Two aromatic amines (ferrostatin-1 and liproxstatin-1) were recently identified from high-throughput screening efforts to uncover potent inhibitors of ferroptosis, the necrotic-like cell death induced by inhibition of glutathione peroxidase 4 (GPX4), deletion of the corresponding gpx4 gene, or starvation of GPX4 of its reducing cosubstrate, glutathione (GSH). We have since demonstrated that these two aromatic amines are highly effective radical-trapping antioxidants (RTAs) in lipid bilayers, suggesting that they subvert ferroptosis by inhibiting lipid peroxidation (autoxidation) and, thus, that this process drives the execution of ferroptosis. Herein, we show that diarylamine RTAs used to protect petroleum-derived products from autoxidation can be potent inhibitors of ferroptosis. The diarylamines investigated include representative examples of additives to engine oils, greases and rubber (4,4'-dialkyldiphenylamines), core structures of dyes and pharmaceuticals (phenoxazines and phenothiazines), and aza-analogues of these three classes of compounds that we have recently shown can be modified to achieve much greater reactivity. We find that regardless of how ferroptosis is induced (GPX4 inhibition, gpx4 deletion or GSH depletion), compounds which possess good RTA activity in organic solution (k > 10 M s) and lipid bilayers (k > 10 M s) are generally potent inhibitors of ferroptosis (in mouse embryonic fibroblasts). Likewise, structural analogs that do not possess RTA activity are devoid of antiferroptotic activity. These results further support the argument that lipid peroxidation (autoxidation) plays a major role in the mechanism of cell death induced by either GPX4 inhibition, gpx4 deletion, or GSH depletion. Moreover, it offers clear direction that ongoing medicinal chemistry efforts on liproxstatin and ferrostatin derivatives, which have been proposed as lead compounds for the treatment and/or prevention of ischemia/reperfusion injury, renal failure, and neurodegeneration, can be widened to include other aminic RTAs. To aid in these efforts, some relevant structure-reactivity relationships are discussed.
Choline is an essential nutrient that is required for synthesis of the main eukaryote phospholipid, phosphatidylcholine. Macrophages are innate immune cells that survey and respond to danger and damage signals. Although it is well-known that energy metabolism can dictate macrophage function, little is known as to the importance of choline homeostasis in macrophage biology. We hypothesized that the uptake and metabolism of choline are important for macrophage inflammation. Polarization of primary bone marrow macrophages with lipopolysaccharide (LPS) resulted in an increased rate of choline uptake and higher levels of PC synthesis. This was attributed to a substantial increase in the transcript and protein expression of the choline transporter-like protein-1 (CTL1) in polarized cells. We next sought to determine the importance of choline uptake and CTL1 for macrophage immune responsiveness. Chronic pharmacological or CTL1 antibody-mediated inhibition of choline uptake resulted in altered cytokine secretion in response to LPS, which was associated with increased levels of diacylglycerol and activation of protein kinase C. These experiments establish a previously unappreciated link between choline phospholipid metabolism and macrophage immune responsiveness, highlighting a critical and regulatory role for macrophage choline uptake via the CTL1 transporter.
Bioorthogonal chemical reactions have emerged as convenient and rapid methods for incorporating unnatural functionality into living systems. Different prototype reactions have been optimized for use in biological settings. Optimization of 3 + 2 dipolar cycloadditions involving nitrones has resulted in highly efficient reaction conditions for bioorthogonal chemistry. Through substitution at the nitrone carbon or nitrogen atom, stereoelectronic tuning of the reactivity of the dipole has assisted in optimizing reactivity. Nitrones have been shown to react rapidly with cyclooctynes with bimolecular rate constants approaching k 2 = 102 M–1 s–1, which are among the fastest bioorthogonal reactions reported (McKay et al. Org. Biomol. Chem. 2012, 10, 3066–3070). Nitrones have also been shown to react with trans-cyclooctenes (TCO) in strain-promoted TCO-nitrone cycloadditions reactions. Copper catalyzed reactions involving alkynes and nitrones have also been optimized for applications in biology. This review provides a comprehensive accounting of the different bioorthogonal reactions that have been developed using nitrones as versatile reactants, and provides some recent examples of applications for probing biological systems.
Recent cell culture and animal studies have suggested that expression of human apo C-III in the liver has a profound impact on the triacylglycerol (TAG)-rich VLDL production under lipid-rich conditions. The apoC-III Gln38Lys variant was identified in subjects of Mexican origin with moderate hypertriglyceridemia. We postulated that Gln38Lys (C3), being a gain-of-function mutation, promotes hepatic VLDL assembly/secretion. To test this hypothesis, we expressed C3 in McA-RH7777 cells and -null mice to contrast its effect with WT apoC-III (C3). In both model systems, C3 expression increased the secretion of VLDL-TAG (by 230%) under lipid-rich conditions. Metabolic labeling experiments with C3 cells showed an increase in de novo lipogenesis (DNL). Fasting plasma concentration of TAG, cholesterol, cholesteryl ester, and FA were increased in C3 mice as compared with C3 mice. Liver of C3 mice also displayed an increase in DNL and expression of lipogenic genes as compared with that in C3 mice. These results suggest that C3 variant is a gain-of-function mutation that can stimulate VLDL production, through enhanced DNL.
