RSC Chemical Biology最新文献

{"title":"Dynamic conformational equilibria in the active states of KRAS and NRAS.","authors":"Enrico Rennella, Chrystèle Henry, Callum J Dickson, Florian Georgescauld, Thomas E Wales, Dirk Erdmann, Simona Cotesta, Michel Maira, Richard Sedrani, Saskia M Brachmann, Nils Ostermann, John R Engen, Lewis E Kay, Kim S Beyer, Rainer Wilcken, Wolfgang Jahnke","doi":"10.1039/d4cb00233d","DOIUrl":"10.1039/d4cb00233d","url":null,"abstract":"<p><p>The design of potent RAS inhibitors benefits from a molecular understanding of the dynamics in KRAS and NRAS and their oncogenic mutants. Here we characterize switch-1 dynamics in GTP-state KRAS and NRAS by ³¹P NMR, by ¹⁵N relaxation dispersion NMR, hydrogen-deuterium exchange mass spectrometry (HDX-MS), and molecular dynamics simulations. In GMPPNP-bound KRAS and NRAS, we see the co-existence of two conformational states, corresponding to an \"inactive\" state-1 and an \"active\" state-2, as previously reported. The KRAS oncogenic mutations G12D, G12C and G12V only slightly affect this equilibrium towards the \"inactive\" state-1, with rank order wt < G12C < G12D < G12V. In contrast, the NRAS Q61R oncogenic mutation shifts the equilibrium fully towards the \"active\" state-2. Our molecular dynamics simulations explain this by the observation of a transient hydrogen bond between the Arg61 side chain and the Thr35 backbone carbonyl oxygen. NMR relaxation dispersion experiments with GTP-bound KRAS Q61R confirm a drastic decrease in the population of state-1, but still detect a small residual population (1.8%) of this conformer. HDX-MS indicates that higher populations of state-1 correspond to increased hydrogen-deuterium exchange rates in some regions and increased flexibility, whereas low state-1 populations are associated with KRAS rigidification. We elucidated the mechanism of action of a potent KRAS G12D inhibitor, MRTX1133. Binding of this inhibitor to the switch-2 pocket causes a complete shift of KRAS G12D towards the \"inactive\" conformation and prevents binding of effector RAS-binding domain (RBD) at physiological concentrations, by signaling through an allosteric network.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":" ","pages":""},"PeriodicalIF":4.2,"publicationDate":"2024-11-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11629925/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142814375","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":"Cultivating the future leaders of chemical biology.","authors":"Anna Rulka, Elizabeth Adams, Akane Kawamura, Stephen Wallace","doi":"10.1039/d4cb90055c","DOIUrl":"https://doi.org/10.1039/d4cb90055c","url":null,"abstract":"<p><p>The inaugural RSC Chemistry Biology Interface Community Leadership Retreat for Early Career Researchers aimed to provide a unique combination of research leadership training, discussion and collaboration opportunities to emerging chemical biologists from the UK and Europe. This article outlines the ethos and reports the outcomes from this new event for the Royal Society of Chemistry and future plans to establish this event as an ongoing feature in the chemical biology conference landscape.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":" ","pages":""},"PeriodicalIF":4.2,"publicationDate":"2024-11-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11583041/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142711256","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":"Red and far-red cleavable fluorescent dyes for self-labelling enzyme protein tagging and interrogation of GPCR co-internalization.","authors":"Kilian Roßmann, Ramona Birke, Joshua Levitz, Ben Jones, Johannes Broichhagen","doi":"10.1039/d4cb00209a","DOIUrl":"10.1039/d4cb00209a","url":null,"abstract":"<p><p>Post-labelling cleavable substrates for self-labelling protein tags, such as SNAP- and Halo-tags, can be used to study cell surface receptor trafficking events by stripping dyes from non-internalized protein pools. Since the complexity of receptor biology requires the use of multiple and orthogonal approaches to simultaneously probe multiple receptor pools, we report the development of four membrane impermeable probes that covalently bind to either the SNAP- or the Halo-tag in the red to far-red range. These molecules bear a disulfide bond to release the non-internalized probe using the reducing agent sodium 2-mercaptoethane sulfonate (MESNA). As such, our approach allows the simultaneous visualization of multiple internalized cell surface proteins in two colors which we showcase using G protein-coupled receptors. We use this approach to detect internalized group II metabotropic glutamate receptor (mGluRs), homo- and heterodimers, and to reveal unidirectional crosstalk between co-expressed glucagon-like peptide 1 (GLP1R) and glucose-dependent insulinotropic polypeptide receptors (GIPR). In these applications, we translate our method to both high resolution imaging and quantitative, high throughput assays, demonstrating the value of our approach for a wide range of applications.