RESUMEN
Ferroptosis is an iron-dependent lipid-peroxidation-driven mechanism of cell death and a promising therapeutic target to eradicate cancer cells. In this study, we discovered that boronic acid-derived salicylidenehydrazone (BASHY) dyes are highly efficient singlet-oxygen photosensitizers (PSs; ΦΔ up to 0.8) that induce ferroptosis triggered by photodynamic therapy. The best-performing BASHY dye displayed a high phototoxicity against the human glioblastoma multiform U87 cell line, with an IC50 value in the low nanomolar range (4.40 nM) and a remarkable phototoxicity index (PI > 22,700). Importantly, BASHY dyes were shown to accumulate in lipid droplets (LDs) and this intracellular partition was found to be essential for the enhanced phototoxicity and the induction of ferroptosis through lipid peroxidation. The safety and phototoxicity of this platform were validated using an in vivo zebrafish model (Danio rerio).
Asunto(s)
Ferroptosis , Fármacos Fotosensibilizantes , Animales , Humanos , Fármacos Fotosensibilizantes/farmacología , Colorantes , Peroxidación de Lípido , Gotas Lipídicas , Pez CebraRESUMEN
Benzodiazaborines (BDABs) have emerged as a valuable tool to produce stable and functional bioconjugates via a click-type transformation. However, the current available methods to install them on peptides lack bioorthogonality, limiting their applications. Here, we report a strategy to install BDABs directly on peptide chains using (2-cyanamidophenyl)boronic acids (2CyPBAs). The resulting BDAB is stabilized through the formation of a key intramolecular B-N bond. This technology was applied in the selective modification of N-terminal cysteine-containing functional peptides.
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Ácidos Borónicos , Cisteína , Ácidos Borónicos/química , Cisteína/química , Péptidos/química , Nitrilos/química , Cianamida/químicaRESUMEN
Immune checkpoint blockade reaches remarkable clinical responses. However, even in the most favorable cases, half of these patients do not benefit from these therapies in the long term. It is hypothesized that the activation of host immunity by co-delivering peptide antigens, adjuvants, and regulators of the transforming growth factor (TGF)-ß expression using a polyoxazoline (POx)-poly(lactic-co-glycolic) acid (PLGA) nanovaccine, while modulating the tumor-associated macrophages (TAM) function within the tumor microenvironment (TME) and blocking the anti-programmed cell death protein 1 (PD-1) can constitute an alternative approach for cancer immunotherapy. POx-Mannose (Man) nanovaccines generate antigen-specific T-cell responses that control tumor growth to a higher extent than poly(ethylene glycol) (PEG)-Man nanovaccines. This anti-tumor effect induced by the POx-Man nanovaccines is mediated by a CD8+ -T cell-dependent mechanism, in contrast to the PEG-Man nanovaccines. POx-Man nanovaccine combines with pexidartinib, a modulator of the TAM function, restricts the MC38 tumor growth, and synergizes with PD-1 blockade, controlling MC38 and CT26 tumor growth and survival. This data is further validated in the highly aggressive and poorly immunogenic B16F10 melanoma mouse model. Therefore, the synergistic anti-tumor effect induced by the combination of nanovaccines with the inhibition of both TAM- and PD-1-inducing immunosuppression, holds great potential for improving immunotherapy outcomes in solid cancer patients.
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Melanoma , Macrófagos Asociados a Tumores , Ratones , Animales , Línea Celular Tumoral , Inmunoterapia , Linfocitos T CD8-positivos , Microambiente TumoralRESUMEN
A set of nine boronic-acid-derived salicylidenehydrazone (BASHY) complexes has been synthesized in good to very good chemical yields in a versatile three-component reaction. In an extension to previous reports on this dye platform, the focus was put on the electronic modification of the "vertical" positions of the salicylidenehydrazone backbone. This enabled the observation of fluorescence quenching by photoinduced electron transfer (PeT), which can be reverted by the addition of acid in organic solvent (OFF-ON fluorescence switching). The resulting emission is observed in the green-to-orange spectral region (maxima at 520-590â nm). In contrast, under physiological pH conditions in water, the PeT process is inherently decativated, thereby enabling the observation of fluorescence in the red-to-NIR region (maxima at 650-680â nm) with appreciable quantum yields and lifetimes. The latter characteristic supported the application of the dyes in fluorescence lifetime imaging (FLIM) of live A549 cells.
