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The pathways for ribosomal RNA (rRNA) maturation diverge greatly among the domains of life. In the Gram-positive model bacterium, Bacillus subtilis, the final maturation steps of the two large ribosomal subunit (50S) rRNAs, 23S and 5S pre-rRNAs, are catalyzed by the double-strand specific ribonucleases (RNases) Mini-RNase III and RNase M5, respectively. Here we present a protocol that allowed us to solve the 3.0 and 3.1 Å resolution cryoelectron microscopy structures of these RNases poised to cleave their pre-rRNA substrates within the B. subtilis 50S particle. These data provide the first structural insights into rRNA maturation in bacteria by revealing how these RNases recognize and process double-stranded pre-rRNA. Our structures further uncover how specific ribosomal proteins act as chaperones to correctly fold the pre-rRNA substrates and, for Mini-III, anchor the RNase to the ribosome. These r-proteins thereby serve a quality-control function in the process from accurate ribosome assembly to rRNA processing.
Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/química , Precursores de RNA/metabolismo , Ribonucleases/química , Subunidades Ribossômicas Maiores de Bactérias/metabolismo , Bacillus subtilis/ultraestrutura , Proteínas de Bactérias/ultraestrutura , Sequência de Bases , Microscopia Crioeletrônica , Modelos Moleculares , Precursores de RNA/ultraestrutura , Ribonucleases/ultraestrutura , Subunidades Ribossômicas Maiores de Bactérias/ultraestrutura , Especificidade por SubstratoRESUMO
Bispecific antibodies (bsAbs) have recently emerged as a promising platform for the treatment of several conditions, most importantly cancer. Based on the combination of two different antigen-binding motifs in a single macromolecule; bsAbs can either display the combined characteristics of their parent antibodies, or new therapeutic features, inaccessible by the sole combination of two distinct antibodies. While bsAbs are traditionally produced by molecular biology techniques, the chemical development of bsAbs holds great promises and strategies have just begun to surface. In this context, we took advantage of a chemical strategy based on the use of the Ugi reaction for the site-selective conjugation of whole antibodies and coupled the resulting conjugates in a bioorthogonal manner with Fab fragments, derived from various antibodies. We thus managed to produce five different bsAbs with 2 : 1 valency, with yields ranging from 20 % to 48 %, and showed that the affinity of the parent antibody was preserved in all bsAbs. We further demonstrated the interest of our strategy by producing two other bsAbs behaving as cytotoxic T cell engagers with IC50 values in the picomolar range inâ vitro.
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Understanding the mechanisms of drug action in malarial parasites is crucial for the development of new drugs to combat infection and to counteract drug resistance. Proteomics is a widely used approach to study host-pathogen systems and to identify drug protein targets. Plasmodione is an antiplasmodial early-lead drug exerting potent activities against young asexual and sexual blood stages inâ vitro with low toxicity to host cells. To elucidate its molecular mechanisms, an affinity-based protein profiling (AfBPP) approach was applied to yeast and P. falciparum proteomes. New (pro-) AfBPP probes based on the 3-benz(o)yl-6-fluoro-menadione scaffold were synthesized. With optimized conditions of both photoaffinity labeling and click reaction steps, the AfBPP protocol was then applied to a yeast proteome, yielding 11 putative drug-protein targets. Among these, we found four proteins associated with oxidoreductase activities, the hypothesized type of targets for plasmodione and its metabolites, and other proteins associated with the mitochondria. In Plasmodium parasites, the MS analysis revealed 44 potential plasmodione targets that need to be validated in further studies. Finally, the localization of a 3-benzyl-6-fluoromenadione AfBPP probe was studied in the subcellular structures of the parasite at the trophozoite stage.
