ACS Chemical Biology最新文献

Chloroquine (CQ) and hydroxychloroquine (HCQ) inhibit autophagy and have shown promise as adjuvant anticancer agents, particularly for targeting therapy-resistant cancer stem cells (CSCs). However, their clinical utility is limited by systemic toxicity and poor tumor selectivity. Here, we report the design, synthesis, and photochemical evaluation of [7-(diethylamino)coumarin-4-yl]methyl (DEACM)-caged CQ and HCQ derivatives as visible-light-activated autophagy inhibitors. Selective caging of the aliphatic amine suppressed biological activity in the dark and enabled rapid release of the parent drugs upon illumination. The lead compound 1C displayed robust light-dependent cytotoxicity across multiple cancer cell lines and, upon photoactivation, recapitulated CQ's effects on LC3-II accumulation. In CSC-enriched tumorspheres, 1C completely abolished sphere formation only if illuminated. Ex vivo and in vivo studies confirmed that visible light penetrates tumor tissue sufficiently to activate 1C and locally release CQ within the tumor. These findings establish the first proof of concept for light-controlled autophagy inhibition and provide a blueprint for spatiotemporally confined anticancer therapies based on photopharmacological modulation of CSCs.

Point mutations in the exonuclease (ExoN) site of nonstructural protein 14 (NSP14) compromise the fitness of betacoronaviruses such as SARS-CoV-2, implicating NSP14 ExoN inhibition as an antiviral strategy. However, there are no advanced compounds that inhibit NSP14's ExoN activity. Building upon the reported crystal structures of two fragments bound to NSP14's ExoN site, we identified a series of 3,5-disubsituted pyrazoles that bound to and inhibited NSP14 ExoN. However, upon resynthesis, we discovered that these putative leads were false positives, perhaps due to contaminating divalent cations, which potently inhibit NSP14 ExoN. Our results provide a cautionary tale to the field about the sensitivity of NSP14 to divalent cations and illustrate the challenges associated with directly targeting the NSP14 ExoN site via fragment merging.

Precise regulation of transcriptional dynamics underlies gene expression programs, governing critical biological processes such as cell fate determination, tissue development, and stress responses. While nascent RNA sequencing technologies offer powerful tools for dissecting these mechanisms, existing methods remain constrained by complex workflows, high cellular input requirements, and cytotoxicity. Here, we present Li-BrU-seq, a systematically optimized 5-bromouridine (BrU)-based profiling strategy designed for low-input samples. Rigorous benchmarking demonstrates that Li-BrU-seq outperforms previous protocols in both enrichment specificity and sensitivity. By streamlining the enrichment workflow, the method enables high-quality transcriptomic profiling from low-input material (500 ng total RNA or ∼25,000 cells). Furthermore, Li-BrU-seq supports flexible temporal resolution ranging from ultrashort pulses to long-term tracking, free from the stress-induced artifacts inherent to 4sU. Additionally, it offers tailored workflows compatible with diverse downstream applications. Li-BrU-seq provides an accessible and versatile platform that expands nascent RNA analysis to low-input, rare, and physiologically sensitive biological systems.

Hepatic fructose utilization depends on ketohexokinase mediated phosphorylation to generate fructose-1-phosphate and commit fructose carbons to additional metabolic steps. Since dysregulated fructose metabolism has been directly connected to the onset and progression of liver disease and cancer, there is considerable interest in identifying the contributions of fructose carbons in bioenergetic pathways. An essential technology for assessing fructose utilization has been the application of isotopically labeled fructose and magnetic resonance with the development of ¹³C hyperpolarized imaging with [2-¹³C]fructose allowing for in vivo assessments. While hyperpolarized imaging of [2-¹³C]fructose has achieved remarkable success in the detection of cancer metabolism, this approach has yet to be utilized to assess fed and fasted states in healthy livers. By challenging mice with a 6 h fast, we demonstrate that hyperpolarized [U-²H, 2-¹³C]fructose in vivo spectroscopy can clearly distinguish direct hepatic gluconeogenesis. Comprehensively, this work aims to establish a foundational methodology for the assessment of hepatic metabolism in vivo.

Hsp90α is an isoform of the heat shock protein 90 (Hsp90) family of molecular chaperones that mediates the folding and activation of ∼400 client proteins. In addition to its intracellular function, Hsp90α is secreted extracellularly (eHsp90α) and has been shown to modulate processes such as cell motility, inflammation, and wound healing. We previously developed the cell-impermeable and Hsp90α-selective inhibitor, NDNA4. NDNA4 manifested weak antiproliferative activity against various cancer cell lines as assessed by an MTS assay. In addition, NDNA4 inhibited cancer cell invasion at nontoxic concentrations and diminished eHsp90α-activated signaling pathways. A key dual-lysine motif located at the charged linker of Hsp90α, termed the F-5 fragment, is the only necessary portion of eHsp90α required for biological activity including induction of cell migration. Interestingly, NDNA4 inhibited both Hsp90α and F-5-induced cancer cell migration despite only being reported to bind the Hsp90α N-terminal ATP-binding site. Synthesis of a biotinylated analogue, NDNA Biotin, allowed pull-down studies to be conducted, which provided evidence that NDNA4 binds F-5 and revealed that the Hsp90α-selective core is required for this interaction. Circular dichroism experiments revealed that NDNA4 binding induces a decrease in the α-helical character of F-5, indicating that a conformational change takes place upon binding. Furthermore, surface plasmon resonance showed NDNA4 dose-dependently binds F-5 with a K_D = 2.66 ± 1.36 μM. Collectively, these results indicate the existence of a previously unrecognized binding site that may be therapeutically relevant. Small-molecule inhibitors that can inhibit eHsp90α's contribution to cancer progression and metastasis represent a new opportunity in drug discovery.

