ACS Chemical Biology最新文献

Thyroid hormone triiodothyronine (T3) is a critical regulator of mammalian development and metabolism, traditionally recognized for its actions. In this study, we initially designed and synthesized a novel T3-based photoaffinity probe in order to identify T3-interacting proteins in live cells. Remarkably, our results demonstrate that T3 can covalently bind to cellular proteins independently of photoirradiation. To validate this covalent labeling, a fluorescein-modified T3 probe (FIT3) was utilized, and a CO/IP combined SILAC approach was applied to profile covalently labeled proteins. Focusing on one putative target, succinate dehydrogenase subunit A (SDHA), site-mapping analysis identified cysteine residues as likely covalent modification sites mediated by a nucleophilic reaction through iodine leaving from T3. Further, two activity-based probes bearing alkyne click handles at distinct positions on the T3 scaffold were further used to expand the profiling of covalent T3 targets. This approach uncovered over 1000 candidate proteins, including ATP1A1, HSP90AB1, and PRDX1, with selected targets validated by Western blotting. These findings reveal a previously unrecognized mode of thyroid hormone action involving covalent protein modification, challenging the classical paradigm of thyroid hormone signaling and offering new insights into hormone biology and potential therapeutic targets.

Deconvolution of the protein targets of hit compounds from phenotypic screens, often conducted in live cells, is critical for understanding mechanism of action and identifying potentially hazardous off-target interactions. While photoaffinity labeling and chemoproteomics are long-established approaches for discovering small-molecule-protein interactions in live cells, there are a relatively small number of photoaffinity labeling strategies that can be applied for chemoproteomic target identification studies. Recently, we reported a novel chemical framework for photoaffinity labeling based on the photo-Brook rearrangement of acyl silanes and demonstrated its ability, when appended to protein-targeting ligands, to label recombinant proteins. Here, we report the application of these probes to live cell photoaffinity workflows, demonstrate their complementarity to current state-of-the-art minimalist diazirine-based photoaffinity probes, and introduce a modular synthetic route to access acyl silane scaffolds with improved labeling properties.

Many viruses use programmed frameshifting and stop-codon misreading to synthesize functional proteins at high levels. The underlying mechanisms involve complex RNA sequence/structure motifs and likely reflect optimization driven by natural selection of inefficient, nonprogrammed processes. Then, it follows from basic evolutionary theory that low levels of proteins generated through gene expression errors could provide viruses with some survival advantage. Here, we devise an experimental demonstration of this possibility. Phage T7 recruits the host thioredoxin as an essential processivity factor for the viral DNA polymerase. We inserted early stop codons in the thioredoxin gene and appended to its end the sequence encoding for a photoconvertible fluorescent protein. Virus replication was not abolished. Single-molecule localization microscopy showed that the phage replicates even when there are only about 10 thioredoxin molecules per host cell on average, a number orders of magnitude below typical cellular protein levels. We show that this seemingly shocking result can be understood in molecular and evolutionary terms as a consequence of the polymerase-thioredoxin complex displaying high kinetic stability and a long residence time, as these are required to ensure high polymerase processivity. More generally, our demonstration that virus replication may be enabled by proteins at exceedingly low copy number suggests that viruses have access to the wide diversity of protein variants harboring phenotypic mutations as a result of gene expression errors. This mechanism could play a role, for instance, in cross-species transmission by enabling virus survival in the new host before adaptations appear at the genetic level.

Interrogating RNA–small molecule interactions inside cells is critical for advancing RNA-targeted drug discovery. In particular, chemical probing technologies that both identify small molecule-bound RNAs and define their binding sites in the complex cellular environment will be key to establishing the on-target activity necessary for successful hit-to-lead campaigns. Using the small molecule metabolite preQ₁ and its cognate riboswitch RNA as a model, herein we describe a chemical probing strategy for filling this technological gap. Building on well-established RNA acylation chemistry employed by in vivo click-selective 2′-hydroxyl acylation analyzed by primer extension (icSHAPE) probes, we developed an icSHAPE-based preQ₁ probe that retains biological activity in a preQ₁ riboswitch reporter assay and successfully enriches the preQ₁ riboswitch from living bacterial cells. Further, we map the preQ₁ binding site on probe-modified riboswitch RNA by mutational profiling (MaP). As the need for rapid profiling of on- and off-target small molecule interactions continues to grow, this chemical probing strategy offers a method to interrogate cellular RNA–small molecule interactions and supports the future development of RNA-targeted therapeutics.

Fusacandin A (1) is a glycolipid natural product that targets β-1,3-glucan synthase and exhibits significant antifungal activity. Its most impressive structural feature is a C-arylglycosyl hydroxybenzyl moiety with a varying degree of O-glycosylation. In this study, the biosynthetic gene cluster (sac) of fusacandin A was identified from Fusarium sacchari, and subsequent investigations of the assembly line revealed two key glycosyltransferases (GTs): a C-GT SacA, which catalyzes regioselective C-glucosylation at the C-6 of 3,5-dihydroxybenzyl alcohol (7) to form aryl-glucoside (8); and an O-GT SacH, which catalyzes a rare iterative O-galactosylation step on 9 to generate fusacandin B (2). Further in vitro biochemical assays and molecular docking experiments revealed the broad substrate tolerance and the key catalytic residues for both GTs. Two unusual esterification steps catalyzed by a C-terminal carnitine O-acyltransferase (cAT) domain of highly reducing polyketide synthase (hrPKS) SacB and a transmembrane acyltransferase (mAT) SacG were also identified, respectively. In addition, the relationship of structural moiety to the antifungal activity of fusacandins was investigated. Our work not only uncovers the assembly logic of these complex and synthetically challenging molecules but also provides valuable glycosyltransferase biocatalysts for the future biomimetic or chemo-enzymatic synthesis of more potent fusacandin derivatives.