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Antibody-drug conjugates (ADCs) are among the fastest-growing classes of therapeutics in oncology. Although ADCs are in the spotlight, they still present significant engineering challenges. Therefore, there is an urgent need to develop more stable and effective ADCs. Most rabbit light chains have an extra disulfide bridge, that links the variable and constant domains, between Cys80 and Cys171, which is not found in the human or mouse. Thus, to develop a new generation of ADCs, we explored the potential of rabbit-derived VL-single-domain antibody scaffolds (sdAbs) to selectively conjugate a payload to Cys80. Hence, a rabbit sdAb library directed towards canine non-Hodgkin lymphoma (cNHL) was subjected to in vitro and in vivo phage display. This allowed the identification of several highly specific VL-sdAbs, including C5, which specifically target cNHL cells in vitro and present promising in vivo tumor uptake. C5 was selected for SN-38 site-selective payload conjugation through its exposed free Cys80 to generate a stable and homogenous C5-DAB-SN-38. C5-DAB-SN-38 exhibited potent cytotoxicity activity against cNHL cells while inhibiting DNA-TopoI activity. Overall, our strategy validates a platform to develop a novel class of ADCs that combines the benefits of rabbit VL-sdAb scaffolds and the canine lymphoma model as a powerful framework for clinically translation of novel therapeutics for cancer.
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Antineoplásicos , Inmunoconjugados , Neoplasias , Animales , Perros , Conejos , Ratones , Humanos , Inmunoconjugados/farmacología , Anticuerpos Monoclonales/farmacología , Irinotecán , Neoplasias/terapia , Antígenos , Antineoplásicos/farmacologíaRESUMEN
An increasing number of novel Ru(II) polypyridyl complexes have been successfully applied as photosensitizers (PSs) for photodynamic therapy (PDT). Despite recent advances in optimized PSs with refined photophysical properties, the lack of tumoral selectivity is often a major hurdle for their clinical development. Here, classical maleimide and versatile NHS-activated acrylamide strategies were employed to site-selectively conjugate a promising Ru(II) polypyridyl complex to the N-terminally Cys-modified Bombesin (BBN) targeting unit. Surprisingly, the decreased cell uptake of these novel Ru-BBN conjugates in cancer cells did not hamper the high phototoxic activity of the Ru-containing bioconjugates and even decreased the toxicity of the constructs in the absence of light irradiation. Overall, although deceiving in terms of selectivity, our new bioconjugates could still be useful for advanced cancer treatment due to their nontoxicity in the dark.
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Antineoplásicos , Complejos de Coordinación , Neoplasias , Fotoquimioterapia , Rutenio , Complejos de Coordinación/farmacología , Complejos de Coordinación/efectos de la radiación , Rutenio/farmacología , Bombesina , Fármacos Fotosensibilizantes/farmacología , Antineoplásicos/farmacología , Neoplasias/tratamiento farmacológicoRESUMEN
N-terminal Cys modification has been intensively studied to produce homogeneous bioconjugates essentially through two modes of reaction: reversible modification with the equilibrium shifted towards the formation of the desired conjugate or stable and irreversible conjugates. Herein, we report a new method of N-terminal cysteine modification using O-salicylaldehyde esters (OSAEs) through fast conjugation and irreversible deconjugation. These reagents can rapidly react with N-terminal Cys at low-micromolar concentration to form thiazolidines with subsequent hydrolysis of the ester moiety to the phenolic derivative. These phenolic thiazolidines can be hydrolyzed at acidic pH (≈4.5) to recover the intact N-terminal Cys. Bioconjugation reactions using OSAEs offer controlled reversibility to as act as a protecting group for N-terminal cysteines, allowing the modification of in-chain residues without perturbing the N-terminal Cys, which can then be deprotected and used as a conjugation site.
