ACS Chemical Biology最新文献

{"title":"Elevenostat Is an HDAC6/Class I-Biased Multi-HDAC Inhibitor Rather than an HDAC11-Selective Chemical Probe.","authors":"Miroslava Vosahlikova, Zsofia Kutilova, Julia Kudlacova, Barbora Havlinova, Marat Meleshin, Zora Novakova, Ivan Snajdr, Mike Schutkowski, Cyril Barinka","doi":"10.1021/acschembio.6c00703","DOIUrl":"https://doi.org/10.1021/acschembio.6c00703","url":null,"abstract":"<p><p>Histone deacetylase 11 (HDAC11) is a lysine defatty-acylase with emerging roles in immunity, metabolism, and disease, creating a need for rigorously validated isoform-selective inhibitors. Elevenostat/JB3-22 has been used as an HDAC11 inhibitor, but its HDAC isoform selectivity has remained insufficiently defined. Here, we re-evaluated Elevenostat using recombinant human HDAC isoforms, orthogonal HDAC11 activity assays, and cellular pharmacodynamic readouts. Elevenostat inhibited HDAC11 only weakly, with IC50 values of 10.2 µM in a fluorogenic assay and 32.1 µM in an HPLC-based fatty-acyl substrate assay. Full HDAC1-11 profiling revealed preferential inhibition of the class IIb enzymes HDAC6 and HDAC10 (IC50 = 3.2 and 8.9 nM, respectively), as well as class I HDACs, whereas HDAC11 was the least sensitive isoform. In cells, Elevenostat increased acetylated α-tubulin and histone acetylation and reduced cell viability, consistent with HDAC6 and class I HDAC engagement. These data demonstrate that Elevenostat/JB3-22 is not an HDAC11-selective chemical probe, but a class I/class IIb-biased multi-HDAC hydroxamate. Biological conclusions based solely on Elevenostat treatment should therefore be interpreted with caution.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":""},"PeriodicalIF":3.6,"publicationDate":"2026-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149177996","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Phospholipase Fluorogenic Probes and Molecular Determinants of Cyclic Guanosine Monophosphate-Adenosine Monophosphate Binding to Cyclic Oligonucleotide-Based Antiphage Signaling System CapV.","authors":"Samson Marvellous Oladeji, Shay Adio, Christopher S Vennard, Ebony Saccento, Herman O Sintim","doi":"10.1021/acschembio.6c00631","DOIUrl":"https://doi.org/10.1021/acschembio.6c00631","url":null,"abstract":"<p><p>Cyclic oligonucleotide-based antiphage signaling systems (CBASS) protect bacteria from bacteriophage infection by producing cyclic nucleotide second messengers that activate downstream effector proteins. Cyclic guanosine monophosphate-adenosine monophosphate (cGAMP)-activated phospholipase in Vibrio (CapV) is a prevalent CBASS effector whose activity is commonly monitored using the commercially available fluorogenic substrate resorufin butyrate; however, this probe is hydrolyzed by both basal and activated CapV, limiting its usefulness in CapV-related studies. Here, we report the rational design and evaluation of a series of resorufin ester probes with varying acyl chain lengths to improve the fluorogenic assay used to monitor CapV (from Acinetobacter baumannii and Vibrio cholerae) activation. While resorufin butyrate and pentanoate exhibited substantial cGAMP-independent hydrolysis, resorufin octanoate and decanoate provided an optimal balance between catalytic turnover and activation-dependent discrimination, displaying over 5-fold and approximately 10-fold increases in hydrolysis, respectively, upon 3',3'-cGAMP activation. Using resorufin octanoate as a fluorogenic reporter, we systematically investigated the effects of cyclic dinucleotide structural modifications on CapV activation. Phosphorothioate analogues with nonbridging sulfur (exo-S linkages) retained partial agonist activity, whereas the corresponding bridging 5'-phosphorothioester (endo-S) analogues was inactive, highlighting the importance of the native phosphodiester moiety in activating CapV. Replacement of the ribose 2'-hydroxyl groups with fluorine preserved CapV activation, whereas 2'-deoxy substitutions reduced activity, with modifications on the adenosine moiety having a greater detrimental effect than those on guanosine. These findings using the fluorogenic probes were validated using phospholipid hydrolysis and activity-based protein labeling assays. Molecular docking and 100 ns molecular dynamics simulations revealed that the observed activity trends arise from differential disruption of key hydrogen-bonding interactions and ligand conformations required for productive CapV activation. Together, these fluorogenic probes provide a robust, high-throughput-compatible platform for studying CapV activation and defining cyclic dinucleotide structure-activity relationships.