RSC Chemical Biology最新文献

{"title":"Structure-guided engineering of a polyphosphate kinase 2 class III from an Erysipelotrichaceae bacterium to produce base-modified purine nucleotides†","authors":"Rachel M. Mitton-Fry, René Rasche, Ann-Marie Lawrence-Dörner, Jannik Eschenbach, Aileen Tekath, Andrea Rentmeister, Daniel Kümmel and Nicolas V. Cornelissen","doi":"10.1039/D5CB00108K","DOIUrl":"10.1039/D5CB00108K","url":null,"abstract":"<p >Nucleobase-modified nucleoside-5′-triphosphates (NTPs) are important building blocks for the enzymatic synthesis of non-coding RNAs and mRNAs with improved properties. Chemical phosphorylation of base-modified nucleotides to NTPs remains challenging. Here, we report the enzymatic phosphorylation of purine-modified nucleoside-5′-monophosphates (NMPs) to the corresponding NTPs by the polyphosphate kinase 2 class III from an <em>Erysipelotrichaceae</em> bacterium (EbPPK2). The enzyme is highly promiscuous, accepting a range of NMPs with purine modifications. EbPPK2 efficiently catalyses the formation of the corresponding di-, tri- and tetraphosphates, typically with &gt;70% conversion to the NTP. Slower conversion was observed for analogues with oxo- or thio-substitutions at the C6-position. To better understand nucleotide binding and catalysis, we determined the crystal structure of EbPPK2 at 1.7 Å resolution bound to a non-hydrolysable ATP analogue and polyphosphate. This enabled structure-guided design of EbPPK2 variants that efficiently convert GMP analogues, while retaining activity for AMP. Apart from being the preferred industrial-scale ATP recycling catalyst, EbPPK2 and variants bear potential to become the favoured enzyme family for purine-modified NTP production.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":" 8","pages":" 1328-1335"},"PeriodicalIF":3.1,"publicationDate":"2025-07-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12257356/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144643776","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Correction: Turn-on fluorescent glucose transport bioprobe enables wash-free real-time monitoring of glucose uptake activity in live cells and small organisms","authors":"Monica S. Hensley, David Hutchings, Aldelrahman Ismail, Micaela Rayne Geborkoff, Thomas Werner and Marina Tanasova","doi":"10.1039/D5CB90030A","DOIUrl":"10.1039/D5CB90030A","url":null,"abstract":"<p >Correction for “Turn-on fluorescent glucose transport bioprobe enables wash-free real-time monitoring of glucose uptake activity in live cells and small organisms” by Monica S. Hensley <em>et al.</em>, <em>RSC Chem. Biol.</em>, 2025, <strong>6</strong>, 987–995, https://doi.org/10.1039/D4CB00239C.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":" 8","pages":" 1353-1353"},"PeriodicalIF":3.1,"publicationDate":"2025-07-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12226788/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144576597","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A dual-functional substrate for quantitation of substrate levels and GCase activity in living cells†","authors":"Ben Tiet, Sha Zhu, Xi Chen, Nadia Anastasi, Nicholas W. See, Matthew C. Deen, Eva Harde and David J. Vocadlo","doi":"10.1039/D5CB00045A","DOIUrl":"10.1039/D5CB00045A","url":null,"abstract":"<p >Loss of function mutations in the gene <em>GBA1</em>, which encodes the lysosomal glycoside hydrolase β-glucocerebrosidase (GCase) cause Gaucher's disease (GD). Moreover, one mutant allele of <em>GBA1</em> is the most common genetic risk factor for the development of Parkinson's disease (PD). To gain a better understanding how these mutations drive development of PD and how GCase is regulated within cells, the field needs chemical reporters of GCase activity that can be used within living cells. Fluorogenic substrates are one method that can be used to quantify enzyme activities within cells yet existing substrates for GCase have limitations. In particular, the inability to monitor cellular uptake of substrate limits the ability to disentangle impairments in uptake of substrate from impairments in lysosomal GCase activity. Here we report on the preparation and biological characterisation of LysoRF-GBA – a new chemical tool which can be used to quantitatively measure both the cellular levels of intact substrate and lysosomal GCase activity within lysosomes. We demonstrate that, by using LysoRF-GBA, endogenous GCase activity can be measured within live neuroblastoma cells. The selectivity of this substrate for GCase, relative to other cellular enzymes, was validated by genetic and pharmacological perturbation of GCase. By using LysoRF-GBA and concomitantly monitoring levels of both cleaved product and intact substrate, we were able to measure GCase engagement with a known pharmacological chaperone and discriminate between pharmacological agents that affect GCase activity from those that impair endocytosis. Further, the ability to monitor intracellular levels of intact LysoRF-GBA also enabled us to measure its time dependent accumulation within cells, providing insight into when steady state levels of this substrate are reached. LysoRF-GBA therefore shows high potential to be exploited as a tool for the discovery of compounds that could beneficially modulate its activity for benefit in diseases including PD.