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Aldehídos , Cisteína , Cisteína/química , Tiazolidinas , Ésteres/químicaRESUMEN
Post-translational modifications of histones are essential in the regulation of chromatin structure and function. Among these modifications, lysine acetylation is one of the most established. Earlier studies relied on the use of chromatin containing heterogeneous mixtures of histones acetylated at multiple sites. Differentiating the individual contribution of single acetylation events towards chromatin regulation is thus of great relevance. However, it is difficult to access homogeneous samples of histones, with a single acetylation, in sufficient quantities for such studies. By engineering histone H3 with a cysteine in proximity of the lysine of interest, we demonstrate that conjugation with maleimide-DBCO followed by a strain-promoted alkyne-azide cycloaddition reaction results in the acetylation of a single lysine in a controlled, site-specific manner. The chemical precision offered by our click-to-acetylate approach will facilitate access to and the study of acetylated histones.
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Histonas , Lisina , Histonas/química , Acetilación , Lisina/química , Cisteína , Procesamiento Proteico-Postraduccional , CromatinaRESUMEN
The site-selective chemical appendage of multiple functionalities on a native peptide backbone is a highly demanding and complex tool of modern chemical biology. Here, novel NHS-activated acrylates were designed to hold various payloads in a single bioconjugation handle that is able to site-selectively and orthogonally target the N-terminal cysteine of peptides.
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Acrilatos , Cisteína , Acrilatos/química , Cisteína/química , Péptidos/química , Medicina EstatalRESUMEN
In this study, we show that fluorescent boronic-acid derived salicylidenehydrazone complexes (BASHY) can function as fluorescent linkers for bioconjugates that were used to monitor the delivery of the proteasome inhibitor bortezomib (Btz) to HT-29 cancer cells. BASHY complexes were structurally optimized to improve the stability of the complex in buffered conditions (ammonium acetate, pH 7 up to t 1/2 = 40 h), photophysically characterized regarding their fluorescence properties and used in confocal microscopy colocalization studies that revealed their intracellular sequestration by lipid droplets. The accumulation in these hydrophobic organelles limited the hydrolysis of the complex and consequently the drug release, a problem that was circumvented by the conjugation of the BASHY-Btz complex with a cell-penetrating peptide GV1001-C. The conjugate exhibited an improved cytoplasmic availability as confirmed by confocal fluorescence microscopy studies and an improved potency against HT-29 cancer cells (IC50 = 100 nM) as compared to the nontargeted complex (IC50 = 450 nM).
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A new family of hypervalent iodine reagents containing transferable primary amine groups is described. Benziodoxolone-based reagents were synthesized on the gram-scale through operationally simple reactions in up to quantitative yields. These bench-stable solids were characterized by X-ray analysis and successfully employed in the α-amination of indanone-based ß-ketoesters in up to 83% yield. Mechanistic studies indicate a substitution mechanism involving an electrophilic amine.
RESUMEN
Antibody-drug conjugates (ADCs) are a new class of therapeutics that combine the lethality of potent cytotoxic drugs with the targeting ability of antibodies to selectively deliver drugs to cancer cells. In this study we show for the first time the synthesis of a reactive-oxygen-species (ROS)-responsive ADC (VL-DAB31-SN-38) that is highly selective and cytotoxic to B-cell lymphoma (CLBL-1 cell line, IC50 value of 54.1â nM). The synthesis of this ADC was possible due to the discovery that diazaborines (DABs) are a very effective ROS-responsive unit that are also very stable in buffer and in plasma. DFT calculations performed on this system revealed a favorable energetic profile (ΔGR=-74.3â kcal mol-1 ) similar to the oxidation mechanism of aromatic boronic acids. DABs' very fast formation rate and modularity enabled the construction of different ROS-responsive linkers featuring self-immolative modules, bioorthogonal functions, and bioconjugation handles. These structures were used in the site-selective functionalization of a VL antibody domain and in the construction of the homogeneous ADC.
