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Histone dopaminylation is a newly identified epigenetic mark that plays a role in the regulation of gene transcription, where an isopeptide bond is formed between the fifth amino acid of H3 (i.e., glutamine) and dopamine. Recently, we developed a chemical probe to specifically label and enrich histone dopaminylation via bioorthogonal chemistry. Given this powerful tool, we found that histone H3 glutamine 5 dopaminylation (H3Q5dop) was highly enriched in colorectal tumors, which could be attributed to the high expression level of its regulator, transglutaminase 2 (TGM2), in colon cancer cells. Due to the enzyme promiscuity of TGM2, nonhistone proteins have also been identified as dopaminylation targets; however, the dopaminylated proteome in cancer cells still remains elusive. Here, we utilized our chemical probe to enrich dopaminylated proteins from colorectal cancer cells in a bioorthogonal manner and performed the chemical proteomics analysis. Therefore, 425 dopaminylated proteins were identified, many of which are involved in nucleic acid metabolism and transcription pathways. More importantly, a number of dopaminylation sites were identified and attributed to the successful application of our chemical probe. Overall, these findings shed light on the significant association between cellular protein dopaminylation and cancer development, further suggesting that targeting these pathways may become a promising anticancer strategy.
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Neoplasias Colorretais , Histonas , Proteína 2 Glutamina gama-Glutamiltransferase , Proteômica , Humanos , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/genética , Proteômica/métodos , Proteína 2 Glutamina gama-Glutamiltransferase/metabolismo , Histonas/metabolismo , Transglutaminases/metabolismo , Transglutaminases/genética , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/genética , Linhagem Celular Tumoral , Proteoma/análise , Proteoma/metabolismo , Processamento de Proteína Pós-Traducional , Glutamina/metabolismo , Glutamina/química , Epigênese GenéticaRESUMO
The designed and ordered co-immobilization of multiple enzymes for vectorial biocatalysis is challenging. Here, a combination of protein phase separation and bioorthogonal linking is used to generate a zeolitic imidazole framework (ZIF-8) containing co-immobilized enzymes. Zn2+ ions induce the clustering of minimal protein modules, such as 6-His tag, proline-rich motif (PRM) and SRC homology 3 (SH3) domains, and allow for phase separation of the coupled aldoketoreductase (AKR) and alcohol dehydrogenase (ADH) at low concentrations. This is achieved by fusing SpyCatcher and PRM-SH3-6His peptide fragments to the C and N termini of AKR, respectively, and the SpyTag to ADH. Addition of 2-methylimidazole results in droplet formation and enables in situ spatial embedding the recombinant AKR and ADH to generate the cascade biocalysis system encapsulated in ZIF-8 (AAE@ZIF). In synthesizing (S)-1-(2-chlorophenyl) ethanol, ater 6 cycles, the yield can still reach 91%, with 99.99% enantiomeric excess (ee) value for each cycle. However, the yield could only reach 72.9% when traditionally encapsulated AKR and ADH in ZIF-8 are used. Thus, this work demonstrates that a combination of protein phase separation and bio-orthogonal linking enables the in situ creation of a stable and spatially organized bi-enzyme system with enhanced channeling effects in ZIF-8.
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3-oxidopyridinium ions are water stable and soluble heteroaromatic betaines that behave as latent dipoles and undergo a wide variety of cycloadditions. Research into the cycloaddition reactions of 3-oxidopyridiniums was spearheaded by Alan R. Katritzky and collaborators from the early 1970s until the late 1980s, but they have yet to be used for bioorthogonal applications. Herein we report that 3-oxidopyridiniums can readily react with 4-dibenzocyclooctynol (DIBO), a common bioorthogonal handle, in a [3+2] cycloaddition. The mechanism was investigated by altering the electronics of the reaction by changing the substituent on the 5 position of the pyridinium. Electron-donating 5-substituents have been shown to significantly increase the rate of the reaction, with bimolecular rate constants ranging from 3.3×10-4 s-1 with 5-trifluoromethyl-N-methyl-3-oxidopyridinium to 1.07â M-1 s-1 with 5-amino-N-methyl-3-oxidopyridinium. 3-oxidopyridiniums' appreciable cycloaddition rates and compatibility with bioorthogonally relevant environments give them the potential to be used in a variety of bioconjugation applications.