Assuntos
Antimaláricos , Plasmodium falciparum , Proteômica , Vitamina K 3 , Antimaláricos/farmacologia , Antimaláricos/química , Plasmodium falciparum/efeitos dos fármacos , Vitamina K 3/farmacologia , Vitamina K 3/química , Vitamina K 3/metabolismo , Proteínas de Protozoários/metabolismo , Marcadores de Fotoafinidade/química , Marcadores de Fotoafinidade/farmacologia , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Sondas Moleculares/química , Sondas Moleculares/farmacologia , Proteoma/análise , Proteoma/metabolismo , Estrutura MolecularRESUMO
Diamine reagents have been used to functionalize a [2]rotaxane building block bearing an activated pentafluorophenyl ester stopper. Upon a first acylation, an intermediate host-guest complex with a terminal amine function is obtained. Dissociation of the intermediate occurs in solution and acylation of the released axle generates a [2]rotaxane with an elongated axle subunit. In contrast, the corresponding [3]rotaxane can be obtained if the reaction conditions are appropriate to stabilize the inclusion complex of the mono-amine intermediate and the pillar[5]arene. This is the case when the stopper exchange is performed under mechanochemical solvent-free conditions. Alternatively, if the newly introduced terminal amide group is large enough to prevent the dissociation, the second acylation provides exclusively a [3]rotaxane. On the other hand, detailed conformational analysis has been also carried out by variable temperature NMR investigations. A complete understanding of the shuttling motions of the pillar[5]arene subunit along the axles of the rotaxanes reported therein has been achieved with the help of density functional theory calculations.
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Invited for the cover of this issue are the group of Iwona and Jean-François Nierengarten from the University of Strasbourg (LIMA, UMR 7042, CNRS) and collaborators from the University of Carthage and the IPHC (University of Strasbourg and CNRS, UMR 7178). The image illustrates the fast motions of a pillar[5]arene subunit along the axle of a rotaxane, reminiscent of those of a guitarist's hand along the neck allowing him to use random parts of a scale with certain sweet spots when improvising a solo. Read the full text of the article at 10.1002/chem.202304131.
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The chemical bioconjugation of proteins has seen tremendous applications in the past decades, with the booming of antibody-drug conjugates and their use in oncology. While genetic engineering has permitted to produce bespoke proteins featuring key (un-)natural amino acid residues poised for site-selective modifications, the conjugation of native proteins is riddled with selectivity issues. Chemoselective strategies are plentiful and enable the precise modification of virtually any residue with a reactive side-chain; site-selective methods are less common and usually most effective on small and medium-sized proteins. In this context, we studied the application of the Ugi multicomponent reaction for the site-selective conjugation of amine and carboxylate groups on proteins, and antibodies in particular. Through an in-depth mechanistic methodology work supported by peptide mapping studies, we managed to develop a set of conditions allowing the highly selective modification of antibodies bearing N-terminal glutamate and aspartate residues. We demonstrated that this strategy did not alter their affinity toward their target antigen and produced an antibody-drug conjugate with subnanomolar potency. Excitingly, we showed that the high site selectivity of our strategy was maintained on other protein formats, especially on anticalins, for which directed mutagenesis helped to highlight the key importance of a single lysine residue.
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Imunoconjugados , Proteínas , Proteínas/química , Lisina/química , Aminoácidos , Anticorpos , Fenômenos QuímicosRESUMO
Antibody-drug conjugates (ADCs) are highly complex proteins mainly due to the structural microvariability of the mAb, along with the additional heterogeneity afforded by the bioconjugation process. Top-down (TD) and middle-down (MD) strategies allow the straightforward fragmentation of proteins to elucidate the conjugated amino acid residues. Nevertheless, these spectra are very crowded with multiple overlapping and unassigned ion fragments. Here we report on the use of dedicated software (ClipsMS) and application of proton transfer charge reduction (PTCR), to respectively expand the fragment ion search space to internal fragments and improve the separation of overlapping fragment ions for a more comprehensive characterization of a recently approved ADC, trastuzumab deruxtecan (T-DXd). Subunit fragmentation allowed between 70 and 90% of sequence coverage to be obtained. Upon addition of internal fragment assignment, the three subunits were fully sequenced, although internal fragments did not contribute significantly to the localization of the payloads. Finally, the use of PTCR after subunit fragmentation provided a moderate sequence coverage increase between 2 and 13%. The reaction efficiently decluttered the fragmentation spectra allowing increasing the number of fragment ions characteristic of the conjugation site by 1.5- to 2.5-fold. Altogether, these results show the interest in the implementation of internal fragment ion searches and more particularly the use of PTCR reactions to increase the number of signature ions to elucidate the conjugation sites and enhance the overall sequence coverage of ADCs, making this approach particularly appealing for its implementation in R&D laboratories.