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Antineoplásicos/farmacología , Compuestos de Boro/farmacología , Inmunoconjugados/farmacología , Linfoma de Células B/tratamiento farmacológico , Especies Reactivas de Oxígeno/metabolismo , Antineoplásicos/química , Antineoplásicos/metabolismo , Compuestos de Boro/química , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Inmunoconjugados/química , Inmunoconjugados/metabolismo , Linfoma de Células B/metabolismo , Linfoma de Células B/patología , Estructura MolecularRESUMEN
The development of bioconjugation chemistry has enabled the combination of various synthetic functionalities to proteins, giving rise to new classes of protein conjugates with functions well beyond what Nature can provide. Despite the progress in bioconjugation chemistry, there are no reagents developed to date where the reactivity can be tuned in a user-defined fashion to address different amino acid residues in proteins. Here, we report that 2-chloromethyl acryl reagents can serve as a simple yet versatile platform for selective protein modification at cysteine or disulfide sites by tuning their inherent electronic properties through the amide or ester linkage. Specifically, the 2-chloromethyl derivatives (acrylamide or acrylate) can be obtained via a simple and easily implemented one-pot reaction based on the coupling reaction between commercially available starting materials with different end-group functionalities (amino group or hydroxyl group). 2-Chloromethyl acrylamide reagents with an amide linkage favor selective modification at the cysteine site with fast reaction kinetics and near quantitative conversations. In contrast, 2-chloromethyl acrylate reagents bearing an ester linkage can undergo two successive Michael reactions, allowing the selective modification of disulfides bonds with high labeling efficiency and good conjugate stability.
RESUMEN
Multiple sclerosis (MS) is a demyelinating disease of the central nervous system that is characterized by the presence of demyelinated regions with accumulated myelin lipid debris. Importantly, to allow effective remyelination, such debris must be cleared by microglia. Therefore, the study of microglial activity with sensitive tools is of great interest to better monitor the MS clinical course. Using a boronic acid-based (BASHY) fluorophore, specific for nonpolar lipid aggregates, we aimed to address BASHY's ability to label nonpolar myelin debris and image myelin clearance in the context of demyelination. Demyelinated ex vivo organotypic cultures (OCSCs) and primary microglia cells were immunostained to evaluate BASHY's co-localization with myelin debris and also to evaluate BASHY's specificity for phagocytosing cells. Additionally, mice induced with experimental autoimmune encephalomyelitis (EAE) were injected with BASHY and posteriorly analyzed to evaluate BASHY+ microglia within demyelinated lesions. Indeed, in our in vitro and ex vivo studies, we showed a significant increase in BASHY labeling in demyelinated OCSCs, mostly co-localized with Iba1-expressing amoeboid/phagocytic microglia. Most importantly, BASHY's presence was also found within demyelinated areas of EAE mice, essentially co-localizing with lesion-associated Iba1+ cells, evidencing BASHY's potential for the in vivo bioimaging of myelin clearance and myelin-carrying microglia in regions of active demyelination.