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":""},"PeriodicalIF":3.6,"publicationDate":"2026-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149177963","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Lipid Chemistry beyond the Bilayer: Roles for Nonbilayer Lipids in Biology, Disease, and Delivery.","authors":"Daniel Milshteyn, Itay Budin","doi":"10.1021/acschembio.6c00629","DOIUrl":"https://doi.org/10.1021/acschembio.6c00629","url":null,"abstract":"<p><p>Cell membranes are composed of both bilayer and nonbilayer lipids, whose molecular shape and interactions support or oppose their packing into flat bilayer sheets. While the role of the former in maintaining membrane structural integrity is clear, a growing body of data highlights the importance of nonbilayer lipids in membrane function, including fission and fusion processes in trafficking, and the dynamics of membrane-associated proteins. In this review, we provide a comprehensive overview of nonbilayer lipids and their roles in membrane biology, disease, and drug delivery. First, we introduce the lipid chemistry that gives rise to intrinsic curvature and curvature elastic stress (CES), and we survey the chemical diversity of nonbilayer lipids across organelles and cell types. Second, we discuss relevant methodological advances for characterizing the physicochemical properties of nonbilayer lipids, including X-ray scattering, lipidomics, and molecular dynamics. We then examine how nonbilayer lipids contribute to membrane fusion and fission and how the CES they generate modulates membrane protein localization and function. Next, we consider how the biophysical principles governing nonbilayer lipid behavior can inform the rational design of lipid nanoparticle formulations for therapeutic delivery. Finally, we explore how cells regulate lipid intrinsic curvature as a distinct homeostatic parameter and discuss the implications of its dysregulation in liver disease, neurodegeneration, and mitochondrial dysfunction. We highlight developments in methods spanning the interface between chemistry and biology that can help investigate the function and regulation of nonbilayer lipid chemistry, with far-reaching potential to answer questions in human health, disease, and drug delivery.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":""},"PeriodicalIF":3.6,"publicationDate":"2026-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149177989","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Chemoenzymatic Proline Isotope Labeling for Solution and Magic-Angle Spinning Protein Nuclear Magnetic Resonance.","authors":"Darja I Rohden, Federico Napoli, Roman J Lichtenecker, Paul Schanda","doi":"10.1021/acschembio.6c00587","DOIUrl":"https://doi.org/10.1021/acschembio.6c00587","url":null,"abstract":"<p><p>The prevalence of proline residues in intrinsically disordered proteins, functional hinges of enzymes, and protein interaction sites highlights the importance of studying their local dynamics and interactions at atomic resolution. While nuclear magnetic resonance (NMR) spectroscopy is, in principle, ideally suited for such investigations, the absence of an amide proton precludes the use of conventional 1H-15N amide-detected experiments. 1H-13C-based approaches, though possible, remain challenging in large proteins. In this work, we present a selective labeling strategy that introduces an isolated 1H2δ-13Cδ-15N spin system in proline residues in otherwise deuterated proteins. Our method leverages the enzymatic conversion of ornithine, synthesized from cost-effective isotopic precursors. We demonstrate the utility of this approach by applying it to a 134 kDa enzyme using magic-angle spinning NMR, highlighting its potential to efficiently probe functionally relevant sites in proteins.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":""},"PeriodicalIF":3.6,"publicationDate":"2026-10-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149163468","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"High-Throughput Screening Identifies Small-Molecule Inhibitors of the Tau-LRP1 Interaction.","authors":"Caiqin Wang, Chen-Ting Ma, Camryn Crotty, Fu-Yue Zeng, Andrey Bobkov, Jonathan A Covel, Erica Keane Rivera, Eduard Sergienko, Kenneth S Kosik, Steven H Olson, Michael R Jackson, Jennifer N Rauch","doi":"10.1021/acschembio.6c00599","DOIUrl":"https://doi.org/10.1021/acschembio.6c00599","url":null,"abstract":"<p><p>The cellular uptake and propagation of tau are central features of tauopathies, including Alzheimer's disease, and are mediated by the endocytic receptor low-density lipoprotein receptor-related protein 1 (LRP1). While prior studies have implicated LRP1 in tau binding and internalization, the biochemical features of this interaction and its suitability for therapeutic targeting remain incompletely defined. Here, we establish a quantitative and scalable framework to interrogate the tau-LRP1 interaction and identify small-molecule modulators. We engineered and purified the LRP1 ligand-binding domain 4 (BD4), a key region mediating tau interaction, and developed multiple orthogonal assays, including fluorescence polarization, split luciferase complementation, and time-resolved FRET, to measure LRP1-BD4 interactions with tau and a known peptide ligand. Across assay formats, we observe consistent binding affinities in the nanomolar range and demonstrate competitive displacement by tau, receptor-associated protein (RAP), and a peptide ligand, supporting overlapping binding interfaces. Leveraging these platforms, we performed small-molecule high-throughput screening and identified a set of candidate inhibitors of the LRP1-BD4-tau interaction. Selected compounds reduced tau uptake in a cellular assay, phenocopying competitive inhibition by tau and a peptide ligand. Together, these studies define the LRP1-BD4-tau interaction as a biochemically tractable and druggable interface and establish an integrated discovery pipeline linking mechanistic characterization to functional cellular outcomes. This work provides a foundation for the development of therapeutic strategies targeting LRP1-mediated tau uptake.