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":" 8","pages":" 1297-1305"},"PeriodicalIF":3.1,"publicationDate":"2025-07-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12223415/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144576596","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A biparatopic HER2-targeting ADC constructed via site-specific glycan conjugation exhibits superior stability, safety, and efficacy†","authors":"Qi Xue, Jianjian Peng, Wenying Dai, Qingsong Wu, Jinbiao Jiao, Yudi Hu, Wanxing Sha, Yang Yang, Wenhao Yu, Siyang Liu, Ting Xu and Jie P. Li","doi":"10.1039/D5CB00096C","DOIUrl":"10.1039/D5CB00096C","url":null,"abstract":"<p >HER2 is overexpressed in approximately 15–20% of cancers and is associated with aggressive disease progression. We developed JSKN003, a bispecific HER2-targeted antibody–drug conjugate (ADC), through site-specific conjugation technology based on <em>N</em>-glycosylation engineering. JSKN003 maintains a biantennary glycan structure and exhibits superior structural homogeneity, optimized hydrophilicity, and reduced aggregation compared to conventional thiol-maleimide chemistry. In preclinical models JSKN003 demonstrated potent antitumor efficacy, inducing tumor regression in multiple HER2-expressing tumors, such as NCI-N87, BxPC-3, and PDX tumor models. Mechanistically, JSKN003 binds specifically to HER2, undergoes efficient internalization, and traffics to the lysosome, where the payload DXd is released, leading to DNA damage and apoptosis. JSKN003 retained its cytotoxic activity against trastuzumab-resistant cells, attributed to efficient payload delivery and blockade of downstream HER2 signaling pathways, demonstrating the potential to overcome clinical trastuzumab resistance. The safety profile of JSKN003 was evaluated in cynomolgus monkeys and was found to be acceptable, with no severe toxicities observed at therapeutic doses. JSKN003 demonstrated excellent antitumor activity and a favorable safety profile in clinical trials, highlighting its potential as a promising therapeutic option for patients with HER2-positive tumors. These findings suggest that JSKN003 could be a valuable therapeutic strategy with excellent efficacy and safety for HER2-expressing tumors in the clinical setting.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":" 8","pages":" 1284-1296"},"PeriodicalIF":3.1,"publicationDate":"2025-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12188319/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144508786","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The SpyBLI cell-free pipeline for the rapid quantification of binding kinetics from crude samples†","authors":"Olga Predeina, Misha Atkinson, Oliver Wissett, Montader Ali, Cristina Visentin, Stefano Ricagno, Anthony H. Keeble, Mark R. Howarth and Pietro Sormanni","doi":"10.1039/D5CB00079C","DOIUrl":"10.1039/D5CB00079C","url":null,"abstract":"<p >Accurate measurements of binding kinetics, encompassing equilibrium dissociation constant (<em>K</em><small>_D</small>), association rate (<em>k</em><small>_on</small>), and dissociation rate (<em>k</em><small>_off</small>), are critical for the development and optimisation of high-affinity binding proteins. However, such measurements require highly purified material and precise ligand immobilisation, limiting the number of binders that can be characterised within a reasonable timescale and budget. Here, we present the SpyBLI method, a rapid and cost-effective biolayer interferometry (BLI) pipeline that leverages the SpyCatcher003–SpyTag003 covalent association, eliminating the need for both binder purification and concentration determination. This approach allows for accurate binding-kinetic measurements to be performed directly from crude mammalian-cell supernatants or cell-free expression blends. We also introduce a linear gene fragment design that enables reliable expression in cell-free systems without any PCR or cloning steps, allowing binding kinetics data to be collected in under 24 hours from receiving inexpensive DNA fragments, with minimal hands-on time. We demonstrate the method's broad applicability using a range of nanobodies and single-chain antibody variable fragments (scFvs), with affinity values spanning six orders of magnitude. By minimising sample preparation and employing highly controlled, ordered sensor immobilisation, our workflow delivers reliable kinetic measurements from crude mixtures without sacrificing precision. We expect that the opportunity to carry out rapid and accurate binding measurements in good throughput should prove especially valuable for binder engineering, the screening of next-generation sequencing–derived libraries, and computational protein design, where large numbers of potential binders for the same target must be rapidly and accurately characterised to enable iterative refinement and candidate selection.