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Imunoconjugados , Prótons , Fluxo de Trabalho , Trastuzumab/química , Imunoconjugados/química , Íons/químicaRESUMO
Nuclear receptors are ligand-activated transcription factors that modulate gene regulatory networks from embryonic development to adult physiology and thus represent major targets for clinical interventions in many diseases. Most nuclear receptors function either as homodimers or as heterodimers. The dimerization is crucial for gene regulation by nuclear receptors, by extending the repertoire of binding sites in the promoters or the enhancers of target genes via combinatorial interactions. Here, we focused our attention on an unusual structural variation of the α-helix, called π-turn that is present in helix H7 of the ligand-binding domain of RXR and HNF4. By tracing back the complex evolutionary history of the π-turn, we demonstrate that it was present ancestrally and then independently lost in several nuclear receptor lineages. Importantly, the evolutionary history of the π-turn motif is parallel to the evolutionary diversification of the nuclear receptor dimerization ability from ancestral homodimers to derived heterodimers. We then carried out structural and biophysical analyses, in particular through point mutation studies of key RXR signature residues and showed that this motif plays a critical role in the network of interactions stabilizing homodimers. We further showed that the π-turn was instrumental in allowing a flexible heterodimeric interface of RXR in order to accommodate multiple interfaces with numerous partners and critical for the emergence of high affinity receptors. Altogether, our work allows to identify a functional role for the π-turn in oligomerization of nuclear receptors and reveals how this motif is linked to the emergence of a critical biological function. We conclude that the π-turn can be viewed as a structural exaptation that has contributed to enlarging the functional repertoire of nuclear receptors.
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Desenvolvimento Embrionário/genética , Receptores Citoplasmáticos e Nucleares/ultraestrutura , Receptores X de Retinoides/genética , Fatores de Transcrição/ultraestrutura , Sequência de Aminoácidos/genética , Sítios de Ligação/genética , Dimerização , Regulação da Expressão Gênica no Desenvolvimento/genética , Redes Reguladoras de Genes/genética , Humanos , Ligantes , Regiões Promotoras Genéticas/genética , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/genética , Receptores X de Retinoides/ultraestrutura , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
Humans are chronically exposed to mixtures of xenobiotics referred to as endocrine-disrupting chemicals (EDCs). A vast body of literature links exposure to these chemicals with increased incidences of reproductive, metabolic, or neurological disorders. Moreover, recent data demonstrate that, when used in combination, chemicals have outcomes that cannot be predicted from their individual behavior. In its heterodimeric form with the retinoid X receptor (RXR), the pregnane X receptor (PXR) plays an essential role in controlling the mammalian xenobiotic response and mediates both beneficial and detrimental effects. Our previous work shed light on a mechanism by which a binary mixture of xenobiotics activates PXR in a synergistic fashion. Structural analysis revealed that mutual stabilization of the compounds within the ligand-binding pocket of PXR accounts for the enhancement of their binding affinity. In order to identify and characterize additional active mixtures, we combined a set of cell-based, biophysical, structural, and in vivo approaches. Our study reveals features that confirm the binding promiscuity of this receptor and its ability to accommodate bipartite ligands. We reveal previously unidentified binding mechanisms involving dynamic structural transitions and covalent coupling and report four binary mixtures eliciting graded synergistic activities. Last, we demonstrate that the robust activity obtained with two synergizing PXR ligands can be enhanced further in the presence of RXR environmental ligands. Our study reveals insights as to how low-dose EDC mixtures may alter physiology through interaction with RXR-PXR and potentially several other nuclear receptor heterodimers.