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":""},"PeriodicalIF":3.6,"publicationDate":"2026-10-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149177932","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Identification and Functional Characterization of UbiA-Type Terpene Synthases in Inonotus obliquus.","authors":"Liqiu Zhang, Yingce Duan, Yimin Zhang, Chunlei Wang, Changtian Li, Chengwei Liu","doi":"10.1021/acschembio.6c00661","DOIUrl":"https://doi.org/10.1021/acschembio.6c00661","url":null,"abstract":"<p><p>Inonotus obliquus (Chaga mushroom) is a medicinal basidiomycete rich in structurally diverse terpenoids, yet the biosynthetic enzymes responsible for sesquiterpene formation remain poorly understood. Here, we identified six UbiA-type terpene synthases (IoUbiA1-6) from the I. obliquus genome and functionally characterized them through heterologous expression in Aspergillus oryzae. GC-MS and NMR analyses demonstrated that IoUbiA1-4 catalyze the biosynthesis of bisabolane-type sesquiterpenes, including (+)-Z-α-bisabolene (1), (-)-β-curcumene (2), and (6R,7E)-α-bisabolene (3). Sequence alignment and mutational analyses revealed two highly conserved catalytic motifs, NQXXXXXXXED and QDXXDXXXD, that are essential for enzymatic activity, as alanine substitution of key residues completely abolished product formation. These results experimentally establish the catalytic significance of the conserved motifs in fungal UbiA-type terpene synthases. In addition, metabolic engineering of the mevalonate pathway in the A. oryzae expression host significantly increased 1 production from 3.99 to 61.13 mg/kg. This study expands the functional diversity of fungal UbiA-type terpene synthases, uncovers the molecular basis for bisabolane-type sesquiterpene biosynthesis in I. obliquus, and provides a foundation for the biotechnological production of valuable sesquiterpenoids.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":""},"PeriodicalIF":3.6,"publicationDate":"2026-10-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149163367","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Second-Generation Acyl Modifications Stabilize RNA, Support Translation, and Suppress Cytokine Responses.","authors":"Wenrui Zhong, Qing Sun, Linglan Fang, Sumon Pratihar, Niek N Sanders, Eric T Kool","doi":"10.1021/acschembio.6c00657","DOIUrl":"https://doi.org/10.1021/acschembio.6c00657","url":null,"abstract":"<p><p>Bio-reversible 2'-OH polyacylation (cloaking) has been described recently as a post-transcriptional modification strategy for stabilizing RNAs while enabling recovery of biological function after delivery into cells. Here, we probe the chemical space and utility of this method by evaluating a series of new acyl adducts for their ability to maintain protein expression levels after being substituted at high levels (ca. 25% of nucleotides) on transcripts. Reagent designs focused on the inclusion of electron-withdrawing groups near the acyl carbonyl to enhance electrophilicity. Multiple new reagents demonstrated efficient messenger RNA acylation and stabilization of the RNA against thermal cleavage for two days at 37 °C. Cellular transfection experiments with these acylated transcripts showed that certain modifications maintained native or near-native levels of translation of a protein-coding RNA, confirming the robustness of the approach across a broad set of chemotypes. We further report that protein-coding RNAs polyacylated with selected reversible acyl groups show transiently reduced proinflammatory cytokine responses relative to unmodified RNA. Together, these findings expand the functional boundaries of bio-reversible 2'-OH acylation and provide guidance for future development of RNA protection and modification strategies.