ABSTRACT
Iron is an essential metal for all organisms, yet disruption of its homeostasis, particularly in labile forms that can contribute to oxidative stress, is connected to diseases ranging from infection to cancer to neurodegeneration. Iron deficiency is also among the most common nutritional deficiencies worldwide. To advance studies of iron in healthy and disease states, we now report the synthesis and characterization of iron-caged luciferin-1 (ICL-1), a bioluminescent probe that enables longitudinal monitoring of labile iron pools (LIPs) in living animals. ICL-1 utilizes a bioinspired endoperoxide trigger to release d-aminoluciferin for selective reactivity-based detection of Fe2+ with metal and oxidation state specificity. The probe can detect physiological changes in labile Fe2+ levels in live cells and mice experiencing iron deficiency or overload. Application of ICL-1 in a model of systemic bacterial infection reveals increased iron accumulation in infected tissues that accompany transcriptional changes consistent with elevations in both iron acquisition and retention. The ability to assess iron status in living animals provides a powerful technology for studying the contributions of iron metabolism to physiology and pathology.
Subject(s)
Acinetobacter Infections/metabolism , Anemia, Iron-Deficiency/metabolism , Firefly Luciferin/analysis , Fluorescent Dyes/analysis , Iron Overload/metabolism , Iron/metabolism , 2,2'-Dipyridyl/pharmacology , Acinetobacter Infections/genetics , Acinetobacter Infections/microbiology , Acinetobacter Infections/pathology , Acinetobacter baumannii/pathogenicity , Acinetobacter baumannii/physiology , Anemia, Iron-Deficiency/genetics , Anemia, Iron-Deficiency/pathology , Animals , Cation Transport Proteins/genetics , Cation Transport Proteins/metabolism , Cations, Divalent , Disease Models, Animal , Ferric Compounds/pharmacology , Firefly Luciferin/analogs & derivatives , Firefly Luciferin/chemical synthesis , Fluorescent Dyes/chemical synthesis , Gene Expression Regulation , Hepcidins/genetics , Hepcidins/metabolism , Homeostasis/genetics , Iron Overload/genetics , Iron Overload/pathology , Iron Regulatory Protein 1/genetics , Iron Regulatory Protein 1/metabolism , Iron Regulatory Protein 2/genetics , Iron Regulatory Protein 2/metabolism , Luminescent Measurements , Mice , Mice, Transgenic , Quaternary Ammonium Compounds/pharmacology , Receptors, Transferrin/genetics , Receptors, Transferrin/metabolism , Signal Transduction , Transferrin/genetics , Transferrin/metabolismABSTRACT
Improved methods for studying intracellular reactive Fe(II) are of significant interest for studies of iron metabolism and disease-relevant changes in iron homeostasis. Here we describe a highly selective reactivity-based probe in which a Fenton-type reaction with intracellular labile Fe(II) leads to unmasking of the aminonucleoside puromycin. Puromycin leaves a permanent and dose-dependent mark on treated cells that can be detected with high sensitivity and precision using a high-content, plate-based immunofluorescence assay. Using this new probe and screening approach, we detected alteration of cellular labile Fe(II) in response extracellular iron conditioning, overexpression of iron storage and/or export proteins, and post-translational regulation of iron export. We also used this new tool to demonstrate that labile Fe(II) pools are larger in cancer cells than in nontumorigenic cells.
Subject(s)
Ferrous Compounds/analysis , Ferrous Compounds/metabolism , Fluorescent Dyes/analysis , Fluorescent Dyes/chemistry , Fluorescent Antibody Technique , Fluorescent Dyes/chemical synthesis , Humans , Molecular Structure , Puromycin/chemistry , Puromycin/pharmacology , Spiro Compounds/analysis , Spiro Compounds/chemical synthesis , Spiro Compounds/chemistryABSTRACT
A generic activation mode for asymmetric LUMO-lowering catalysis has been developed using the long-established principles of oxy-allyl cation chemistry. Here, the enantioselective conversion of racemic α-tosyloxy ketones to optically enriched α-indolic carbonyls has been accomplished using a new amino alcohol catalyst in the presence of electron-rich indole nucleophiles. Kinetic studies reveal that the rate-determining step in this S(N)1 pathway is the catalyst-mediated α-tosyloxy ketone deprotonation step to form an enantiodiscriminant oxy-allyl cation prior to the stereodefining nucleophilic addition event.
