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Sequence-function space of radical SAM cyclophane synthases reveal conserved active site residues that influence substrate specificity. 自由基 SAM 环烷合成酶的序列-功能空间揭示了影响底物特异性的保守活性位点残基。
IF 4.2
RSC Chemical Biology Pub Date : 2024-10-23 DOI: 10.1039/d4cb00227j
Chin-Soon Phan, Brandon I Morinaka
{"title":"Sequence-function space of radical SAM cyclophane synthases reveal conserved active site residues that influence substrate specificity.","authors":"Chin-Soon Phan, Brandon I Morinaka","doi":"10.1039/d4cb00227j","DOIUrl":"10.1039/d4cb00227j","url":null,"abstract":"<p><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 <i>Pandoraea</i> 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":null,"pages":null},"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}
引用次数: 0
Induced degradation of SNAP-fusion proteins. 诱导 SNAP 融合蛋白降解。
IF 4.2
RSC Chemical Biology Pub Date : 2024-10-21 DOI: 10.1039/d4cb00184b
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, Doris Hellerschmied
{"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, Doris Hellerschmied","doi":"10.1039/d4cb00184b","DOIUrl":"https://doi.org/10.1039/d4cb00184b","url":null,"abstract":"<p><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":null,"pages":null},"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}
引用次数: 0
Fluorescent probes for investigating the internalisation and action of bioorthogonal ruthenium catalysts within Gram-positive bacteria. 用于研究生物正交钌催化剂在革兰氏阳性细菌体内的内化和作用的荧光探针。
IF 4.2
RSC Chemical Biology Pub Date : 2024-10-15 DOI: 10.1039/d4cb00187g
Nicole Schubert, James W Southwell, Melissa Vázquez-Hernández, Svenja Wortmann, Sylvia Schloeglmann, Anne-Kathrin Duhme-Klair, Patrick Nuernberger, Julia E Bandow, Nils Metzler-Nolte
{"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, Nils Metzler-Nolte","doi":"10.1039/d4cb00187g","DOIUrl":"https://doi.org/10.1039/d4cb00187g","url":null,"abstract":"<p><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 <i>Bacillus subtilis</i>. Two fluorophores based on 7-aminocoumarin were synthesised and characterised to establish their luminescence properties. The introduction of an allyl carbamate (R<sub>2</sub>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 <i>Bacillus subtilis</i> cells.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":null,"pages":null},"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}
引用次数: 0
Discovery and design of molecular glue enhancers of CDK12-DDB1 interactions for targeted degradation of cyclin K. 发现和设计 CDK12-DDB1 相互作用的分子胶增强剂,以定向降解细胞周期蛋白 K。
IF 4.2
RSC Chemical Biology Pub Date : 2024-10-11 DOI: 10.1039/d4cb00190g
Pompom Ghosh, Maximilian Schmitz, Thiyagamurthy Pandurangan, Solomon Tadesse Zeleke, Sean Chin Chan, John Mosior, Luxin Sun, Vinayak Palve, Dylan Grassie, Kanchan Anand, Sylvia Frydman, William R Roush, Ernst Schönbrunn, Matthias Geyer, Derek Duckett, Andrii Monastyrskyi
{"title":"Discovery and design of molecular glue enhancers of CDK12-DDB1 interactions for targeted degradation of cyclin K.","authors":"Pompom Ghosh, Maximilian Schmitz, Thiyagamurthy Pandurangan, Solomon Tadesse Zeleke, Sean Chin Chan, John Mosior, Luxin Sun, Vinayak Palve, Dylan Grassie, Kanchan Anand, Sylvia Frydman, William R Roush, Ernst Schönbrunn, Matthias Geyer, Derek Duckett, Andrii Monastyrskyi","doi":"10.1039/d4cb00190g","DOIUrl":"https://doi.org/10.1039/d4cb00190g","url":null,"abstract":"<p><p>The CDK12 inhibitor SR-4835 promotes the proteasomal degradation of cyclin K, contingent on the presence of CDK12 and the CUL4-RBX1-DDB1 E3 ligase complex. The inhibitor displays molecular glue activity, which correlates with its enhanced ability to inhibit cell growth. This effect is achieved by facilitating the formation of a ternary complex that requires the small molecule SR-4835, CDK12, and the