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":" ","pages":""},"PeriodicalIF":4.2,"publicationDate":"2024-11-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11599839/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142751975","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":"Rational engineering of an antimalarial peptide with enhanced proteolytic stability and preserved parasite invasion inhibitory activity.","authors":"Abhisek Kar, Akash Narayan, Vishal Malik, Kalyaneswar Mandal","doi":"10.1039/d4cb00229f","DOIUrl":"10.1039/d4cb00229f","url":null,"abstract":"<p><p>We describe rational chemical engineering to enhance the proteolytic stability of a chimeric peptide using a combination of unique strategies that involve the incorporation of a series of d-amino acids into the parent l-peptide sequence and restricting the conformational freedom of the peptide by covalent stitching. We hypothesize that replacing a stretch of sequence of an unstructured peptide motif with d-amino acids would increase its proteolytic stability without significantly affecting its affinity to the target protein. Also, considering the C_β-C_β distances, replacing an appropriate pair of residues with cysteine to form an additional disulfide bond in the molecule would provide additional stability to the engineered peptide. To verify this hypothesis, we have implemented these strategies to a previously reported peptidic inhibitor RR, against <i>P. falciparum</i> invasion into red blood cells (RBCs) and designed two novel heterochiral chimeric peptides, RR-I and RR-II. We have demonstrated that these peptides exhibit remarkable inhibitory activity with dramatically enhanced proteolytic stability. Finally, we have designed a cyclic analog, RR-III, to enhance the stability of the peptide against endopeptidases. The RR-III peptide exhibits the same inhibitory activity as RR-II while demonstrating impressive resistance to enzymatic degradation and prolonged stability in human plasma. These developments hold promise for a new generation of peptide-based therapeutics, showcasing the potential of residue selection for tailored modifications, as demonstrated in this work.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":" ","pages":""},"PeriodicalIF":4.2,"publicationDate":"2024-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11576825/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142689219","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 platform of ADAPTive scaffolds: development of CDR-H3 β-hairpin mimics into covalent inhibitors of the PD1/PDL1 immune checkpoint†","authors":"Sarah H. Naylon, Alexis D. Richaud, Guangkuan Zhao, Linda Bui, Craig P. Dufresne, Chunjing J. Wu, Medhi Wangpaichitr, Niramol Savaraj and Stéphane P. Roche","doi":"10.1039/D4CB00174E","DOIUrl":"10.1039/D4CB00174E","url":null,"abstract":"<p >Aberrant and dysregulated protein–protein interactions (PPIs) drive a significant number of human diseases, which is why they represent a major class of targets in drug discovery. Although a number of high-affinity antibody-based drugs have emerged in this therapeutic space, the discovery of smaller PPI inhibitors is lagging far behind, underscoring the need for novel scaffold modalities. To bridge this gap, we introduce a biomimetic platform technology – adaptive design of antibody paratopes into therapeutics (<em>ADAPT</em>) – that enables the paratope-forming binding loops of antibodies to be crafted into large β-hairpin scaffolds (<em>ADAPTins</em>). In this study, we describe a novel strategy for engineering native CDR-H3 “hot loops” with varying sequences, lengths, and rigidity into <em>ADAPTins</em>, ultimately transforming these compounds into irreversible covalent inhibitors. A proof-of-concept was established by creating a series of <em>ADAPTin</em> blockers of the PD1:PDL1 immune checkpoint PPI (blocking activity EC<small>₅₀</small> &lt; 0.3 μM) which were subsequently modified into potent covalent PD1 inhibitors. The compelling rate of stable and folded <em>ADAPTins</em> above physiological temperature (21 out of 29) obtained across six different scaffolds suggests that the platform technology could provide a novel opportunity for high-quality peptide display and biological screening.