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The field of bioorthogonal chemistry is rapidly growing, presenting successful applications of organic and transition metal-catalysed reactions in cells and living systems (in vivo). The development of such reactions typically proceeds through many iterative steps focused on biocompatibility and fast reaction kinetics to ensure product formation. However, obtaining kinetic data, even under simulated biological (biomimetic) conditions, remains a challenge due to substantial concentrations of salts and biomolecules hampering the use of typically employed solution-phase analytical techniques. In this study, we explored the suitability of gas evolution as a probe to study kinetics under biomimetic conditions. As proof of concept, we show that the progress of two transition metal-catalysed bioorthogonal chemical reactions can be accurately monitored, regardless of the complexity of the medium. As such, we introduce a protocol to gain more insight into the performance of a catalytic system under biomimetic conditions to further progress iterative catalyst development for in vivo applications.
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Biomimética , Catálise , Cinética , Biomimética/métodos , Gases/química , Elementos de Transição/química , Materiais Biomiméticos/químicaRESUMO
Site-selective chemical modifications of proteins have emerged as a potent technology in chemical biology, materials science, and medicine, facilitating precise manipulation of proteins with tailored functionalities for basic biology research and developing innovative therapeutics. Compared to traditional recombinant expression methods, one of the prominent advantages of chemical protein modification lies in its capacity to decorate proteins with a wide range of functional moieties, including non-genetically encoded ones, enabling the generation of novel protein conjugates with enhanced or previously unexplored properties. Among these, approaches for dual or multiple protein modifications are increasingly garnering attention, as it has been found that single modifications of proteins are inadequate to meet current demands. Therefore, in light of the rapid developments in this field, this review provides a timely and comprehensive overview of the latest advancements in chemical and biological approaches for protein dual functionalization. It further discusses their advantages, limitations, and potential future directions in this relatively nascent area.
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Increasing the selectivity of chemotherapies by converting them into prodrugs that can be activated at the tumour site decreases their side effects and allows discrimination between cancerous and non-cancerous cells. Herein, the use of metabolic glycoengineering (MGE) to selectively label MCF-7 breast cancer cells with tetrazine (Tz) activators for subsequent activation of prodrugs containing the trans-cyclooctene (TCO) moiety by a bioorthogonal reaction is demonstrated. Three novel Tz-modified monosaccharides, Ac4ManNTz 7, Ac4GalNTz 8, and Ac4SiaTz 16, were used for expression of the Tz activator within sialic-acid rich breast cancer cells' surface glycans through MGE. Tz expression on breast cancer cells (MCF-7) was evaluated versus the non-cancerous L929 fibroblasts showing a concentration-dependant effect and excellent selectivity with ≥35-fold Tz expression on the MCF-7 cells versus the non-cancerous L929 fibroblasts. Next, a novel TCO-N-mustard prodrug and a TCO-doxorubicin prodrug were analyzed in vitro on the Tz-bioengineered cells to probe our hypothesis that these could be activated via a bioorthogonal reaction. Selective prodrug activation and restoration of cytotoxicity were demonstrated for the MCF-7 breast cancer cells versus the non-cancerous L929 cells. Restoration of the parent drug's cytotoxicity was shown to be dependent on the level of Tz expression where the Ac4ManNTz 7 and Ac4GalNTz 8 derivatives (20 µM) lead to the highest Tz expression and full restoration of the parent drug's cytotoxicity. This work suggests the feasibility of combining MGE and tetrazine ligation for selective prodrug activation in breast cancer.