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":""},"PeriodicalIF":3.6,"publicationDate":"2026-10-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149147322","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Masked Bisphosphonate Inhibitors of Acid Sphingomyelinase: Improved Cell Penetration and Potency.","authors":"Christian Kappe, Marcel Rühling, Rama Machlah, Fabian Schumacher, Maria Russi, Eyad Naser, Alexander Carpinteiro, Burkhard Kleuser, Martin Fraunholz, Christoph Arenz","doi":"10.1021/acschembio.6c00541","DOIUrl":"https://doi.org/10.1021/acschembio.6c00541","url":null,"abstract":"<p><p>Inhibitors of acid sphingomyelinase (ASM) with nanomolar in cellulo activity were developed by masking the amino bisphosphonate inhibitor ARC39. ASM is a key enzyme catalyzing the hydrolysis of sphingomyelin to ceramide, which plays a role in various physiological and pathological processes. Inhibitors of ASM have been proposed as potential treatment for disorders such as cancer and metastasis, acute lung injury, sepsis, atherosclerosis, and major depression. Despite the vital interest in ASM as a drug target, potent inhibitors of this enzyme are still urgently needed. The masked ARC39 derivatives developed in this study are 100 times more potent than the parent compound. Their properties such as cell uptake, stability, etc. were thoroughly investigated.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":""},"PeriodicalIF":3.6,"publicationDate":"2026-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149126123","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Structure-Dependent Effects of Lipid Conjugation on Cytosolic Accumulation in Escherichia coli and Mammalian Cells.","authors":"Dylan J Coffin, Sobika Bhandari, Liora E Wittle, Karl L Ocius, George M Ongwae, Marcos M Pires","doi":"10.1021/acschembio.6c00550","DOIUrl":"10.1021/acschembio.6c00550","url":null,"abstract":"<p><p>Lipidation is a widely used strategy to promote membrane permeation, yet whether the factors governing lipid-driven accumulation are shared across mammalian and Gram-negative envelopes remains unresolved. Here, we apply the chloroalkane azide-based membrane penetration (CHAMP) assay to a systematically designed library of lipid conjugates in both HeLa and E. coli cells. CHAMP, developed by our group, pairs a minimally disruptive azide tag with a cytosolically anchored HaloTag to quantify cytosolic accumulation directly. The two systems show divergent trends: most lipid modifications reduce E. coli cytosolic accumulation, whereas larger, more hydrophobic conjugates, including medium-chain, cyclized, and heteroatom-containing lipids, are preferentially internalized by mammalian cells. By systematically editing the compound scaffold and, separately, perturbing individual layers of the bacterial envelope, we resolve how charge, scaffold composition, and specific envelope barriers shape these patterns. Together, these results indicate that lipidation is a context-dependent permeation strategy rather than a general one. By showing that hydrophobic modifications frequently hinder cytosolic entry into E. coli, this work offers a permeability-based hypothesis for the scarcity of lipidated antibacterials with cytosolic targets and delineates where lipophilicity-driven optimization is likely to fail in the diderm envelope.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":""},"PeriodicalIF":3.6,"publicationDate":"2026-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149126102","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Protein Age Bias in Target Degradation by PROTACs","authors":"Bingbing X. Li,&nbsp;Xiangshu Xiao","doi":"10.1021/acschembio.6c00591","DOIUrl":"10.1021/acschembio.6c00591","url":null,"abstract":"<p>Targeted protein degradation (TPD) by PROteolysis TArgeting Chimeras (PROTACs) has emerged as a powerful chemical biology and therapeutic modality, yet many degraders exhibit incomplete target clearance and characteristic rebound kinetics despite continuous exposure. The mechanistic basis for this behavior remains poorly understood. Here, we uncover protein age as a previously unrecognized determinant of PROTAC efficacy. Using _CG-SLENP, a chemical genetics strategy that selectively labels newly synthesized and pre-existing proteins within the same living cell, we directly resolve PROTAC-induced degradation of distinct intracellular protein populations. Applying this approach to the bromodomain protein BRD4, we show that two mechanistically and structurally distinct PROTACs, dBET6 and MZ-1, preferentially degrade pre-existing BRD4, while newly synthesized BRD4 is degraded substantially more slowly and incompletely. This age-dependent degradation bias is observed in live-cell imaging, across compound concentrations and time scales, and for both reporter and endogenous BRD4. BRD9-targeting PROTAC VZ185 exhibited similar degradation preference. These findings reveal that PROTAC-mediated degradation is governed not only by target engagement and ternary complex formation but also by the dynamic balance between protein synthesis and degradation. By identifying temporal proteostasis as a critical parameter in TPD, this work provides a mechanistic framework for incomplete degradation and rebound kinetics and therefore establishes protein maturation state as an important consideration for degrader design and evaluation.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":"21 9","pages":"2477–2487"},"PeriodicalIF":3.6,"publicationDate":"2026-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148910243","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}