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":" 12","pages":" 1259-1270"},"PeriodicalIF":4.2,"publicationDate":"2024-11-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11562385/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142649250","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 nanoengineered tandem nitroreductase: designing a robust prodrug-activating nanoreactor.","authors":"Mariia Zmyslia, Michael J Capper, Michael Grimmeisen, Kerstin Sartory, Benedikt Deuringer, Mohamed Abdelsalam, Kaiwei Shen, Manfred Jung, Wolfgang Sippl, Hans-Georg Koch, Laurine Kaul, Regine Süss, Jesko Köhnke, Claudia Jessen-Trefzer","doi":"10.1039/d4cb00127c","DOIUrl":"10.1039/d4cb00127c","url":null,"abstract":"<p><p>Nitroreductases are important enzymes for a variety of applications, including cancer therapy and bioremediation. They often require encapsulation to improve stability and activity. We focus on genetically encoded encapsulation of nitroreductases within protein capsids, like encapsulins. Our study showcases the encapsulation of nitroreductase NfsB as functional dimers within encapsulins, which enhances protein activity and stability in diverse conditions. Mutations within the pore region are beneficial for activity of the encapsulated enzyme, potentially by increasing diffusion rates. Cryogenic electron microscopy reveals the overall architecture of the encapsulated dimeric NfsB within the nanoreactor environment and identifies multiple pore states in the shell. These findings highlight the potential of encapsulins as versatile tools for enhancing enzyme performance across various fields.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":" ","pages":""},"PeriodicalIF":4.2,"publicationDate":"2024-11-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11532998/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142592479","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":"Lipid–polymer hybrid-vesicles interrupt nucleation of amyloid fibrillation†‡","authors":"Newton Sen, Stephanie Krüger and Wolfgang H. Binder","doi":"10.1039/D4CB00217B","DOIUrl":"10.1039/D4CB00217B","url":null,"abstract":"<p >Solubility and aggregation of proteins are crucial factors for their functional and further biological roles. Aggregation of proteins <em>in vivo</em>, such as the amyloid beta (Aβ<small>_1–40</small>) peptide into fibrils, is significantly modulated by membrane lipids, abundantly present in cells. We developed a model membrane system, composed of lipid hybrid-vesicles bearing embedded hydrophilic polymers to <em>in vitro</em> study the aggregation of the Aβ<small>_1–40</small> peptide. Focus is to understand and inhibit the primordial, nucleation stages of their fibrillation by added hybrid-vesicles, composed of a natural lipid and amphiphilic polymers. These designed hybrid-vesicles are based on 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), displaying embedded hydrophilic (EO)<small>_<em>m</em></small>P<small>_<em>n</em></small>A_<strong>EG</strong> polymers (<em>m</em> = 2 or 3; P<small>_<em>n</em></small> = 10 to 52 with <em>M</em><small>_n</small> = 2800–9950 gmol<small>⁻¹</small>) in amounts ranging from 5–20 mol%, anchored to the POPC vesicles <em>via</em> hydrophobic hexadecyl-, glyceryl- and cholesteryl-moieties, affixed to the polymers as end-groups. All investigated hybrid-vesicles significantly delay fibrillation of the Aβ<small>_1–40</small> peptide as determined by thioflavin T (ThT) assays. We observed that the hybrid-vesicles interacted with early aggregating species of Aβ<small>_1–40</small> peptide, irrespective of their composition or size. A substantial perturbation of both primary (<em>k</em><small>₊</small><em>k</em><small>_<em>n</em></small>) and secondary (<em>k</em><small>₊</small><em>k</em><small>₂</small>) nucleation rates of Aβ<small>_1–40</small> by the POPC–polymer vesicles compared to POPC vesicles was observed, particularly for the cholesteryl-anchored polymers, interfering with the fragmentation and elongation steps of Aβ<small>_1–40</small>. Furthermore, morphological differences of the aggregates were revealed by transmission electron microscopy (TEM) images supported the inhibitory kinetic signatures.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":" 12","pages":" 1248-1258"},"PeriodicalIF":4.2,"publicationDate":"2024-10-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11575630/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142683251","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":"Sequence-function space of radical SAM cyclophane synthases reveal conserved active site residues that influence substrate specificity†","authors":"Chin-Soon Phan and Brandon I. Morinaka","doi":"10.1039/D4CB00227J","DOIUrl":"10.1039/D4CB00227J","url":null,"abstract":"<p >Radical SAM cyclophane synthases catalyze C–C, C–N, and C–O crosslinking reactions in the biosynthesis of bioactive peptide natural products. Here, we studied an uncharacterized rSAM enzyme, HtkB from <em>Pandoraea</em> sp., and found this enzyme to catalyze the formation of a HisC2-to-LysCβ crosslink. We used a combination of ColabFold and mutagenesis studies to show that residues D214 in HtkB and H204 in HaaB (another cyclophane synthase) are important for substrate specificity. Mutation of these residues changes the specificity and lowers substrate recognition on the wild-type motifs. This result opens opportunities to alter the specificity and promiscuity for rSAM peptide modifying enzymes.