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Antineoplásicos , Neoplasias da Mama , Pró-Fármacos , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Pró-Fármacos/síntese química , Humanos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/síntese química , Feminino , Estrutura Molecular , Ensaios de Seleção de Medicamentos Antitumorais , Relação Estrutura-Atividade , Células MCF-7 , Relação Dose-Resposta a Droga , Proliferação de Células/efeitos dos fármacos , Engenharia Metabólica , Sobrevivência Celular/efeitos dos fármacosRESUMO
BACKGROUND: Radiotheranostics differs from the vast majority of other cancer therapies in its capacity for simultaneous imaging and therapy, and it is becoming more widely implemented. A balance between diagnostic and treatment requirements is essential for achieving effective radiotheranostics. Herein, we propose a proof-of-concept strategy aiming to address the profound differences in the specific requirements of the diagnosis and treatment of radiotheranostics. RESULTS: To validate the concept, we designed an s-tetrazine (Tz) conjugated prostate-specific membrane antigen (PSMA) ligand (DOTA-PSMA-Tz) for 68Ga or 177Lu radiolabeling and tumor radiotheranostics, a trans-cyclooctene (TCO) modified Pd@Au nanoplates (Pd@Au-PEG-TCO) for signal amplification, respectively. We then demonstrated this radiotheranostic strategy in the tumor-bearing mice with the following three-step procedures: (1) i.v. injection of the [68Ga]Ga-PSMA-Tz for diagnosis; (2) i.v. injection of the signal amplification module Pd@Au-PEG-TCO; (3) i.v. injection of the [177Lu]Lu-PSMA-Tz for therapy. Firstly, this strategy was demonstrated in 22Rv1 tumor-bearing mice via positron emission tomography (PET) imaging with [68Ga]Ga-PSMA-Tz. We observed significantly higher tumor uptake (11.5 ± 0.8%ID/g) with the injection of Pd@Au-PEG-TCO than with the injection [68Ga]Ga-PSMA-Tz alone (5.5 ± 0.9%ID/g). Furthermore, we validated this strategy through biodistribution studies of [177Lu]Lu-PSMA-Tz, with the injection of the signal amplification module, approximately five-fold higher tumor uptake of [177Lu]Lu-PSMA-Tz (24.33 ± 2.53% ID/g) was obtained when compared to [177Lu]Lu-PSMA-Tz alone (5.19 ± 0.26%ID/g) at 48 h post-injection. CONCLUSION: In summary, the proposed strategy has the potential to expand the toolbox of pretargeted radiotherapy in the field of theranostics.
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Neoplasias Colorretais , Compostos Radiofarmacêuticos , Masculino , Animais , Camundongos , Radioisótopos de Gálio , Distribuição Tecidual , Linhagem Celular Tumoral , Neoplasias Colorretais/patologiaRESUMO
Protein O-GlcNAcylation is a ubiquitous posttranslational modification of cytosolic and nuclear proteins involved in numerous fundamental regulation processes. Investigation of O-GlcNAcylation by metabolic glycoengineering (MGE) has been carried out for two decades with peracetylated N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine derivatives modified with varying reporter groups. Recently, it has been shown that these derivatives can result in non-specific protein labeling termed S-glyco modification. Here, we report norbornene-modified GlcNAc derivatives with a protected phosphate at the anomeric position and their application in MGE. These derivatives overcome two limitations of previously used O-GlcNAc reporters. They do not lead to detectable S-glyco modification, and they efficiently react in the inverse-electron-demand Diels-Alder (IEDDA) reaction, which can be carried out even within living cells. Using a derivative with an S-acetyl-2-thioethyl-protected phosphate, we demonstrate the protein-specific detection of O-GlcNAcylation of several proteins and the protein-specific imaging of O-GlcNAcylation inside living cells by Förster resonance energy transfer (FRET) visualized by confocal fluorescence lifetime imaging microscopy (FLIM).
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Acetilglucosamina , Glicoproteínas , Imagem Molecular , Norbornanos , Processamento de Proteína Pós-Traducional , Glicosilação , Engenharia Metabólica , Norbornanos/química , Acetilglucosamina/análogos & derivados , Acetilglucosamina/química , Transferência Ressonante de Energia de Fluorescência , Glicoproteínas/análise , Humanos , Células HeLaRESUMO
The field of bioorthogonal chemistry has revolutionized our ability to interrogate and manipulate biological systems at the molecular level. However, the range of chemical reactions that can operate efficiently in biological environments without interfering with the native cellular machinery, remains limited. In this context, the rapidly growing area of photocatalysis offers a promising avenue for developing new type of bioorthogonal tools. The inherent mildness, tunability, chemoselectivity, and external controllability of photocatalytic transformations make them particularly suitable for applications in biological and living systems. This minireview summarizes recent advances in bioorthogonal photocatalytic technologies, with a particular focus on their potential to enable the selective generation of designed products within biologically relevant or living settings.