adaptor protein DDB1, leading to the subsequent ubiquitination and degradation of cyclin K. We have successfully solved the structure of the ternary complex, enabling the <i>de novo</i> design of molecular glues that transform four different CDK12 scaffold inhibitors, including the clinical pan-CDK inhibitor dinaciclib, into cyclin K degraders. These results not only deepen our understanding of CDK12's role in cell regulation but also underscore significant progress in designing molecular glues for targeted protein degradation in cancers associated with dysregulated cyclin K activity.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":null,"pages":null},"PeriodicalIF":4.2,"publicationDate":"2024-10-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11494886/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142516697","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}
引用次数: 0
Characterization of conformational states of the homodimeric enzyme fluoroacetate dehalogenase by 19F-13C two-dimensional NMR. 利用 19F-13C 二维核磁共振分析同源二聚体氟乙酸脱卤酶的构象状态。
IF 4.2
RSC Chemical Biology Pub Date : 2024-10-10 DOI: 10.1039/d4cb00176a
Motasem Suleiman, Geordon A Frere, Ricarda Törner, Lauren Tabunar, Gaurav Vijay Bhole, Keith Taverner, Nobuyuki Tsuchimura, Dmitry Pichugin, Roman J Lichtenecker, Oleksandr Vozny, Patrick Gunning, Haribabu Arthanari, Adnan Sljoka, Robert S Prosser
{"title":"Characterization of conformational states of the homodimeric enzyme fluoroacetate dehalogenase by <sup>19</sup>F-<sup>13</sup>C two-dimensional NMR.","authors":"Motasem Suleiman, Geordon A Frere, Ricarda Törner, Lauren Tabunar, Gaurav Vijay Bhole, Keith Taverner, Nobuyuki Tsuchimura, Dmitry Pichugin, Roman J Lichtenecker, Oleksandr Vozny, Patrick Gunning, Haribabu Arthanari, Adnan Sljoka, Robert S Prosser","doi":"10.1039/d4cb00176a","DOIUrl":"https://doi.org/10.1039/d4cb00176a","url":null,"abstract":"<p><p>Tryptophan plays a critical role in proteins by contributing to stability, allostery, and catalysis. Using fluorine (<sup>19</sup>F) nuclear magnetic resonance (NMR), protein conformational dynamics and structure-activity relationships (SARs) can be studied <i>via</i> fluorotryptophan reporters. Tryptophan analogs such as 4-, 5-, 6-, or 7-fluorotryptophan can be routinely incorporated into proteins during heterologous expression by arresting endogenous tryptophan biosynthesis. Building upon the large <sup>19</sup>F chemical shift dispersion associated with 5-fluorotryptophan, we introduce an approach to the incorporation of <sup>13</sup>C-enriched 5-fluorotryptophan using a direct biosynthetic precursor, 5-fluoroanthranilic acid-(phenyl-<sup>13</sup>C<sub>6</sub>). The homodimeric enzyme fluoroacetate dehalogenase (FAcD), a thermophilic alpha/beta hydrolase responsible for the hydrolysis of a C-F bond in fluoroacetate, was expressed and biosynthetically labeled with (phenyl-<sup>13</sup>C<sub>6</sub>) 5-fluorotryptophan. The resulting two-dimensional <sup>19</sup>F-<sup>13</sup>C (transverse relaxation optimized spectroscopy) TROSY heteronuclear correlation spectra provide complete resolution of all 9 tryptophan residues in the apo enzyme and FAcD saturated with the substrate analog bromoacetate. The (<sup>19</sup>F,<sup>13</sup>C) correlation spectra also reveal a multitude of minor resonances in the apo sample. The role of each tryptophan residue in allosteric communication was validated with computational rigidity transmission allostery analysis, which in this case explores the relative interprotomer communication between all possible tryptophan pairs.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":null,"pages":null},"PeriodicalIF":4.2,"publicationDate":"2024-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11465415/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142477056","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}