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":" 12","pages":" 1195-1200"},"PeriodicalIF":4.2,"publicationDate":"2024-10-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11499958/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142509942","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":"Induced degradation of SNAP-fusion proteins†","authors":"Savina Abraham Pol, Sara Liljenberg, Jack Barr, Gina Simon, Luis Wong-Dilworth, Danielle L. Paterson, Vladimir P. Berishvili, Francesca Bottanelli, Farnusch Kaschani, Markus Kaiser, Mariell Pettersson and Doris Hellerschmied","doi":"10.1039/D4CB00184B","DOIUrl":"10.1039/D4CB00184B","url":null,"abstract":"<p >Self-labeling protein tags are an efficient means to visualize, manipulate, and isolate engineered fusion proteins with suitable chemical probes. The SNAP-tag, which covalently conjugates to benzyl–guanine and –chloropyrimidine derivatives is used extensively in fluorescence microscopy, given the availability of suitable SNAP-ligand-based probes. Here, we extend the applicability of the SNAP-tag to targeted protein degradation. We developed a set of SNAP PROteolysis TArgeting Chimeras (SNAP-PROTACs), which recruit the VHL or CRBN-ubiquitin E3 ligases to induce the degradation of SNAP-fusion proteins. Endogenous tagging enabled the visualization and the selective depletion of a SNAP-clathrin light chain fusion protein using SNAP-PROTACs. The addition of PROTACs to the SNAP-tag reagent toolbox facilitates the comprehensive analysis of protein function with a single gene tagging event.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":" 12","pages":" 1232-1247"},"PeriodicalIF":4.2,"publicationDate":"2024-10-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11494418/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142509941","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":"Fluorescent probes for investigating the internalisation and action of bioorthogonal ruthenium catalysts within Gram-positive bacteria†","authors":"Nicole Schubert, James W. Southwell, Melissa Vázquez-Hernández, Svenja Wortmann, Sylvia Schloeglmann, Anne-Kathrin Duhme-Klair, Patrick Nuernberger, Julia E. Bandow and Nils Metzler-Nolte","doi":"10.1039/D4CB00187G","DOIUrl":"10.1039/D4CB00187G","url":null,"abstract":"<p >Bioorthogonal reactions are extremely useful for the chemical modification of biomolecules, and are already well studied in mammalian cells. In contrast, very little attention has been given to the feasibility of such reactions in bacteria. Herein we report modified coumarin dyes for monitoring the internalisation and activity of bioorthogonal catalysts in the Gram-positive bacterial species <em>Bacillus subtilis</em>. Two fluorophores based on 7-aminocoumarin were synthesised and characterised to establish their luminescence properties. The introduction of an allyl carbamate (R<small>₂</small>N-COOR′) group onto the nitrogen atom of two 7-aminocoumarin derivatives with different solubility led to decreased fluorescence emission intensities and remarkable blue-shifts of the emission maxima. Importantly, this allyl carbamate group could be uncaged by the bioorthogonal, organometallic ruthenium catalyst investigated in this work, to yield the fluorescent product under biologically-relevant conditions. The internalisation of this catalyst was confirmed and quantified by ICP-OES analysis. Investigation of the bacterial cytoplasm and extracellular fractions separately, following incubation of the bacteria with the two caged dyes, facilitated their localisation, as well as that of their uncaged form by catalyst addition. In fact, significant differences were observed, as only the more lipophilic dye was located inside the cells and importantly remained there, seemingly avoiding efflux mechanisms. However, the uncaged form of this dye is not retained, and was found predominantly in the extracellular space. Finally, a range of siderophore-conjugated derivatives of the catalyst were investigated for the same transformations. Even though uptake was observed, albeit less significant than for the non-conjugated version, the fact that similar intracellular reaction rates were observed regardless of the iron content of the medium supports the notion that their uptake is independent of the iron transporters utilised by Gram-positive <em>Bacillus subtilis</em> cells.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":" 12","pages":" 1201-1213"},"PeriodicalIF":4.2,"publicationDate":"2024-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11477652/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142477058","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}