ACS Chemical Biology最新文献_第9页

Development of Oligo-PROTACs That Target C/EBPα. 靶向C/EBPα的寡聚protacs的研制

IF 3.6 2区生物学

ACS Chemical Biology Pub Date : 2026-08-31 DOI: 10.1021/acschembio.6c00462

Junming Cai, Xianmei Qi, Adam T Smiley, Douglas G Brownfield

{"title":"Development of Oligo-PROTACs That Target C/EBPα.","authors":"Junming Cai, Xianmei Qi, Adam T Smiley, Douglas G Brownfield","doi":"10.1021/acschembio.6c00462","DOIUrl":"10.1021/acschembio.6c00462","url":null,"abstract":"CCAAT/enhancer-binding protein alpha (C/EBPα) is a critical lineage-defining transcription factor and tumor suppressor. Despite its clinical significance in conditions like acute myeloid leukemia and hepatocellular carcinoma (HCC), C/EBPα has remained undruggable due to its intrinsically disordered regions and lack of small-molecule binding pockets. In this study, we describe the development of a novel Oligonucleotide-based PROteolysis-TArgeting Chimera (Oligo-PROTAC or O'PROTAC) strategy designed to achieve targeted degradation of endogenous C/EBPα. We designed and synthesized a 10-nucleotide double-stranded DNA decoy derived from a C/EBPα-binding motif and conjugated it via variable linkers to the E3 ligase ligands pomalidomide or VH032. A total of six candidates were synthesized and evaluated in 3T3-J2 and Huh7 cell lines. We demonstrate that these O'PROTACs successfully localize to the nucleus and achieve dose-dependent degradation of C/EBPα, with the most potent candidates exhibiting sub-micromolar DC50 via lipofection delivery. Notably, this degradation occurred without inducing significant cytotoxicity. We observe a transcriptional shift consistent with loss of C/EBPα, including downregulation of cooperative factors involved in liver lineage specification and their downstream target genes. This work establishes O'PROTACs as a viable modality for targeting C/EBPα, offering a novel strategy for mechanistic investigation and future therapeutic targeting.","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":""},"PeriodicalIF":3.6,"publicationDate":"2026-08-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148872312","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}

引用次数: 0

Pre-miR-21 Conformational Equilibrium Modulates the Efficacy of the Maturation Inhibitor L50 Pre-miR-21构象平衡调节成熟抑制剂L50的功效。

IF 3.6 2区生物学

ACS Chemical Biology Pub Date : 2026-08-21 Epub Date: 2026-07-17 DOI: 10.1021/acschembio.6c00207

Yuhei Nishimura, Yuji Tokunaga, Yutaka Kofuku, Yutaro Shiraishi, Shunsuke Imai, Jing Gong, Kazuhiro Ohara, Junya Okude, Takumi Ueda, Takuya Torizawa, Ichio Shimada, Koh Takeuchi

{"title":"Pre-miR-21 Conformational Equilibrium Modulates the Efficacy of the Maturation Inhibitor L50","authors":"Yuhei Nishimura, Yuji Tokunaga, Yutaka Kofuku, Yutaro Shiraishi, Shunsuke Imai, Jing Gong, Kazuhiro Ohara, Junya Okude, Takumi Ueda, Takuya Torizawa, Ichio Shimada, Koh Takeuchi","doi":"10.1021/acschembio.6c00207","DOIUrl":"10.1021/acschembio.6c00207","url":null,"abstract":"Thecyclic peptide L50 is a promising anticancer candidatethatinhibits miR-21 maturation by targeting the Dicer cleavage site ofpre-miR-21. A recent study revealed that pre-miR-21 exists in equilibriumbetween an Ade-29-deprotonated Bulged state and a Dicer-susceptibleAde-29-protonated Paired state. However, the impact of this equilibriumon L50 efficacy remains unclear. Here, we addressed this questionby conducting biochemical and structural analyses under various pHconditions. We found that L50 exhibits reduced inhibitory activityand affinity for pre-miR-21 in the Paired state. The analysis of themolecular interface suggested that changes in the L50 binding modeduring the conformational transition of pre-miR-21 to the Paired statelead to the loss of key interactions required for high-affinity binding.These findings explain the limited efficacy of L50 in inhibiting miR-21maturation, provide insights into its optimization, and highlightthe importance of considering RNA conformational equilibrium in RNA-targetingdrug development.","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":"21 8","pages":"1917–1926"},"PeriodicalIF":3.6,"publicationDate":"2026-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148467797","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}