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Receptor de Pregnano X/química , Receptores X de Retinoides/química , Xenobióticos , Animais , Linhagem Celular , Cristalografia por Raios X , Dimerização , Polarização de Fluorescência , Regulação da Expressão Gênica , Humanos , Ligantes , Luciferases/genética , Luciferases/metabolismo , Modelos Químicos , Receptor de Pregnano X/metabolismo , Receptores X de Retinoides/metabolismo , Xenobióticos/química , Xenobióticos/metabolismo , Xenobióticos/farmacologia , XenopusRESUMO
Monoclonal antibodies (mAbs) currently represent the main class of therapeutic proteins. mAbs approved by regulatory agencies are selected from IgG1, IgG2, and IgG4 subclasses, which possess different interchain disulfide connectivities. Ion mobility coupled to native mass spectrometry (IM-MS) has emerged as a valuable approach to tackle the challenging characterization of mAbs' higher order structures. However, due to the limited resolution of first-generation IM-MS instruments, subtle conformational differences on large proteins have long been hard to capture. Recent technological developments have aimed at increasing available IM resolving powers and acquisition mode capabilities, namely, through the release of high-resolution IM-MS (HR-IM-MS) instruments, like cyclic IM-MS (cIM-MS). Here, we outline the advantages and drawbacks of cIM-MS for better conformational characterization of intact mAbs (â¼150 kDa) in native conditions compared to first-generation instruments. We first assessed the extent to which multipass cIM-MS experiments could improve the separation of mAbs' conformers. These initial results evidenced some limitations of HR-IM-MS for large native biomolecules which possess rich conformational landscapes that remain challenging to decipher even with higher IM resolving powers. Conversely, for collision-induced unfolding (CIU) approaches, higher resolution proved to be particularly useful (i) to reveal new unfolding states and (ii) to enhance the separation of coexisting activated states, thus allowing one to apprehend gas-phase CIU behaviors of mAbs directly at the intact level. Altogether, this study offers a first panoramic overview of the capabilities of cIM-MS for therapeutic mAbs, paving the way for more widespread HR-IM-MS/CIU characterization of mAb-derived formats.
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Anticorpos Monoclonais , Espectrometria de Massas em Tandem , Anticorpos Monoclonais/química , Conformação Molecular , Imunoglobulina G/química , DissulfetosRESUMO
Post-translational modifications (PTMs) not only substantially increase structural heterogeneity of proteins but can also alter the conformation or even biological functions. Monitoring of these PTMs is particularly important for therapeutic products, including monoclonal antibodies (mAbs), since their efficacy and safety may depend on the PTM profile. Innovative analytical strategies should be developed to map these PTMs as well as explore possible induced conformational changes. Cation-exchange chromatography (CEX) coupled with native mass spectrometry has already emerged as a valuable asset for the characterization of mAb charge variants. Nevertheless, questions regarding protein conformation cannot be explored using this approach. Thus, we have combined CEX separation with collision-induced unfolding (CIU) experiments to monitor the unfolding pattern of separated mAbs and thereby pick up subtle conformational differences without impairing the CEX resolution. Using this novel strategy, only four CEX-CIU runs had to be recorded for a complete CIU fingerprint either at the intact mAb level or after enzymatic digestion at the mAb subunit level. As a proof of concept, CEX-CIU was first used for an isobaric mAb mixture to highlight the possibility to acquire individual CIU fingerprints of CEX-separated species without compromising CEX separation performances. CEX-CIU was next successfully applied to conformational characterization of mAb glyco-variants, in order to derive glycoform-specific information on the gas-phase unfolding, and CIU patterns of Fc fragments, revealing increased resistance of sialylated glycoforms against gas-phase unfolding. Altogether, we demonstrated the possibilities and benefits of combining CEX with CIU for in-depth characterization of mAb glycoforms, paving the way for linking conformational changes and resistance to gas-phase unfolding charge variants.