引用次数: 0
Fluorescence lifetime imaging unravels the pathway of glioma cell death upon hypericin-induced photodynamic therapy. 荧光寿命成像揭示了金丝桃素诱导的光动力疗法导致胶质瘤细胞死亡的途径。
IF 4.2
RSC Chemical Biology Pub Date : 2024-10-08 DOI: 10.1039/d4cb00107a
Miriam C Bassler, Jonas Hiller, Frank Wackenhut, Sven Zur Oven-Krockhaus, Philipp Frech, Felix Schmidt, Christoph Kertzscher, Tim Rammler, Rainer Ritz, Kai Braun, Marcus Scheele, Alfred J Meixner, Marc Brecht
{"title":"Fluorescence lifetime imaging unravels the pathway of glioma cell death upon hypericin-induced photodynamic therapy.","authors":"Miriam C Bassler, Jonas Hiller, Frank Wackenhut, Sven Zur Oven-Krockhaus, Philipp Frech, Felix Schmidt, Christoph Kertzscher, Tim Rammler, Rainer Ritz, Kai Braun, Marcus Scheele, Alfred J Meixner, Marc Brecht","doi":"10.1039/d4cb00107a","DOIUrl":"https://doi.org/10.1039/d4cb00107a","url":null,"abstract":"<p><p>Malignant primary brain tumors are a group of highly aggressive and often infiltrating tumors that lack adequate therapeutic treatments to achieve long time survival. Complete tumor removal is one precondition to reach this goal. A promising approach to optimize resection margins and eliminate remaining infiltrative so-called guerilla cells is photodynamic therapy (PDT) using organic photosensitizers that can pass the disrupted blood-brain-barrier and selectively accumulate in tumor tissue. Hypericin fulfills these conditions and additionally offers outstanding photophysical properties, making it an excellent choice as a photosensitizing molecule for PDT. However, the actual hypericin-induced PDT cell death mechanism is still under debate. In this work, hypericin-induced PDT was investigated by employing the three distinct fluorescent probes hypericin, resorufin and propidium iodide (PI) in fluorescence-lifetime imaging microscopy (FLIM). This approach enables visualizing the PDT-induced photodamaging and dying of single, living glioma cells, as an <i>in vitro</i> tumor model for glioblastoma. Hypericin PDT and FLIM image acquisition were simultaneously induced by 405 nm laser irradiation and sequences of FLIM images and fluorescence spectra were recorded to analyze the PDT progression. The reproducibly observed cellular changes provide insight into the mechanism of cell death during PDT and suggest that apoptosis is the initial mechanism followed by necrosis after continued irradiation. These new insights into the mechanism of hypericin PDT of single glioma cells may help to adjust irradiation doses and improve the implementation as a therapy for primary brain tumors.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":null,"pages":null},"PeriodicalIF":4.2,"publicationDate":"2024-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11474773/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142477057","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}
引用次数: 0
Characterization of the Baeyer–Villiger monooxygenase in the pathway of the bacterial pyrrolizidine alkaloids, legonmycins† 细菌吡咯里西啶生物碱--莱昂霉素途径中 Baeyer-Villiger 单加氧酶的特征。
IF 4.2
RSC Chemical Biology Pub Date : 2024-09-30 DOI: 10.1039/D4CB00186A
Shan Wang, Fleurdeliz Maglangit, Qing Fang, Kwaku Kyeremeh and Hai Deng
{"title":"Characterization of the Baeyer–Villiger monooxygenase in the pathway of the bacterial pyrrolizidine alkaloids, legonmycins†","authors":"Shan Wang, Fleurdeliz Maglangit, Qing Fang, Kwaku Kyeremeh and Hai Deng","doi":"10.1039/D4CB00186A","DOIUrl":"10.1039/D4CB00186A","url":null,"abstract":"<p >The Baeyer–Villiger monooxygenase (BVMO), LgnC, plays a crucial role in the biosynthesis of bacterial pyrrolizidine alkaloids, legonmycins. It processes bicyclic indolizidine substrates generated from the coordinative action of two non-ribosomal peptide synthetases (LgnB and LgnD) and the