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":" 8","pages":" 1313-1327"},"PeriodicalIF":3.1,"publicationDate":"2025-06-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12247212/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144627374","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Regulation of bacterial phosphorelay systems","authors":"Daniel M. Foulkes, Daniel M. Cooper, Catherine Westland and Dominic P. Byrne","doi":"10.1039/D5CB00016E","DOIUrl":"10.1039/D5CB00016E","url":null,"abstract":"<p >In terms of biomass, bacteria are the most successful organisms on earth. This is partly attributed to their tremendous adaptive capabilities, which allows them to sense and rapidly organise responses to changing environmental stimuli. Using complex signalling mechanisms, bacteria can relay cellular information to fine-tune their metabolism, maintain homeostasis, and trigger virulence processes during infection. Across all life, protein phosphorylation represents the most abundant signalling mechanism, which is controlled by a versatile class of enzymes called protein kinases and their cognate phosphatases. For many years, histidine kinase (HK)-containing two-component systems (TCSs) were considered the canonical instruments of bacterial sensing. However, advances in metagenomics has since proven that bacterial phosphorelay is in fact orchestrated by a functionally diverse array of integrated protein kinase types, including Ser, Thr, Tyr and Arg-targeting enzymes. In this review, we provide an up-to-date appraisal of bacterial kinase signalling, with an emphasis on how these sensing pathways are regulated to modulate kinase output. Finally, we explore how selective kinase inhibitors may be exploited to control infections and combat the looming health emergency of multidrug resistant bacteria.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":" 8","pages":" 1252-1269"},"PeriodicalIF":3.1,"publicationDate":"2025-06-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12189002/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144508787","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Visualizing stress granule dynamics with an RNA guanine quadruplex targeted ruthenium(ii) peptide conjugate.","authors":"Rhianne C Curley, Lorcan Holden, Tia E Keyes","doi":"10.1039/d5cb00008d","DOIUrl":"10.1039/d5cb00008d","url":null,"abstract":"<p><p>Stress granules (SGs) are membraneless ribonucleoprotein assemblies that form in response to cellular stress. They have been linked to cell survival and cancer progression, though many questions remain regarding their structure, function and therapeutic potential. Live-cell fluorescence imaging is key to advancing understanding of SGs, but there are very few small-molecule probes reported that selectively image these organelles. RNA G-quadruplex (rG4) folding is believed to play a role in initiation of SG formation. Thus, to create a probe for SGs, we conjugated a G4 binding domain peptide from RNA helicase associated with AU-rich element (RHAU) to a luminescent [Ru(bpy)₂(PIC-COOH)]²⁺, Ru-RHAU. Ru-RHAU is designed to target rG4s and thus SGs in live cells. Studies <i>in cellulo</i> demonstrate that Ru-RHAU can induce SG formation in a concentration and time dependent manner and immunolabelling confirmed the complex remains associated with rG4s in the SGs. The SG stimulation is attributed to stabilization of rG4 by Ru-RHAU consistent with rG4's role in SG formation. Ru-RHAU shows low cytotoxicity under imaging conditions, facilitating prolonged observation in live cells. Interestingly, under more intense photoirradiation, Ru-RHAU induces phototoxicity through an apoptotic pathway. Ru-RHAU is a versatile tool for probing SG dynamics and function in cellular stress responses and has heretofore unconsidered potential in phototherapeutic applications targeting SGs.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":" ","pages":""},"PeriodicalIF":4.2,"publicationDate":"2025-06-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12188320/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144508788","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Advances in programmable DNA nanostructures enabling stimuli-responsive drug delivery and multimodal biosensing.","authors":"Yao Hong, Wenyue Ma, Meixia Wang, Hong-Hui Wang","doi":"10.1039/d5cb00057b","DOIUrl":"10.1039/d5cb00057b","url":null,"abstract":"<p><p>Recent advancements in DNA nanotechnology have unlocked unprecedented opportunities to address critical challenges in precision medicine, particularly in targeted drug delivery and biomedical imaging. Conventional nanocarriers often suffer from poor spatiotemporal control, suboptimal tumor accumulation, and non-specific biodistribution. To overcome these limitations, DNA-engineered nanostructures-including tile-based assemblies, origami frameworks, spherical nucleic acids, and stimuli-responsive hydrogels-have emerged as programmable platforms capable of dynamically responding to tumor microenvironmental cues (<i>e.g.