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Anticorpos Monoclonais , Cromatografia , Anticorpos Monoclonais/química , Espectrometria de Massas/métodos , Conformação Proteica , Cromatografia por Troca IônicaRESUMO
Bispecific antibodies as T cell engagers designed to display binding capabilities to both tumor-associated antigens and antigens on T cells are considered promising agents in the fight against cancer. Even though chemical strategies to develop such constructs have emerged, a method that readily converts a therapeutically applied antibody into a bispecific construct by a fully non-genetic process is not yet available. Herein, we report the application of a biogenic, tyrosine-based click reaction utilizing chemoenzymatic modifications of native IgG1 antibodies to generate a synthetic bispecific antibody construct that exhibits tumor-killing capability at picomolar concentrations. Control experiments revealed that a covalent linkage of the different components is required for the observed biological activities. In view of the highly potent nature of the constructs and the modular approach that relies on convenient synthetic methods utilizing therapeutically approved biomolecules, our method expedites the production of potent bispecific antibody constructs with tunable cell killing efficacy with significant impact on therapeutic properties.
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Anticorpos Biespecíficos , Neoplasias , Humanos , Linfócitos T , Química Click , Neoplasias/tratamento farmacológico , Anticorpos Biespecíficos/química , Antígenos de Neoplasias/metabolismoRESUMO
Peptide and protein bioconjugation sees ever-growing applications in the pharmaceutical sector. Novel strategies and reagents that can address the chemo- and regioselectivity issues inherent to these biomolecules, while delivering stable and functionalizable conjugates, are therefore needed. Herein, we introduce the crosslinking ethynylbenziodazolone (EBZ) reagent JW-AM-005 for the conjugation of peptides and proteins through the selective linkage of cysteine residues. This easily accessed compound gives access to peptide dimers or stapled peptides under mild and tuneable conditions. Applied to the antibody fragment of antigen binding (Fab) species, JW-AM-005 delivered rebridged proteins in a one-pot three-reaction process with high regioselectivity, outperforming the standard reagents commonly used for this transformation.
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Cisteína , Iodo , Cisteína/química , Reagentes de Ligações Cruzadas/química , Iodo/química , Proteínas/química , Peptídeos , Indicadores e ReagentesRESUMO
Proteo3Dnet is a web server dedicated to the analysis of mass spectrometry interactomics experiments. Given a flat list of proteins, its aim is to organize it in terms of structural interactions to provide a clearer overview of the data. This is achieved using three means: (i) the search for interologs with resolved structure available in the protein data bank, including cross-species remote homology search, (ii) the search for possibly weaker interactions mediated through Short Linear Motifs as predicted by ELM-a unique feature of Proteo3Dnet, (iii) the search for protein-protein interactions physically validated in the BioGRID database. The server then compiles this information and returns a graph of the identified interactions and details about the different searches. The graph can be interactively explored to understand the way the core complexes identified could interact. It can also suggest undetected partners to the experimentalists, or specific cases of conditionally exclusive binding. The interest of Proteo3Dnet, previously demonstrated for the difficult cases of the proteasome and pragmin complexes data is, here, illustrated in the context of yeast precursors to the small ribosomal subunits and the smaller interactome of 14-3-3zeta frequent interactors. The Proteo3Dnet web server is accessible at http://bioserv.rpbs.univ-paris-diderot.fr/services/Proteo3Dnet/.