Trans‐cyclooctenes (TCOs) represent interesting and highly reactive dipolarophiles for organic transformations including bioorthogonal chemistry. Herein we show that TCOs react rapidly with nitrones and that these reactions are bioorthogonal. Kinetic analysis of acyclic and cyclic nitrones with strained‐trans‐cyclooctene (s‐TCO) shows fast reactivity and demonstrates the utility of this cycloaddition reaction for bioorthogonal labelling. Labelling of the bacterial peptidoglycan layer with unnatural d‐amino acids tagged with nitrones and s‐TCO‐Alexa488 is demonstrated. These new findings expand the bioorthogonal toolbox, and allow TCO reagents to be used in bioorthogonal applications beyond tetrazine ligations for the first time and open up new avenues for bioorthogonal ligations with diverse nitrone reactants.
We present optimized micelle-assisted aqueous copper(i)-catalyzed alkyne–nitrone cycloaddition involving rearrangement (CuANCR) reactions applicable to bioorthogonal applications, namely membrane-associated peptide modification.
Choline is an essential nutrient and in macrophages, mainly supports phosphatidylcholine (PC) synthesis. Cellular uptake and incorporation of choline into PC is critical for LPS-induced macrophage inflammation. Here, we examined choline metabolism in the context of IL-4 polarization of mouse macrophages in vitro and in vivo. Like LPS, IL-4 increased the levels of choline transporter-like protein 1, the rate of choline uptake, and incorporation into PC. Targeted lipidomics analysis revealed higher PC content in IL-4-polarized macrophages, with enrichment in low-saturated species. Pharmacological inhibition of choline metabolism significantly suppressed the transcription of certain hallmark IL-4 genes (Retnla) but not others (Chil3, Mrc1, Arg1). Blocking choline metabolism diminished the expression and secretion of RELMα protein (encoded by Retnla), while also limiting PD-L2 up-regulation and increasing PD-L1 expression. In vivo administration of RSM-932a, a choline kinase inhibitor, caused a dramatic shift in the peritoneal immune cell profile and up-regulated macrophage CD86 and PD-L1, while down-regulating CD206 and PD-L2. Strikingly, blocking choline metabolism lowered RELMα expression in multiple cell-types and tissues in naive mice as well as mice infected with the helminth pathogens Heligmosomoides polygyrus and Nippostrongylus brasiliensis. There were no changes in pathogen burden or clearance in the two separate helminth models. In contrast, in dextran sulfate sodium-induced colitis, loss of colon length as a marker of inflammation was mitigated by choline metabolism inhibition. These data demonstrate a critical link between choline and macrophage effector functions and suggest that targeting choline metabolism could be leveraged to fine-tune immunopathology.
Choline is an essential nutrient and a critical component of the membrane phospholipid phosphatidylcholine (PC), the neurotransmitter acetylcholine, while also contributing to the methylation pathway. In the liver specifically, PC is the major membrane constituent and can be synthesized by the cytidine diphosphate-choline or the phosphatidylethanolamine N-methyltransferase pathway. With the continuing global rise in the rates of obesity and nonalcoholic fatty liver disease, we sought to explore how excess fatty acids on primary hepatocytes and diet-induced obesity affect choline uptake and metabolism. Our results demonstrate that hepatocytes chronically treated with palmitate, but not oleate or a mixture, had decreased choline uptake, which was associated with lower choline incorporation into PC and lower expression of choline transport proteins. Interestingly, a reduction in the rate of degradation spared PC levels in response to palmitate when compared with control. The effects of palmitate treatment were independent of endoplasmic reticulum stress, which counterintuitively augmented choline transport and transporter expression. In a model of obesity-induced hepatic steatosis, male mice fed a 60% high-fat diet for 10 weeks had significantly diminished hepatic choline uptake compared with lean mice fed a control diet. Although the transcript and protein expression of various choline metabolic enzymes fluctuated slightly, we observed reduced protein expression of choline transporter-like 1 (CTL1) in the liver of mice fed a high-fat diet. Polysome profile analyses revealed that in livers of obese mice, the CTL1 transcript, despite being more abundant, was translated to a lesser extent compared with lean controls. Finally, human liver cells demonstrated a similar response to palmitate treatment. Conclusion: Our results suggest that the altered fatty acid milieu seen in obesity-induced fatty liver disease progression may adversely affect choline metabolism, potentially through CTL1, but that compensatory mechanisms work to maintain phospholipid homeostasis. (Hepatology Communications 2020;4:876-889).
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