standalone type II thioesterase-like enzyme (LgnA). It has been demonstrated that the enzyme selectively inserts molecular oxygen into the carbon–carbon bond adjacent to the carbonyl group in legonindolizidines to form bicyclic 1,3-oxazepine carbamate intermediates. After ring opening and contraction, the most advanced products, prelegonmycins, are formed. However, factors controlling the final hydroxylation step and how the enzyme handles the substrates have remained elusive. In this study, we show that the final hydroxylation at the activated carbon of the electron-rich pyrrole system is attributed to either spontaneous oxidation or the action of an endogenous redox reagent. Substrate docking on the structural model of LgnC combined with site-directed mutagenesis allows the identification of several key amino acids that are essential for substrate/intermediate binding and a mechanism of LgnC-catalysed transformation is proposed.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":null,"pages":null},"PeriodicalIF":4.2,"publicationDate":"2024-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11457151/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142394113","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}
引用次数: 0
CyaY and TusA regulate ISC- and SUF-mediated l-cysteine desulfurase activity† CyaY 和 TusA 可调控 ISC 和 SUF 介导的 l-半胱氨酸脱硫酶活性。
IF 4.2
RSC Chemical Biology Pub Date : 2024-09-27 DOI: 10.1039/D4CB00225C
Paolo Olivieri, Jason C. Crack, Angelika Lehmann, Nick E. Le Brun and Silke Leimkühler
{"title":"CyaY and TusA regulate ISC- and SUF-mediated l-cysteine desulfurase activity†","authors":"Paolo Olivieri, Jason C. Crack, Angelika Lehmann, Nick E. Le Brun and Silke Leimkühler","doi":"10.1039/D4CB00225C","DOIUrl":"10.1039/D4CB00225C","url":null,"abstract":"<p >CyaY, the frataxin homolog of <em>Escherichia coli</em>, plays an important role in ISC iron–sulfur cluster assembly through interactions with the cysteine desulfurase IscS, which regulate the supply of sulfur. IscS is not exclusive for ISC Fe–S cluster assembly, as it functions as a hub for the supply of sulfur to a number of other sulfur-requiring pathways, such as for the biosynthesis of Moco and thiolated tRNAs. How the balance of sulfur supply to the various competing pathways is achieved is not fully understood, but a network of protein–protein interactions plays a key role. For example, IscU and TusA compete for binding to IscS and thus for sulfur supply to ISC and Moco/tRNA biosynthesis. Here, we show that TusA can displace CyaY from IscS and can form hetero-complexes involving IscS, CyaY and TusA. Displacement of CyaY from IscS raised the question of whether it can interact with the SUF pathway. The SUF cysteine desulfurase SufS functions as a complex with SufE. Native mass spectrometry studies showed that the SufS dimer can bind up to four SufE molecules, two at high affinity, and two at low affinity, sites. Titration of SufSE (or SufS alone) with CyaY demonstrated binding, probably at the lower affinity site in competition with SufE. Binding of CyaY dramatically reduced the activity of SufSE <em>in vitro</em>, and over-expression of CyaY also significantly affected total cellular desulfurase activity and Fe–S cluster assembly, with the greatest effect observed in mutant strains in which SufS was the principal desulfurase. These data point to a physiological role for CyaY in regulating the desulfurase activity of IscS and SufS and, hence, both the <em>E.coli</em> iron–sulfur assembly systems. They also demonstrate that TusA can displace the regulatory CyaY protein from IscS–CyaY complexes, facilitating sulfur delivery from IscS to other essential cellular processes, and increasing the likelihood of SufSE–CyaY interactions.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":null,"pages":null},"PeriodicalIF":4.2,"publicationDate":"2024-09-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11446229/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142381952","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}