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Conformação Proteica , Mapeamento de Interação de Proteínas/métodos , Software , Proteínas 14-3-3/metabolismo , Internet , Espectrometria de Massas , Domínios e Motivos de Interação entre Proteínas , Mapas de Interação de Proteínas , Proteômica , Subunidades Ribossômicas Menores de Eucariotos/metabolismoRESUMO
BACKGROUND: Nuclear receptors are transcription factors of central importance in human biology and associated diseases. Much of the knowledge related to their major functions, such as ligand and DNA binding or dimerization, derives from functional studies undertaken in classical model animals. It has become evident, however, that a deeper understanding of these molecular functions requires uncovering how these characteristics originated and diversified during evolution, by looking at more species. In particular, the comprehension of how dimerization evolved from ancestral homodimers to a more sophisticated state of heterodimers has been missing, due to a too narrow phylogenetic sampling. Here, we experimentally and phylogenetically define the evolutionary trajectory of nuclear receptor dimerization by analyzing a novel NR7 subgroup, present in various metazoan groups, including cnidarians, annelids, mollusks, sea urchins, and amphioxus, but lost in vertebrates, arthropods, and nematodes. RESULTS: We focused on NR7 of the cephalochordate amphioxus B. lanceolatum. We present a complementary set of functional, structural, and evolutionary analyses that establish that NR7 lies at a pivotal point in the evolutionary trajectory from homodimerizing to heterodimerizing nuclear receptors. The crystal structure of the NR7 ligand-binding domain suggests that the isolated domain is not capable of dimerizing with the ubiquitous dimerization partner RXR. In contrast, the full-length NR7 dimerizes with RXR in a DNA-dependent manner and acts as a constitutively active receptor. The phylogenetic and sequence analyses position NR7 at a pivotal point, just between the basal class I nuclear receptors that form monomers or homodimers on DNA and the derived class II nuclear receptors that exhibit the classical DNA-independent RXR heterodimers. CONCLUSIONS: Our data suggest that NR7 represents the "missing link" in the transition between class I and class II nuclear receptors and that the DNA independency of heterodimer formation is a feature that was acquired during evolution. Our studies define a novel paradigm of nuclear receptor dimerization that evolved from DNA-dependent to DNA-independent requirements. This new concept emphasizes the importance of DNA in the dimerization of nuclear receptors, such as the glucocorticoid receptor and other members of this pharmacologically important oxosteroid receptor subfamily. Our studies further underline the importance of studying emerging model organisms for supporting cutting-edge research.
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Receptores de Glucocorticoides , Receptores do Ácido Retinoico , Animais , DNA , Dimerização , Humanos , Cetosteroides , Ligantes , Filogenia , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Glucocorticoides/genética , Receptores do Ácido Retinoico/química , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/metabolismo , Receptores X de Retinoides/química , Receptores X de Retinoides/genética , Receptores X de Retinoides/metabolismoRESUMO
In the quest to market increasingly safer and more potent biotherapeutic proteins, the concept of the multi-attribute method (MAM) has emerged from biopharmaceutical companies to boost the quality-by-design process development. MAM strategies rely on state-of-the-art analytical workflows based on liquid chromatography coupled to mass spectrometry (LC-MS) to identify and quantify a selected series of critical quality attributes (CQA) in a single assay. Here, we aimed at evaluating the repeatability and robustness of a benchtop LC-MS platform along with bioinformatics data treatment pipelines for peptide mapping-based MAM studies using standardized LC-MS methods, with the objective to benchmark MAM methods across laboratories, taking nivolumab as a case study. Our results evidence strong interlaboratory consistency across LC-MS platforms for all CQAs (i.e., deamidation, oxidation, lysine clipping and glycosylation). In addition, our work uniquely highlights the crucial role of bioinformatics postprocessing in MAM studies, especially for low-abundant species quantification. Altogether, we believe that MAM has fostered the development of routine, robust, easy-to-use LC-MS platforms for high-throughput determination of major CQAs in a regulated environment.
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Anticorpos Monoclonais , Anticorpos Monoclonais/química , Espectrometria de Massas/métodos , Cromatografia Líquida/métodos , Glicosilação , Mapeamento de Peptídeos/métodosRESUMO
We have prepared a hetero-tetrametallic assembly consisting of three ytterbium ions coordinated to a central [Ru(bpm)3]2+ (bpm = 2,2'-bipyrimidine) motif. Irradiation into the absorption band of the peripheral ytterbium ions at 980 nm engenders emission of the 3MLCT state of the central [Ru(bpm)3]2+ core at 636 nm, which represents the first example of f â d molecular upconversion (UC). Time-resolved measurements reveal a slow rise of the UC emission, which was modeled with a mathematical treatment of the observed kinetics according to a cooperative photosensitization mechanism using a virtual Yb centered doubly excited state followed by energy transfer to the Ru centered 1MLCT state.