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The use of highly potent but very toxic antibiotics such as colistin has become inevitable due to the rise of antimicrobial resistance. We aimed for a chemically-triggered, controlled release of colistin at the infection site to lower its systemic toxicity by harnessing the power of click-to-release reactions. Kinetic experiments with nine tetrazines and three dienophiles demonstrated a fast release via an inverse-electron-demand Diels-Alder reaction between trans-cyclooctenes (TCO) and the amine-functionalized tetrazine Tz7. The antibiotic activity of colistin against Escherichia coli was masked by TCO units, but restored upon reaction with d-Ubi-Tz, a tetrazine functionalised with the bacterial binding peptide d-Ubi29-41. While standard TCO did not improve toxicity against human proximal tubular kidney HK-2 cells, the installation of an aspartic acid-modified TCO masking group reduced the overall charge of the peptide and entry to the kidney cells, thereby dramatically lowering its toxicity. The analog Col-(TCO-Asp)1 had favourable pharmacokinetic properties in mice and was successfully activated locally in the lung by d-Ubi-Tz in an in vivo infection model, whereas it remained inactive and non-harmful without the chemical trigger. This study constitutes the first example of a systemically acting two-component antibiotic with improved drug tolerability.
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Although the clearance of senescent cells has been proven to slow down the aging process and promote anti-cancer chemotherapy, the development of senolytics remains challenging. Herein, we report a senolytic strategy enabled by senescent cell-sensitive bioorthogonal tetrazine ligation. Our design is based on linking dihydrotetrazine (Tz) to a galactose (Gal) moiety that serves both as a recognition moiety for senescence-associated ß-galactosidase and a caging group for the control of tetrazine activity. Gal-Tz enables efficient click-release of a fluorescent hemicyanine and doxorubicin from a trans-cyclooctene-caged prodrug to detect and eliminate senescent HeLa and A549 cells over non-senescent counterparts with a 16.44 senolytic index. Furthermore, we leverage the strategy for the selective activation and delivery of proteolysis-targeting chimeras (PROTACs) as senolytics. PROTAC prodrug TCO-ARV-771 can be selectively activated by Gal-Tz and delivered into senescent HeLa and A549 cells to induce the degradation of bromodomain-containing protein 4. Senolytic PROTACs may offer an efficient way for intervention on cell senescence thanks to their unique capacity to degrade target proteins in a sub-stoichiometric and catalytic fashion. The results of this study establish the bioorthogonal tetrazine ligation approach as a viable strategy for selective removal of senescent cells.
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Compostos Heterocíclicos , Pró-Fármacos , Humanos , Linhagem Celular Tumoral , Senoterapia , Pró-Fármacos/farmacologia , Senescência CelularRESUMO
Developing fluorogenic probes for simultaneous live cell labeling of multiple targets is crucial for understanding complex cellular events. The emerging [4+1] cycloaddition between tetrazine and isonitriles holds promise as a bioorthogonal tool, yet existing tetrazine probes lack reactivity and fluorogenicity. Here, we present the development of a series of tetrazine-functionalized bioorthogonal probes. By incorporating pyrazole adducts into the fluorophore scaffolds, the post-reacted probes displayed remarkable fluorescence turn-on ratios, up to 3184-fold. Moreover, these modifications are generalizable to various fluorophores, enabling a broad emission range from 473 to 659â nm. Quantum chemical calculations further elucidate the turn-on mechanisms. These probes enable the simultaneous labeling of multiple targets in live cells, without the need for a washing step. Consequently, our findings pave the way for advanced multiplex imaging and detection techniques for cellular studies.
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Corantes Fluorescentes , Imagem Óptica , Linhagem Celular Tumoral , Reação de Cicloadição , Imagem Óptica/métodosRESUMO
Cells rely heavily on the uptake of exogenous nutrients for survival, growth, and differentiation. Yet quantifying the uptake of small molecule nutrients at the single cell level is difficult. Here we present a new approach to studying the nutrient uptake in live single cells using Inverse Electron-Demand Diels Alder (IEDDA) chemistry. We have modified carboxyfluorescein-diacetate-succinimidyl esters (CFSE)-a quenched fluorophore that can covalently react with proteins and is only turned on in the cytosol of a cell following esterase activity-with a tetrazine. This tetrazine serves as a second quencher for the pendant fluorophore. Upon reaction with nutrients modified with an electron-rich or strained dienophile in an IEDDA reaction, this quenching group is destroyed, thereby enabling the probe to fluoresce. This has allowed us to monitor the uptake of a variety of dienophile-containing nutrients in live primary immune cell populations using flow cytometry and live-cell microscopy.