引用次数: 0
Bispecific FpFs: a versatile tool for preclinical antibody development† 双特异性 FpFs:临床前抗体开发的多功能工具。
IF 4.2
RSC Chemical Biology Pub Date : 2024-09-27 DOI: 10.1039/D4CB00130C
Matthew Collins, Nkiru Ibeanu, Wiktoria Roksana Grabowska, Sahar Awwad, Peng T. Khaw, Steve Brocchini and Hanieh Khalili
{"title":"Bispecific FpFs: a versatile tool for preclinical antibody development†","authors":"Matthew Collins, Nkiru Ibeanu, Wiktoria Roksana Grabowska, Sahar Awwad, Peng T. Khaw, Steve Brocchini and Hanieh Khalili","doi":"10.1039/D4CB00130C","DOIUrl":"10.1039/D4CB00130C","url":null,"abstract":"<p >We previously described FpFs <strong><img></strong> (Fab–PEG–Fab) as binding mimetics of IgGs. FpFs are prepared with di(bis-sulfone) conjugation reagents <strong><img></strong> that undergo disulfide rebridging conjugation with the accessible disulfide of each Fab (Scheme 1). We have now prepared bispecific FpFs <strong><img></strong> (bsFpF and Fab<small><sub>1</sub></small>–PEG–Fab<small><sub>2</sub></small>) as potential bispecific antibody mimetics with the intent that bsFpFs could be used in preclinical antibody development since sourcing bispecific antibodies may be challenging during preclinical research. The di(bis-sulfone) reagent <strong><img></strong> was first used to prepare a bsFpF <strong><img></strong> by the sequential conjugation of a first Fab and then a second Fab to another target (Scheme 2). Seeking to improve bsFpF synthesis, the asymmetric conjugation reagent, bis-sulfone bis-sulfide <strong><img><img>,</strong> with different thiol conjugation reactivities at each terminus (Scheme 4) was examined and the bsFpFs appeared to be formed at similar conversion to the di(bis-sulfone) reagent <strong><img></strong>. To explore the advantages of using common intermediates in the preparation of bsFpF families, we investigated bsFpF synthesis with a protein conjugation–ligation approach (Scheme 5). Reagents with a bis-sulfone moiety for conjugation on one PEG terminus and a ligation moiety on the other terminus were examined. Bis-sulfone PEG <em>trans</em>-cyclooctene (TCO) <strong><img><img></strong> and bis-sulfone PEG tetrazine (Tz) <strong><img><img></strong> were used to prepare several bsFpFs targeting various therapeutic targets (TNF-α, IL6R, IL17, and VEGF) and tissue affinity targets (hyaluronic acid and collagen II). Surface plasmon resonance (SPR) binding studies indicated that there was little difference between the dissociation rate constant (<em>k</em><small><sub>d</sub></small>) for the unmodified Fab, mono-conjugated PEG–Fab and the corresponding Fab in a bsFpF. The Fab association rate (<em>k</em><small><sub>a</sub></small>) in the bsFpF was slower than for PEG–Fab, which may be because of mass differences that influence SPR results. These observations suggest that each Fab will bind to its target independently of the other Fab and that bsFpF binding profiles can be estimated using the corresponding PEG–Fab conjugates.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":null,"pages":null},"PeriodicalIF":4.2,"publicationDate":"2024-09-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11427889/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142356031","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}
引用次数: 0
Metal chelation as an antibacterial strategy for Pseudomonas aeruginosa and Acinetobacter baumannii 金属螯合作为铜绿假单胞菌和鲍曼不动杆菌的抗菌策略。
IF 4.2
RSC Chemical Biology Pub Date : 2024-09-24 DOI: 10.1039/D4CB00175C
Martina M. Golden, Amelia C. Heppe, Cassandra L. Zaremba and William M. Wuest
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