illustrative and inspirational examples from the literature for their construction in different complex settings. Next, I will provide an overview of our work on the macrocyclic APD-CLD natural products, rakicidin A and BE-43547A1, involving the development of an efficient macrocyclization strategy and the development of methods to construct the hallmark APD group: a modified enamide. The synthesis of the meroterpenoid strongylophorine-26 is discussed next, where we developed an oxidative quinone methoxylation to build a vinylogous ester group in the final step of the synthesis and employed FeCl3-mediated cascade reactions for the rapid assembly of the overall scaffold to enable a short semisynthesis from isocupressic acid. An efficient core scaffold assembly was also in focus in our synthesis of the alkaloid streptazone A with the signature enaminone system being assembled through a rhodium-catalyzed Pauson-Khand reaction. Sequential, site-selective redox manipulations were developed to arrive at strepatzone A and additional members of the natural product family. Finally, I discuss our work to prepare analogs of complex polyether ionophores featuring functionalized tetronic acids as cation-binding groups. A method for the construction of a suitably protected chloromethylidene-modified tetronate is presented which enabled its installation in the full structure through a C-acylation reaction. This work exemplifies how components of abundant polyether ionophores can be recycled and used to access new structures which may possess enhanced biological activities.

Triculamin: An Unusual Lasso Peptide with Potent Antimycobacterial Activity.

Lasso peptides are ribosomally synthesized and post-translationally modified peptides (RiPPs) produced by microorganisms. Here we show that the two natural products triculamin and alboverticillin, originally isolated in 1967 and 1958, respectively, with potent and specific activity against mycobacteria are in fact the same lasso peptide. We solved the structure using 2D NMR spectroscopy and expanded on the previously reported bioactivity. Through genome sequencing, we identify the responsible biosynthetic gene clusters, which curiously revealed that, unlike any known lasso peptides, their precursor peptides appear to have a follower instead of a leader peptide.

Organocatalytic Asymmetric Multicomponent Cascade Reaction for the Synthesis of Contiguously Substituted Tetrahydronaphthols.

Isobenzopyrylium ions are unique, highly reactive, aromatic intermediates which are largely unexplored in asymmetric catalysis despite their high potential synthetic utility. In this study, an organocatalytic asymmetric multicomponent cascade via dienamine catalysis, involving a cycloaddition, a nucleophilic addition, and a ring-opening reaction, is disclosed. The reaction furnishes chiral tetrahydronaphthols containing four contiguous stereocenters in good to high yield, high diastereoselectivity (up to >20:1), and excellent enantioselectivity (93-98% ee). The obtained products are important synthetic intermediates, and it is demonstrated that they can be used for the generation of frameworks such as octahydrobenzo[h]isoquinoline and [2.2.2]octane scaffolds. Furthermore, mechanistic experiments involving oxygen-18-labeling studies and density functional theory calculations provide a vivid picture of the reaction mechanism. Finally, the bioactivity of 16 representative tetrahydronaphthol compounds has been evaluated in U-2OS cancer cells with some compounds showing a unique profile and a clear morphological change.

Photophysics of a protein-bound derivative of malachite green that sensitizes the production of singlet oxygen.

Genetically encodable proteins that photosensitize the production of singlet oxygen, O2(a1Δg), will play an increasingly important role in elucidating mechanisms of cellular processes modulated by reactive oxygen species, ROS, and changes in redox balance. In the development of such tools, it is essential to characterize the oxygen-dependent photophysics of the protein-encased chromophore. Of the O2(a1Δg)-photosensitizing systems recently developed, a protein-bound derivative of Malachite Green has several desirable features: (1) it absorbs light at wavelengths longer than those typically absorbed by endogenous molecules, and (2) the chromophore becomes a viable sensitizer only when bound to the activating protein. However, we now demonstrate that the photophysics of this Malachite Green system is not simple. Our data indicate that, with an increase in the concentration of ground-state oxygen, O2(X3Σg-), the yield of O2(a1Δg) does not increase in a proportional manner. Moreover, the lifetime of O2(a1Δg) decreases as the O2(X3Σg-) concentration is increased. One mechanism that could account for our observations involves the concomitant photo-initiated formation of O2(a1Δg) and the superoxide radical anion. We propose that the superoxide ion acts as a dynamic diffusion-dependent quencher to influence the O2(a1Δg) lifetime and as a static quencher within the protein enclosure to influence the measured O2(a1Δg) yield. Thus, in the least, caution should be exercised when using this Malachite Green system to probe mechanisms of ROS-mediated processes. Our results contribute to a better understanding of the general photophysics of protein-bound O2(a1Δg) sensitizers which, in turn, facilitates the further development of these useful mechanistic tools.

Divalent Metal Transporter 1 Knock-Down Modulates IL-1ß Mediated Pancreatic Beta-Cell Pro-Apoptotic Signaling Pathways through the Autophagic Machinery.

Pro-inflammatory cytokines promote cellular iron-import through enhanced divalent metal transporter-1 (DMT1) expression in pancreatic ß-cells, consequently cell death. Inhibition of ß-cell iron-import by DMT1 silencing protects against apoptosis in animal models of diabetes. However, how alterations of signaling networks contribute to the protective action of DMT1 knock-down is unknown. Here, we performed phosphoproteomics using our sequential enrichment strategy of mRNA, protein, and phosphopeptides, which enabled us to explore the concurrent molecular events in the same set of wildtype and DMT1-silenced ß-cells during IL-1ß exposure. Our findings reveal new phosphosites in the IL-1ß-induced proteins that are clearly reverted by DMT1 silencing towards their steady-state levels. We validated the levels of five novel phosphosites of the potential protective proteins using parallel reaction monitoring. We also confirmed the inactivation of autophagic flux that may be relevant for cell survival induced by DMT1 silencing during IL-1ß exposure. Additionally, the potential protective proteins induced by DMT1 silencing were related to insulin secretion that may lead to improving ß-cell functions upon exposure to IL-1ß. This global profiling has shed light on the signal transduction pathways driving the protection against inflammation-induced cell death in ß-cells after DMT1 silencing.