</i>, pH, enzymatic activity, redox gradients) for triggered drug release. In this review, we comprehensively analyze these architectures with emphasis on their modular design strategies, <i>in vivo</i> stability improvements <i>via</i> polyethylene glycol (PEG) functionalization, and multi-ligand targeting capabilities against cancer-specific biomarkers. In addition to therapeutic uses, these nanostructures also enable highly sensitive detection of circulating tumor DNA and exosomes using fluorescence resonance energy transfer (FRET) probes, electrochemiluminescence amplification circuits, SERS substrates, and cell variable region sensing technology. They also allow for real-time monitoring of dynamic intercellular interactions, overcoming the constraints of traditional sensing methods. This review systematically elaborates on the structural characteristics of DNA assemblies and summarizes the innovative applications of these nanostructures in multimodal detection, offering a more comprehensive perspective for early cancer diagnosis and precision treatment. Despite promising preclinical results, key translational challenges persist, including scalable manufacturing bottlenecks, immune compatibility optimization, and rigorous assessment of long-term nanotoxicity. Future integration with artificial intelligence-driven design tools may catalyze the development of next-generation theranostic nanodevices, ultimately bridging the gap between synthetic biology and clinical oncology.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":" ","pages":""},"PeriodicalIF":4.2,"publicationDate":"2025-06-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12203123/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144530222","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Quantitative neuropeptide analysis by mass spectrometry: advancing methodologies for biological discovery","authors":"Angel Erbey Ibarra, Wenxin Wu, Haoran Zhang and Lingjun Li","doi":"10.1039/D5CB00082C","DOIUrl":"10.1039/D5CB00082C","url":null,"abstract":"<p >Neuropeptides are critical endogenous signaling molecules involved in a wide range of biological processes, including neurotransmission, hormonal regulation, immune responses, and stress management. Despite their importance, the field of neuropeptide research has been historically hampered by significant technical challenges. These include their low abundance in biological systems, diverse and complex post-translational modifications, dynamic expression patterns, and susceptibility to degradation. As such, traditional proteomics approaches often fall short of accurately characterizing neuropeptides, underscoring the need for specialized methodologies to unlock their biological and translational potential. This review evaluates state-of-the-art quantitative mass spectrometry (MS)-based peptidomics, emphasizing their impact on neuropeptide analysis. We highlight how strategies in label-free and label-based quantitation, tandem MS acquisition, and mass spectrometry imaging provide unprecedented sensitivity and throughput for capturing the landscape of neuropeptides and their modifications. Importantly, the review bridges technological innovation with practical applications, highlighting how these approaches have been utilized to uncover novel neuropeptides and elucidate their roles in systems biology and disease pathways.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":" 8","pages":" 1214-1232"},"PeriodicalIF":3.1,"publicationDate":"2025-06-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12168976/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144318303","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Correction: Proximity-induced SuFEx increases the potency of cytosolic nucleotidase inhibitors and reveals a rare example of covalently targeted histidine","authors":"Mikolaj Chrominski, Marcin Warminski, Mateusz Kozarski, Dorota Kubacka, Joanna Panecka-Hofman, Tomasz Spiewla, Mikolaj Zmudzinski, Jacek Jemielity and Joanna Kowalska","doi":"10.1039/D5CB90025E","DOIUrl":"10.1039/D5CB90025E","url":null,"abstract":"<p >Correction for ‘Proximity-induced SuFEx increases the potency of cytosolic nucleotidase inhibitors and reveals a rare example of covalently targeted histidine’ by Mikolaj Chrominski <em>et al.</em>, <em>RSC Chem. Biol.</em>, 2025, <strong>6</strong>, 942–947, https://doi.org/10.1039/d5cb00005j.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":" 7","pages":" 1184-1184"},"PeriodicalIF":4.2,"publicationDate":"2025-06-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12159541/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144303153","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}