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Corantes Fluorescentes , Corantes Fluorescentes/química , Humanos , Fluoresceínas/química , Animais , Nutrientes/metabolismo , Succinimidas/química , Citometria de Fluxo , Sobrevivência Celular , Reação de Cicloadição , Camundongos , Estrutura MolecularRESUMO
Cancer has been the most deadly disease, and 13 million cancer casualties are estimated to occur each year by 2030. Gold nanoparticles (AuNPs)-based photothermal therapy (PTT) has attracted great interest due to its high spatiotemporal controllability and noninvasiveness. Due to the trade-off between particle size and photothermal efficiency of AuNPs, rational design is needed to realize aggregation of AuNPs into larger particles with desirable NIR adsorption in tumor site. Exploiting the bioorthogonal "Click and Release" (BCR) reaction between iminosydnone and cycloalkyne, aggregation of AuNPs can be achieved and attractively accompanied by the release of chemotherapeutic drug purposed to photothermal synergizing. We synthesize iminosydnone-lonidamine (ImLND) as a prodrug and choose dibenzocyclooctyne (DBCO) as the trigger of BCR reaction. A PEGylated AuNPs-based two-component nanoplatform consisting of prodrug-loaded AuNPs-ImLND and tumor-targeting peptide RGD-conjugated AuNPs-DBCO-RGD is designed. In the therapeutic regimen, AuNPs-DBCO-RGD are intravenously injected first for tumor-specific enrichment and retention. Once the arrival of AuNPs-ImLND injected later at tumor site, highly photothermally active nanoaggregates of AuNPs are formed via the BCR reaction between ImLND and DBCO. The simultaneous release of lonidamine further enhanced the therapeutic performance by sensitizing cancer cells to PTT.
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Indazóis , Nanopartículas Metálicas , Nanopartículas , Neoplasias , Pró-Fármacos , Humanos , Ouro , Terapia Fototérmica , Nanopartículas Metálicas/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Pró-Fármacos/uso terapêutico , Oligopeptídeos/uso terapêutico , Linhagem Celular TumoralRESUMO
The site-specific activation of bioorthogonal prodrugs has provided great opportunities for reducing the severe side effects of chemotherapy. However, the precise control of activation location, sustained drug production at the target site, and high bioorthogonal reaction efficiency in vivo remain great challenges. Here, we propose the construction of tumor cell membrane reactors in vivo to solve the above problems. Specifically, tumor-targeted liposomes with efficient membrane fusion capabilities are generated to install the bioorthogonal trigger, the amphiphilic tetrazine derivative, on the surface of tumor cells. These predecorated tumor cells act as many living reactors, transforming the tumor into a "drug factory" that in situ activates an externally delivered bioorthogonal prodrug, for example intratumorally injected transcyclooctene-caged doxorubicin. In contrast to the rapid elimination of cargo that is encapsulated and delivered by liposomes, these reactors permit stable retention of bioorthogonal triggers in tumor for 96â h after a single dose of liposomes via intravenous injection, allowing sustained generation of doxorubicin. Interestingly, an additional supplement of liposomes will compensate for the trigger consumed by the reaction and significantly improve the efficiency of the local reaction. This strategy provides a solution to the efficacy versus safety dilemma of tumor chemotherapy.
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Compostos Heterocíclicos , Neoplasias , Pró-Fármacos , Humanos , Pró-Fármacos/uso terapêutico , Lipossomos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Doxorrubicina/uso terapêuticoRESUMO
Carbon disulfide (CS2) is an environmental contaminant, which is deadly hazardous to the workers under chronic or acute exposure. However, the toxicity mechanisms of CS2 are still unclear due to the scarcity of biocompatible donors, which can release CS2 in cells. Here we developed the first bioorthogonal CS2 delivery system based on the "click-and-release" reactions between mesoionic 1,3-thiazolium-5-thiolates (TATs) and strained cyclooctyne exo-BCN-OH. We successfully realized intracellular CS2 release and investigated the causes of CS2-induced hepatotoxicity, including oxidative stress, proteotoxic stress and copper-dependent cell death. It is found that CS2 can be copper vehicles bypassing copper transporters after reacting with nucleophiles in cytoplasm, and extra copper supplementation will exacerbate the loss of homeostasis of cells and ultimately cell death. These findings inspired us to explore the anticancer activity of CS2 in combination with copper by introducing a copper chelating group in our CS2 delivery system.