引用次数: 0

Direct Small Molecule Modulation of LILRB4 (ILT3) Restores Antitumor Immunity In Vivo and in Patient-Derived Cells 直接小分子调节LILRB4 （ILT3）在体内和患者源性细胞中恢复抗肿瘤免疫。

IF 3.6 2区生物学

ACS Chemical Biology Pub Date : 2026-08-21 Epub Date: 2026-07-25 DOI: 10.1021/acschembio.6c00486

Somaya A. Abdel-Rahman, Zamara Mariam, Giuseppe Deganutti, Moustafa T. Gabr

{"title":"Direct Small Molecule Modulation of LILRB4 (ILT3) Restores Antitumor Immunity In Vivo and in Patient-Derived Cells","authors":"Somaya A. Abdel-Rahman, Zamara Mariam, Giuseppe Deganutti, Moustafa T. Gabr","doi":"10.1021/acschembio.6c00486","DOIUrl":"10.1021/acschembio.6c00486","url":null,"abstract":"Small molecule targeting of suppressivemyeloid immune checkpointsremains a major challenge in cancer immunotherapy, particularly fornonenzymatic receptors lacking conventional druggable active sites.Leukocyte immunoglobulin-like receptor B4 (LILRB4/ILT3) is an immunosuppressivemyeloid checkpoint implicated in tumor immune evasion, T-cell dysfunction,and resistance to immunotherapy across both solid and hematologicmalignancies. Here, we report the discovery and characterization of GL-4512, a direct small molecule modulator of LILRB4 identifiedthrough a Dianthus-based temperature-related intensity change (TRIC)screening platform. Orthogonal biophysical studies, including microscalethermophoresis, surface plasmon resonance, and cellular thermal shiftassays, confirmed direct target engagement with nanomolar affinity.Extensive microsecond molecular dynamics simulations combined withsite-directed mutagenesis identified a previously unrecognized ligandablepocket within the flexible extracellular domain of LILRB4. Functionally, GL-4512 disrupted the immunosuppressive LILRB4-SCG2 signalingaxis and suppressed downstream SHP1/SHP2 and STAT3 signaling. In patient-derivedcolorectal cancer and acute myeloid leukemia coculture systems, pharmacologicalinhibition of LILRB4 restored antitumor immune activity, enhancedIFN-γ and IL-2 production, increased cytotoxic T-cell activation,and reduced tumor cell viability. GL-4512 additionallydemonstrated favorable pharmacokinetic and safety properties supportingoral in vivo administration. In immunocompetent CT26 syngeneic colorectaltumors, once-daily oral treatment significantly suppressed tumor growthand enhanced intratumoral immune activation. Collectively, these findingsestablish LILRB4 as a tractable target for direct small molecule immunomodulationand support therapeutic targeting of suppressive myeloid immune checkpointsfor cancer using nonbiologic modalities.","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":"21 8","pages":"2040–2052"},"PeriodicalIF":3.6,"publicationDate":"2026-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148589129","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}

引用次数: 0

Ionizable Silyl Lipids with Distinct Membrane Fluidity and mRNA Delivery Profiles 具有不同膜流动性和mRNA传递谱的可电离硅基脂类。

IF 3.6 2区生物学

ACS Chemical Biology Pub Date : 2026-08-21 Epub Date: 2026-07-16 DOI: 10.1021/acschembio.6c00306

Leah A. Patterson, Wendy N. Tran, Yuliia Humeniuk, David A. Coppage, Ebube Agwaramgbo, Olivia Downey, Henriette O’Geen, David J. Segal, Annaliese K. Franz

{"title":"Ionizable Silyl Lipids with Distinct Membrane Fluidity and mRNA Delivery Profiles","authors":"Leah A. Patterson, Wendy N. Tran, Yuliia Humeniuk, David A. Coppage, Ebube Agwaramgbo, Olivia Downey, Henriette O’Geen, David J. Segal, Annaliese K. Franz","doi":"10.1021/acschembio.6c00306","DOIUrl":"10.1021/acschembio.6c00306","url":null,"abstract":"We report the synthesis and evaluationof ionizable silyl-lipidswith single or double alkyl chains to modulate RNA encapsulation efficiency(EE), mRNA transfection, particle size, and membrane fluidity in lipidnanoparticles (LNPs). Incorporation of silyl lipid tails with varyingtail substituents enables control over LNP properties, with distinctdifferences in membrane fluidity and transfection efficiency correlatingwith structure. Single-chain (MA) silyl lipids formed particles withmore fluid membranes, while double-chain (DA) silyl lipids (similarto ALC–0315) formed particles with more rigid membranes, asdetermined using a C-Laurdan fluorescence assay. We show that particlescomposed of MA silyl lipids can have higher EE and distinct particlemorphology (as determined using cryo-TEM) compared to DA lipids. Thetransfection efficiency was dependent on the particle composition,with MA lipids performing well even without the addition of helperlipids. Overall, this work highlights the potential of single- anddouble-chain ionizable silyl lipids to rationally modulate membranefluidity and optimize LNP performance and demonstrates that phenyl-containingcationic silyl lipids can significantly enhance mRNA transfectionefficiency relative to controls.","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":"21 8","pages":"1937–1951"},"PeriodicalIF":3.6,"publicationDate":"2026-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148467823","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}