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Itérbio , Transferência de Energia , ÍonsRESUMO
Multispecific antibodies, which target multiple antigens at once, are emerging as promising therapeutic entities to offer more effective treatment than conventional monoclonal antibodies (mAbs). However, these highly complex mAb formats pose significant analytical challenges. We report here on the characterization of a trispecific antibody (tsAb), which presents two isomeric forms clearly separated and identified with size exclusion chromatography coupled to native mass spectrometry (SEC-nMS). Previous studies showed that these isomers might originate from a proline cis/trans isomerization in one Fab subunit of the tsAb. We combined several innovative ion mobility (IM)-based approaches to confirm the isomeric nature of the two species and to gain new insights into the conformational landscape of both isomers. Preliminary SEC-nIM-MS measurements performed on a low IM resolution instrument provided the first hints of the coexistence of different conformers, while complementary collision-induced unfolding (CIU) experiments evidenced distinct gas-phase unfolding behaviors upon activation for the two isomers. As subtle conformational differences remained poorly resolved on our early generation IM platform, we performed high-resolution cyclic IM (cIM-MS) to unambiguously conclude on the coexistence of two conformers. The cis/trans equilibrium was further tackled by exploiting the IMn slicing capabilities of the cIM-MS instrument. Altogether, our results clearly illustrate the benefits of combining state-of-the-art nMS and IM-MS approaches to address challenging issues encountered in biopharma. As engineered antibody constructs become increasingly sophisticated, CIU and cIM-MS methodologies undoubtedly have the potential to integrate the drug development analytical toolbox to achieve in-depth conformational characterization of these products.
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Anticorpos Monoclonais , Antineoplásicos Imunológicos , Anticorpos Monoclonais/química , Cromatografia em Gel , Espectrometria de Massas/métodosRESUMO
Thermal decomposition is a very efficient synthesis strategy to obtain nanosized metal oxides with controlled structures and properties. For the iron oxide nanoparticle synthesis, it allows an easy tuning of the nanoparticle's size, shape, and composition, which is often explained by the LaMer theory involving a clear separation between nucleation and growth steps. Here, the events before the nucleation of iron oxide nanocrystals are investigated by combining different complementary in situ characterization techniques. These characterizations are carried out not only on powdered iron stearate precursors but also on a preheated liquid reaction mixture. They reveal a new nucleation mechanism for the thermal decomposition method: instead of a homogeneous nucleation, the nucleation occurs within vesicle-like-nanoreactors confining the reactants. The different steps are: 1) the melting and coalescence of iron stearate particles, leading to "droplet-shaped nanostructures" acting as nanoreactors; 2) the formation of a hitherto unobserved iron stearate crystalline phase within the nucleation temperature range, simultaneously with stearate chains loss and Fe(III) to Fe(II) reduction; 3) the formation of iron oxide nuclei inside the nanoreactors, which are then ejected from them. This mechanism paves the way toward a better mastering of the metal oxide nanoparticles synthesis and the control of their properties.
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Nanopartículas Metálicas , Óxidos , Meios de Cultura , Compostos Férricos/química , Ferro , Nanopartículas Metálicas/química , Óxidos/química , EstearatosRESUMO
Cleavable linkers have become the subject of intense study in the field of chemical biology, particularly because of their applications in the construction of antibody-drug conjugates (ADC), where they facilitate lysosomal cleavage and liberation of drugs from their carrier protein. Due to lysosomes' acidic nature, acid-labile motifs have attracted much attention, leading to the development of hydrazone and carbonate linkers among several other entities. Continuing our efforts in designing new moieties, we present here a family of cyclic acetals that exhibit excellent plasma stability and acid lability, notably in lysosomes. Incorporated in ADC, they led to potent constructs with picomolar potency in vitro and similar in vivo efficacy as the commercially available ADC Kadcyla in mouse xenograft models.