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Dissulfeto de Carbono , Cobre , Humanos , Dissulfeto de Carbono/química , Dissulfeto de Carbono/metabolismo , Cobre/química , Cobre/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Química Click , Estresse Oxidativo/efeitos dos fármacos , Estrutura Molecular , Sistemas de Liberação de MedicamentosRESUMO
As an effective and non-invasive treatment modality for cancer, photodynamic therapy (PDT) has attracted considerable interest. With the recent advances in the photosensitizing agents, the fiber-optic systems, and other aspects, its application is extended to a wide range of superficial and localized cancers. However, for the few clinically used photosensitizers, most of them suffer from the drawback of causing prolonged photosensitivity after the treatment. As a result, post-PDT management is also a crucial issue. Herein, a facile bioorthogonal approach is reported that can effectively suppress this common side effect of PDT in nude mice. It involves the use of an antidote that contains a black-hole quencher BHQ-3 conjugated with a bicyclo[6.1.0]non-4-yne (BCN) moiety and a tetrazine-substituted boron dipyrromethene-based photosensitizer. By using tumor-bearing nude mice as an animal model, it is demonstrated that after PDT with this photosensitizer, the administration of the antidote can effectively quench the photodynamic activity of the residual photosensitizer by bringing the BHQ-3 quencher close to the photosensitizing unit through a rapid click reaction. It results in substantial reduction in skin damage upon light irradiation. The overall results demonstrate that this simple and facile strategy can provide an effective means for minimizing the photosensitivity after PDT.
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Neoplasias , Fotoquimioterapia , Animais , Camundongos , Fármacos Fotossensibilizantes/uso terapêutico , Fotoquimioterapia/métodos , Antídotos , Camundongos Nus , Neoplasias/tratamento farmacológicoRESUMO
Tumor whole cell, carrying a complete set of tumor-associated antigens and tumor-specific antigens, has shown great potential in the construction of tumor vaccines but is hindered by the complex engineering means and limited efficacy to cause immunity. Herein, we provided a strategy for the self-mineralization of autologous tumor cells with palladium ions in microfluidic droplets, which endowed the engineered cells with both immune and catalytic functions, to establish a bioorthogonally catalytic tumor whole-cell vaccine. This vaccine showed strong inhibition both in the occurrence and recurrence of tumor by invoking the immediate antitumor immunity and building a long-term immunity.
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Vacinas Anticâncer , Neoplasias , Humanos , Microfluídica , Imunoterapia , Neoplasias/terapia , Antígenos de NeoplasiasRESUMO
Reactive amino acid side chains play important roles in the binding of peptides to specific targets. In addition, their reactivity enables selective peptide conjugation and functionalization for pharmaceutical purposes. Diverse reactive amino acids are incorporated into nonribosomal peptides, which serve as a source for drug candidates. Notable examples include (poly)unsaturated (enamine, alkyne, and furyl) and halogenated residues, strained carbacycles (cyclopropyl and cyclopropanol), small heterocycles (oxirane and aziridine), and reactive N-N functionalities (hydrazones, diazo compounds, and diazeniumdiolates). Their biosynthesis requires diverse biocatalysts for sophisticated reaction mechanisms. Several avenues have been identified for their incorporation into peptides, the recruitment by adenylation domains or ligases, on-line modifications, and enzymatic tailoring reactions. Combined with protein engineering approaches, this knowledge provides new opportunities in synthetic biology and bioorthogonal chemistry.
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Aminoácidos , Peptídeos , Aminoácidos/metabolismo , Aminoácidos/química , Peptídeos/metabolismo , Peptídeos/química , Biossíntese de Peptídeos Independentes de Ácido Nucleico , Engenharia de Proteínas/métodos , Peptídeo Sintases/metabolismo , Peptídeo Sintases/químicaRESUMO
Messenger RNA (mRNA) display is being increasingly adopted for peptide drug candidate discovery. While many conditions have been reported for the affinity enrichment step and in some cases for peptide modification, there is still limited understanding about the versatility of peptide-puromycin-mRNA/cDNA (complementary DNA) complexes. This work explores the chemical stability of mRNA/cDNA hybrid complexes under a range of different fundamental chemical conditions as well as with peptide modification conditions reported in an mRNA display setting. We further compare the stability of full complexes originating from two different mRNA display systems (RaPID and cDNA-TRAP). Overall, these complexes were found to be stable under a broad range of conditions, with some edge conditions benefitting from encoding directly in cDNA rather than mRNA. This should allow for more and broader exploitation of late-stage peptide modification chemistry in mRNA display, with confidence regarding the stability of encoding, and potentially better hit-finding campaigns as a result.