Hydrogen sulfide (H₂S) is an important gasotransmitter that has shown many physiological effects, ranging from anti-inflammation to antioxidation. To advance research on H₂S, donor compounds that can slowly release H₂S in biological conditions while producing minimal bioactive byproducts are essential. Herein, we report the evaluation of thiosaccharides as hydrolysis-based H₂S donors. These compounds were found to slowly produce H₂S over days, in aqueous buffers and in cells. Their H₂S release rates could be affected by the hydroxyl protection groups of thiosaccharides, with faster release by electron-donating groups and slower release by electron-withdrawing groups. We also demonstrated the vasodilatory effect of 1-thioglucose using arterial rings isolated from adult ewes, which is likely due to H₂S release. Altogether, thiosaccharides might be suitable slow-releasing H₂S donors for biological applications.

Mincle, a member of the C-type lectin receptor (CLR) family, detects various glycolipids and glycerolipids such as trehalose dimycolate (TDM) from Mycobacterium tuberculosis, leading to the activation of the innate immune system. In this study, we developed new fluorescence-labeled molecular probes, TDE-Fluor-Ligand and TDE-Reactive-Probe, based on the structure of trehalose diester to elucidate the intracellular behavior of Mincle and its ligands. TDE-Fluor-Ligand was prepared for the ligand analysis, and TDE-Reactive-Probe was specifically designed to label Mincle by turn-on fluorescent affinity labeling. Live-cell imaging analysis using these probes revealed that TDE-Fluor-Ligand internalizes into the cell in a Mincle-dependent manner. Furthermore, imaging analysis using TDE-Reactive-Probe successfully detected Mincle in cells in a Mincle expression-dependent manner.

Glycopeptide antibiotics (GPAs) are clinically important antibiotics characterized by a rigid, highly cross-linked structure. The cross-links in GPAs are installed by the activity of several cytochrome P450 (Oxy) enzymes, which are recruited to their peptide substrates by a unique domain, the X-domain. Given that this cross-linking cascade is the source of both the antibiotic activity and the synthetic complexity of GPAs, it remains a central point for exploring the tolerance of the Oxy enzymes for altered peptide substrates. In this study, we have investigated the ability of the Oxy enzymes to cross-link peptides with changes to their amide backbone, specifically a [Ψ[CH₂NH]Tpg] methylene linkage that was inspired by synthetic efforts showing that such analogues can recover antibiotic activity toward resistant bacteria. Our results show that the Oxy enzymes are extremely sensitive to the presence of a methylene linkage in their peptide substrates, which suggests that these backbone carbonyl groups play a crucial role in maintaining the correct binding of peptide substrates to the P450 enzymes within the GPA cross-linking cascade.

The m⁶A modification plays key roles in RNA metabolism and function and is implicated in various human diseases. In this study, we reported a novel molecular glue strategy for transcript-specific m⁶A editing using synthetic bifunctional molecules containing an RNA-targeting moiety and a ligand that recruit an endogenous m⁶A erasing enzyme. Through cyclic γ-AApeptide library screening, we identified a novel peptidomimetic binder to the long noncoding RNA MALAT1 A2577 region, which has a high m⁶A level. We developed a bifunctional molecular glue by coupling the identified MALAT1-binding cyclic γ-AApeptide to fluorescein, a reported binder to the m⁶A eraser FTO. We demonstrated that this bifunctional molecular glue successfully recruited FTO to the target RNA site, achieved the m⁶A erasing, disrupted HNRNPC-MALAT1 binding, and destabilized MALAT1. We anticipate that this novel molecular glue strategy will offer a new direction in developing molecules to regulate RNA modifications.

N⁶-Methyladenosine (m⁶A) on mature mRNA has been extensively characterized, yet its precise mapping and functions in nuclear noncoding RNAs remain elusive. To address this issue, we recently developed Nuclear-m⁶A-label-seq, a metabolic labeling-based method for transcriptome-wide nuclear m⁶A profiling at single-base resolution. This approach builds on the prototypical m⁶A-label-seq principle, in which an allyl group, instead of methyl group, is metabolically installed at N⁶-position at supposed RNA m⁶A-generating adenosines and the resultant N⁶-allyl adenosine is subsequently converted into 1, N⁶-cyclized adenosine (cyc-A) by mild iodination reaction. During RNA reverse transcription, HIV reverse transcriptase is employed to introduce a base misincorporation at cyc-A sites while enabling a template switch to incorporate adapter sequences to the complementary DNA end in a single step. Through this strategy, library construction is shortened to about 6 h, and the required cell-labeling total RNA input is reduced to 5 μg of total nuclear RNA, representing a 100-fold reduction compared to the prototypical protocol. Both polyadenylated and nonpolyadenylated nuclear transcripts are captured through the sequential nuclear RNA isolation and rRNA depletion. Following high-throughput sequencing, reads from human cells are aligned with the complete T2T-CHM13 genome, enabling accurate mapping of repetitive regions. Aligned reads are then analyzed using the user-friendly rMATS-DVR pipeline to identify high-confidence m⁶A sites based on cyc-A-induced misincorporation patterns. Here, we provide a detailed step-by-step protocol for Nuclear-m⁶A-label-seq, which stands for a direct and high-resolution approach for profiling the nuclear m⁶A epitranscriptome.