引用次数: 0

Alkyne Tags for Multiplexed, Optical Photothermal Infrared Imaging of Cellular Metabolites 用于细胞代谢产物多路光学光热红外成像的炔标签。

IF 3.6 2区生物学

ACS Chemical Biology Pub Date : 2026-08-21 Epub Date: 2026-07-22 DOI: 10.1021/acschembio.6c00300

Halley M. Lin-Jones, Tuyet Hoang, Yong Li, Katherine M. Townsend, Shelbee K. Botello, Eric O. Potma, Jennifer A. Prescher

引用次数: 0

Identification of the Site of the Fe–S Cluster in CPSF30 via Metal-Coupled Oxidation-Mass Spectrometry 金属耦合氧化-质谱法鉴定CPSF30中Fe-S簇的位置

IF 3.6 2区生物学

ACS Chemical Biology Pub Date : 2026-08-21 Epub Date: 2026-08-05 DOI: 10.1021/acschembio.6c00449

Matthew S. Hursey, Jordan D. Pritts, Danté T. Johnson, Melanie M. Chestnut, Abigail D. Reitz, Jasper G. Ballot, David P. Goldberg, Lisa M. Jones, Veronika A. Szalai, Sarah L. J. Michel

{"title":"Identification of the Site of the Fe–S Cluster in CPSF30 via Metal-Coupled Oxidation-Mass Spectrometry","authors":"Matthew S. Hursey, Jordan D. Pritts, Danté T. Johnson, Melanie M. Chestnut, Abigail D. Reitz, Jasper G. Ballot, David P. Goldberg, Lisa M. Jones, Veronika A. Szalai, Sarah L. J. Michel","doi":"10.1021/acschembio.6c00449","DOIUrl":"https://doi.org/10.1021/acschembio.6c00449","url":null,"abstract":"Cleavage and polyadenylation specificity factor 30 (CPSF30)isa zinc finger protein that plays a key role in pre-mRNA processingby recognizing specific AU-rich sequence elements to facilitate polyadenylation.CPSF30 contains five highly conserved domains made up of three cysteinesand a single histidine residue (CCCH, where C = cysteine; H = histidine).These domains coordinate both Zn and an essential 2Fe-2S cluster requiredfor high-affinity RNA binding. The location of the 2Fe-2S clusterwithin the five CCCH domains and its function have proven elusive.Here, we apply metal-catalyzed oxidation mass spectrometry (MCO-MS)to identify the 2Fe-2S cluster site within CPSF30. By first determiningits redox properties and then initiating Fenton chemistry, we localizedthe Fe–S cluster to the second CCCH domain. The effect of redoxstate on RNA binding was then assessed, and it was determined thatthe Fe–S site is a structural domain. The effect of redox statusin THP-1 cells on CPSF30 was then investigated, and the redox propertiesof the cluster were linked to protein abundance under hypoxic andnormoxic stress. Together, this work describes a new approach to identifyFe–S sites in complex metalloproteins that house multiple metalcofactors, suggests a mechanism for metal-mediated RNA recognitionby CPSF30, and reveals redox-mediated control of CPSF30 in cells.","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":"21 8","pages":"2012–2020"},"PeriodicalIF":3.6,"publicationDate":"2026-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148769419","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}

引用次数: 0

Clickable Substrate Transport (CST): A High-Throughput Functional Assay for Solute Carrier Proteins 可点击底物运输（CST）：溶质载体蛋白的高通量功能分析。

IF 3.6 2区生物学

ACS Chemical Biology Pub Date : 2026-08-21 Epub Date: 2026-07-13 DOI: 10.1021/acschembio.6c00367

Abigail L. Macmillan-Jones, Phillip Biallas, Linda Kitching, Marcus Ladds, Alice Lanne, Dvora Meyer, Tiziana Monteverde, Francesco Ricci, Carsten Schultz-Fademrecht, Hannah Warren, Sabrina Winfield, Jo Francis