Subject(s)
Allyl Compounds/chemistry , Indoles/chemical synthesis , Ketones/chemistry , Oxygen/chemistry , Catalysis , Cations/chemistry , Indoles/chemistry , Molecular Structure , StereoisomerismABSTRACT
Hypercontractility of the cardiac sarcomere may be essential for the underlying pathological hypertrophy and fibrosis in genetic hypertrophic cardiomyopathies. Aficamten (CK-274) is a novel cardiac myosin inhibitor that was discovered from the optimization of indoline compound 1. The important advancement of the optimization was discovery of an Indane analogue (12) with a less restrictive structure-activity relationship that allowed for the rapid improvement of drug-like properties. Aficamten was designed to provide a predicted human half-life (t1/2) appropriate for once a day (qd) dosing, to reach steady state within two weeks, to have no substantial cytochrome P450 induction or inhibition, and to have a wide therapeutic window in vivo with a clear pharmacokinetic/pharmacodynamic relationship. In a phase I clinical trial, aficamten demonstrated a human t1/2 similar to predictions and was able to reach steady state concentration within the desired two-week window.
Subject(s)
Cardiac Myosins/drug effects , Cardiomyopathy, Hypertrophic/drug therapy , Drug Discovery , Dose-Response Relationship, Drug , Humans , Molecular Structure , Structure-Activity RelationshipABSTRACT
The first enantioselective aldehyde α-benzylation using electron-deficient aryl and heteroaryl substrates has been accomplished. The productive merger of a chiral imidazolidinone organocatalyst and a commercially available iridium photoredox catalyst in the presence of household fluorescent light directly affords the desired homobenzylic stereogenicity in good to excellent yield and enantioselectivity. The utility of this methodology has been demonstrated via rapid access to an enantioenriched drug target for angiogenesis suppression.
Subject(s)
Aldehydes/chemistry , Aldehydes/chemical synthesis , Imidazolidines/chemistry , Catalysis , Crystallography, X-Ray , Iridium/chemistry , Models, Molecular , Molecular Structure , Oxidation-Reduction , Photochemistry , StereoisomerismABSTRACT
Aberrant histone methylation profile is reported to correlate with the development and progression of NAFLD during obesity. However, the identification of specific epigenetic modifiers involved in this process remains poorly understood. Here, we identify the histone demethylase Plant Homeodomain Finger 2 (Phf2) as a new transcriptional co-activator of the transcription factor Carbohydrate Responsive Element Binding Protein (ChREBP). By specifically erasing H3K9me2 methyl-marks on the promoter of ChREBP-regulated genes, Phf2 facilitates incorporation of metabolic precursors into mono-unsaturated fatty acids, leading to hepatosteatosis development in the absence of inflammation and insulin resistance. Moreover, the Phf2-mediated activation of the transcription factor NF-E2-related factor 2 (Nrf2) further reroutes glucose fluxes toward the pentose phosphate pathway and glutathione biosynthesis, protecting the liver from oxidative stress and fibrogenesis in response to diet-induced obesity. Overall, our findings establish a downstream epigenetic checkpoint, whereby Phf2, through facilitating H3K9me2 demethylation at specific gene promoters, protects liver from the pathogenesis progression of NAFLD.
Subject(s)
Demethylation , Histone Demethylases/metabolism , Histones/metabolism , Homeodomain Proteins/metabolism , NF-E2-Related Factor 2/metabolism , Non-alcoholic Fatty Liver Disease/pathology , Nuclear Proteins/metabolism , Obesity/pathology , Transcription Factors/metabolism , Animals , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors , Cells, Cultured , Enzyme Activation , Glucose/metabolism , Glutathione/biosynthesis , Humans , Liver/pathology , Male , Methylation , Mice , Mice, Inbred C57BL , Mice, Knockout , Nuclear Proteins/genetics , Oxidative Stress/genetics , Oxidative Stress/physiology , Pentose Phosphate Pathway/physiology , Promoter Regions, Genetic/genetics , Transcription Factors/geneticsABSTRACT
We report that varying the contact force in force spectroscopy results in a significant shift in DNA unbinding forces, measured from short oligonucleotides using a PicoForce microscope. The contact force between a 30-mer complementary DNA-coated probe and surface was varied from 100 pN to 10 nN, resulting in a significant shift in the most abundant unbinding force measured between the duplex. When contact forces were set at 200 pN or less, which is generally considered to be a low contact force region for biomolecular force spectroscopy studies, the shift in DNA unbinding forces was significant with changes in contact force. The effect of the salt concentration on the DNA unbinding forces was also examined for a range of salt concentrations from 5 to 500 mM because the presence of salt ions is necessary to facilitate the hybridization process. Although an increase in salt concentration resulted in the facilitation of DNA multiple binding events during force spectroscopy measurements, no significant shift in unbinding forces was observed. Our experiment demonstrates that the wide variation in DNA unbinding forces reported in the literature (50-600 pN) for short oligonucleotides can be accounted for by the different contact forces used and shows little or no effect of the salt concentration used in those studies. Furthermore, this study demonstrates the importance of reporting contact forces in force spectroscopy measurements for quantitative comparisons between different biomolecular systems, especially for noncovalent-type interactions.