Glioblastoma rapidly acquires resistance to conventional genotoxic therapy. This behavior is closely associated with the enhancement of stem-cell-like character during disease progression. Farnesyl diphosphate synthase (FDPS) plays an important role in maintaining such stem-cell-like features. This finding has stimulated interest in FDPS as a neuro-oncology drug target; however, the lack of CNS-permeable inhibitors has hampered further development. In this study we explored the utility of taxodione, a diterpenoid described as an FDPS inhibitor and predicted to penetrate the blood-brain-barrier. The effects of taxodione were compared to its congener 7-(2′-oxohexyl)-taxodione and a known FDPS inhibitor in U87MG glioblastoma cells. Taxodione was the only treatment that significantly reduced the size of tumor spheroids in a temporal and dose-dependent manner. This activity was associated with FDPS inhibition and the transcriptional downregulation of other mevalonate pathway genes. Consistent with this putative mechanism of action, taxodione sensitized glioblastoma cells to subnanomolar concentrations of paclitaxel.

Terminal alkyne-containing biomolecules are key compounds utilized in bioorthogonal chemistry via azide–alkyne cycloaddition click chemistry. Various synthetic strategies for the introduction of the terminal alkyne to biomolecules have been developed; however, an enzymatic terminal alkyne-modifying system is not well-explored because the biosynthetic systems for terminal alkynes are rare. Recently, BesA, a member of the ATP-grasp enzyme family, has been reported to exclusively utilize terminal alkyne-containing l-propargylglycine and l-glutamic acid as substrates in the synthesis of γ-l-glutamyl-l-propargylglycine. Because of its use of the terminal alkyne for click chemistry, a BesA-based catalytic system is regarded as a potentially attractive biocatalyst for the enrichment of terminal alkyne-containing biomolecules. Toward developing BesA-based biocatalysts, it is important to understand the structure-based mechanism of action of BesA, especially recognition of the terminal alkyne. Here, we elucidate the structural basis of BesA for synthesis of γ-l-glutamyl-l-propargylglycine. The X-ray crystal analysis of BesA unveiled a narrow substrate-binding cleft, beside Y33, R50, R365, and R404 as conserved residues among BesA enzymes from Streptomyces, as the active site for binding of two amino acids, l-propargylglycine and l-glutamic acid. In particular, the region beside Y33 is likely to accommodate the terminal alkyne of l-propargylglycine via CH−π interaction based on the dipeptide-docking simulation of BesA and the results of the activity assay of the BesA Y33A variant. Furthermore, we demonstrate a BesA-catalyzed conjugation system for the synthesis of non-natural alkyne-containing dipeptides. The BesA R50A variant showed a little activity for ligation between l-propargylglycine and 1-methyl-l-glutamate, affording 1-methyl-l-glutamyl-l-propargylglycine. Moreover, the BesA wild-type showed activity for ligation of l-homopropargylglycine and l-glutamic acid, yielding γ-l-glutamyl-l-homopropargylglycine. Structural comparison of BesA with proteins that possibly bind the alkynes shows the significance of Tyr in recognition of the alkynes. These findings highlight the usefulness of BesA-based biocatalytic systems in expanding the chemical space of alkyne-containing peptides applicable for click chemistry as well as understanding alkyne recognition by proteins.

N-terminal acetyltransferase D (NatD) is a highly selective enzyme that acetylates the α-N-terminal amine of histones H4 and H2A, which share the SGRGK motif. Elevated NatD expression has been observed in lung, colorectal, breast, and bone cancer tissues, and is correlated with poor patient survival in these cancer types. In non-small cell lung cancer, NatD depletion reduces progression by repressing the epithelial-to-mesenchymal transition (EMT). Hence, NatD is a potential epigenetic target for lung cancer. To unravel the functions of NatD, a cell-potent and selective NatD inhibitor is needed to investigate the acetyltransferase activity of NatD in cancer progression. We previously reported potent and selective NatD bisubstrate inhibitors, designed by covalently linking coenzyme A to peptide substrates via an acetyl and propionyl linker. However, these inhibitors are not cell-active, limiting their application for cellular studies. Here, we designed cell-permeable bisubstrate analogs by attaching cell-penetrating peptides (CPP) to the bisubstrate inhibitor. The inhibitor displayed a K_i value of 23 nM and effectively reduced cellular Nα-acetylation on histone H4, leading to reduced migration in lung cancer cells by modulating the expression of E-cadherin, N-cadherin, and vimentin. Our findings demonstrate that the CPP-conjugated NatD inhibitor serves as a valuable chemical probe for elucidating the biological roles of NatD in lung cancer, laying the groundwork for future therapeutic strategies targeting NatD.

Matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) is widely valued for its speed and sensitivity in biomolecular analysis, yet the inherently nonquantitative nature hampers its use in many applications including high-throughput screening. Here, we introduce an iodo-based labeling strategy that enables accurate quantification of peptides and peptide libraries using high-resolution MALDI FT-ICR MS. The peptides are coupled at the N-terminus with benzoic acid (BA) or 4-iodobenzoic acid (IBA) to generate the analyte and its internal standard, respectively, differing only by a single iodine substitution. This new labeling strategy was first validated using a simple four-peptide mixture, and subsequently applied to quantitatively evaluate glycine-zipper peptide libraries containing up to 125 members for the discovery of bacterial-binding peptides. Screening of these libraries against Gram-negative Escherichia coli and Gram-positive Bacillus subtilis revealed peptides with strong and selective interactions with the bacteria. This universally applicable, cost-effective, and straightforward approach for peptide quantification significantly enhances the reliability and accuracy of high-throughput peptide screening via MALDI FT-ICR MS.