{"title":"Clickable Substrate Transport (CST): A High-Throughput Functional Assay for Solute Carrier Proteins","authors":"Abigail L. Macmillan-Jones, Phillip Biallas, Linda Kitching, Marcus Ladds, Alice Lanne, Dvora Meyer, Tiziana Monteverde, Francesco Ricci, Carsten Schultz-Fademrecht, Hannah Warren, Sabrina Winfield, Jo Francis","doi":"10.1021/acschembio.6c00367","DOIUrl":"10.1021/acschembio.6c00367","url":null,"abstract":"Solutecarrier proteins (SLCs) are the largest familyof membrane-associatedtransport proteins, facilitating the transport of a diverse rangeof substrates vital for cellular fitness and function. While the therapeuticpotential of modulating SLC proteins is evident, with clinical-stagedrugs targeting this superfamily well-documented, progress in druggingless well-described members has been hampered by challenges in workingwith these proteins in an in vitro setting. In this work, we presenta novel strategy to enable the discovery and development of smallmolecules against a variety of transporters. Using the glucose transporterSLC5a1 as a model system, we have developed a cellular assay thatreports on the transport of a substrate of interest through the applicationof a fluorogenic dye and bioorthogonal chemistry. This assay is robust,amenable to scale, and applicable across a range of substrates andtargets of interest. We anticipate that this work will facilitatethe discovery of novel modulators for this therapeutically significantprotein family through expanding the limited catalog of availablescreening assays for these traditionally challenging targets.","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":"21 8","pages":"1972–1979"},"PeriodicalIF":3.6,"publicationDate":"2026-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148434237","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}

引用次数: 0

Chemically Responsive Protein Switches for the Precise Control of Biological Activities 精确控制生物活性的化学反应蛋白开关。

IF 3.6 2区生物学

ACS Chemical Biology Pub Date : 2026-08-21 Epub Date: 2026-07-20 DOI: 10.1021/acschembio.6c00360

Jaime Franco Pinto, Naama Drahy, Séverine Divoux, Franck Perez, Arnaud Gautier

{"title":"Chemically Responsive Protein Switches for the Precise Control of Biological Activities","authors":"Jaime Franco Pinto, Naama Drahy, Séverine Divoux, Franck Perez, Arnaud Gautier","doi":"10.1021/acschembio.6c00360","DOIUrl":"10.1021/acschembio.6c00360","url":null,"abstract":"Controlling the proximity or interaction of proteinswith smallmolecules enables researchers to chemically regulate cellular functions.Here, we leveraged CATCHFIRE (chemically assisted tethering of chimeraby fluorogenic induced recognition)─a technology enabling thechemical induction of dimerization in a reversible manner─tocreate chemically responsive protein switches for the precise andreversible control of various biological activities. CATCHFIRE allowedus to chemically induce the assembly and thus function of varioussplit enzymes─including luciferases, proteases, and DNA recombinases.We extended this approach to develop CATCH–ON, a chemicallyinducible gene expression system relying on the chemically induceddimerization of the DNA-binding domain GAL4 and the truncated transcriptionfactor p65Δ. CATCH–ON allowed us to precisely regulatethe expression of cellular enzymes such as proteases, DNA recombinases,or suicide switches, as well as to control the secretion of therapeuticallyrelevant proteins such as insulin. We showed that the CATCH–ONsystem is fast-acting, reversible, titratable, nontoxic, and compatiblewith other chemically induced dimerization systems, opening excitingpossibilities for its application in basic research, biotechnology,and cell therapy.","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":"21 8","pages":"1958–1971"},"PeriodicalIF":3.6,"publicationDate":"2026-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148534556","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}

引用次数: 0

TEO-GalNAc: MS Tag-Free Chemoenzymatic Enrichment and Relative Assessment of Tn/T Antigen Sialylation Occupancy via Ketone-Oxime Chemistry TEO-GalNAc: MS无标记化学酶富集及酮肟化学对Tn/T抗原唾液化占用的相对评价。

IF 3.6 2区生物学

ACS Chemical Biology Pub Date : 2026-08-21 Epub Date: 2026-07-22 DOI: 10.1021/acschembio.6c00467

Li Liu, Yinping Tian, Zhuo Zhang, Xuyang Yue, Liuqing Wen, Mingliang Ye, Hongqiang Qin

{"title":"TEO-GalNAc: MS Tag-Free Chemoenzymatic Enrichment and Relative Assessment of Tn/T Antigen Sialylation Occupancy via Ketone-Oxime Chemistry","authors":"Li Liu, Yinping Tian, Zhuo Zhang, Xuyang Yue, Liuqing Wen, Mingliang Ye, Hongqiang Qin","doi":"10.1021/acschembio.6c00467","DOIUrl":"10.1021/acschembio.6c00467","url":null,"abstract":"Tn/Tantigens and their sialylated forms (STn/ST) are tumor-associatedtruncated O-glycans whose selective enrichment and MS analysis remainchallenging due to limited enrichment efficiency and the MS-incompatiblebulky tags in conventional chemoenzymatic enrichment methods. Here,we develop TEO-GalNAc (MS Tag-free Enrichment of O-GalNAc), a chemoenzymatic strategy that integrates ST6GalNAc1-catalyzedlabeling with a ketone-modified CMP-sialic acid donor, reversibleoxime capture, and mild acid-catalyzed tag removal prior to MS analysis.Using TEO-GalNAc, we identified 489 unique Tn/T glycopeptides fromhuman serum and 1,068 unique Tn/T glycopeptides from Jurkat cell lysates,demonstrating the applicability of the workflow to complex biologicalsamples. Importantly, this approach enables relative assessment ofGalNAc-associated sialylation occupancy by comparing results withor without preservation of endogenous sialic acids prior to enzymatictagging. Using this analytical framework, we observed differentiallabeling patterns between nonmetastatic MCF-7 and metastatic MDA-MB-231cells, including distinct signals associated with several glycoproteinssuch as MUC1, GLU2B, MESD, and SEL1L. By enabling tag-removable chemoenzymaticenrichment and MS-compatible analysis of Tn/T-associated glycopeptides,TEO-GalNAc provides a complementary analytical strategy for studyingtruncated O-glycosylation in complex biological samples.","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":"21 8","pages":"2021–2031"},"PeriodicalIF":3.6,"publicationDate":"2026-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148560211","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}

引用次数: 0

Rational Thermostabilization of the Fe(II)/α-Ketoglutarate-Dependent Halogenase BesD While Preserving Halogenation Selectivity 铁(II)/α-酮戊二酸盐依赖卤化酶BesD的合理热稳定化同时保持卤化选择性

IF 3.6 2区生物学

ACS Chemical Biology Pub Date : 2026-08-21 Epub Date: 2026-07-27 DOI: 10.1021/acschembio.6c00487

Teppei Niide, Keita Miyawaki, Hyuga Miyamoto, Yumi Miyake, Yoshihiro Toya, Hiroshi Shimizu

{"title":"Rational Thermostabilization of the Fe(II)/α-Ketoglutarate-Dependent Halogenase BesD While Preserving Halogenation Selectivity","authors":"Teppei Niide, Keita Miyawaki, Hyuga Miyamoto, Yumi Miyake, Yoshihiro Toya, Hiroshi Shimizu","doi":"10.1021/acschembio.6c00487","DOIUrl":"https://doi.org/10.1021/acschembio.6c00487","url":null,"abstract":"Fe(II)/α-ketoglutarate (αKG)-dependent halogenasesthat catalyze site-selective C–H halogenation of free substrateswithout carrier proteins are attractive biocatalysts for diversifyingpharmaceuticals and agrochemicals. However, their application remainslimited by the narrow diversity of natural halogenases, poor stability,and restricted substrate scope. Protein stabilization is a commonstrategy to enhance mutational tolerance during enzyme engineering;however, Fe(II)/αKG-dependent halogenases are structurally closelyrelated to hydroxylases, and consensus-based stabilizing mutationsrisk shifting activity toward competing hydroxylation. To addressthis challenge, a workflow was designed to improve structural stabilitywhile preserving substrate and product specificity by combining computationalidentification of substrate-recognition residues with Rosetta-basedstabilization. This approach was applied to the l-lysine4-chlorinase BesD from Streptomyces cattleya as a model enzyme. The resulting variants exhibited a T50 increase of more than 45 °C with no loss of substratespecificity or regioselective chlorination activity and served asstable seed enzymes for subsequent substrate scope expansion. Thisstrategy, which systematically excludes substrate recognition- and/orreaction-selectively related residues from the mutation space to preservenative enzyme function, may provide a versatile platform for stabilizingenzymes without substantially compromising catalytic activity.","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":"21 8","pages":"2032–2039"},"PeriodicalIF":3.6,"publicationDate":"2026-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148769785","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}

引用次数: 0