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Ácidos Borónicos/química , Colorantes/química , Enfermedades Desmielinizantes/patología , Diagnóstico por Imagen , Colorantes Fluorescentes/química , Microglía/patología , Vaina de Mielina/patología , Fagocitosis , Animales , Línea Celular , Endocitosis , Fluorescencia , Humanos , Macrófagos , Ratones Endogámicos C57BL , Ratas WistarRESUMEN
Dynamic covalent chemistry (DCvC) has emerged as a versatile synthetic tool for devising stable, stimuli-responsive linkers or conjugates. The interplay of binding affinity, association and dissociation constants exhibits a strong influence on the selectivity of the reaction, the conversion rate, as well as the stability in aqueous solutions. Nevertheless, dynamic covalent interactions often exhibit fast binding and fast dissociation events or vice versa, affecting their conversion rates or stabilities. To overcome the limitation in linker design, we reported herein dual responsive dynamic covalent peptide tags combining a pH responsive boronate ester with fast association and dissociation rates, and a redox-active disulfide with slow formation and dissociation rate. Precoordination by boronic acid-catechol interaction improves self-sorting and selectivity in disulfide formation into heterodimers. The resulting bis-peptide conjugate exhibited improved complex stability in aqueous solution and acidic tumor-like extracellular microenvironment. Furthermore, the conjugate responds to pH changes within the physiological range as well as to redox conditions found inside cancer cells. Such tags hold great promise, through cooperative effects, for controlling the stability of bioconjugates under dilution in aqueous media, as well as designing intelligent pharmaceutics that react to distinct biological stimuli in cells.
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Ácidos BorónicosRESUMEN
Correction for 'A 2-formylphenylboronic acid (2FPBA)-maleimide crosslinker: a versatile platform for Cys-peptide-hydrazine conjugation and interplay' by João P. M. António et al., Org. Biomol. Chem., 2021, 19, 6221-6226, DOI: 10.1039/D1OB00917F.
RESUMEN
In this work, we describe the preparation of a heterobifunctional 2-formylphenylboronic acid (2-FPBA)-maleimide crosslinker and explore its versatility in the preparation of various bioconjugates. We demonstrate the straightforward attachment of hydrazine payloads to cysteine residues in peptides, as well as the crosslinking of different thiol-bearing peptides or payloads with N-terminal cysteine peptides. Importantly, the dynamic nature of the 2-FPBA handle enables an interplay between the thiazolidine and diazaborine forms, which allows obtaining various products controlled by (and in some cases independent of) the order of addition of the components.
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Phenylketonuria (PKU) is a genetic disease caused by deficient activity of human phenylalanine hydroxylase (hPAH) that, when untreated, can lead to severe psychomotor impairment. Protein misfolding is recognized as the main underlying pathogenic mechanism of PKU. Therefore, the use of stabilizers of protein structure and/or activity is an attractive therapeutic strategy for this condition. Here, we report that 3-hydroxyquinolin-2(1H)-one derivatives can act as protectors of hPAH enzyme activity. Electron paramagnetic resonance spectroscopy demonstrated that the 3-hydroxyquinolin-2(1H)-one compounds affect the coordination of the non-heme ferric center at the enzyme active-site. Moreover, surface plasmon resonance studies showed that these stabilizing compounds can be outcompeted by the natural substrate l-phenylalanine. Two of the designed compounds functionally stabilized hPAH by maintaining protein activity. This effect was observed on the recombinant purified protein and in a cellular model. Besides interacting with the catalytic iron, one of the compounds also binds to the N-terminal regulatory domain, although to a different location from the allosteric l-Phe binding site, as supported by the solution structures obtained by small-angle X-ray scattering.
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Fenilalanina Hidroxilasa/metabolismo , Quinolonas/química , Quinolonas/farmacología , Dominio Catalítico , Espectroscopía de Resonancia por Spin del Electrón , Fluorometría , Células HEK293 , Humanos , Enfermedades Metabólicas/metabolismo , Modelos Moleculares , Fenilalanina/metabolismo , Fenilcetonurias/metabolismo , Resonancia por Plasmón de Superficie , TripsinaRESUMEN
Widely used reagents in the peptide functionalization toolbox, Michael acceptors and N-hydroxysuccinimide (NHS) activated esters, are combined in NHS-activated acrylamides for efficient chemoselective amino-sulfhydryl stapling on native peptides and proteins. NHS-activated acrylamides allow for a fast functionalization of N-terminal cysteines (k2 =1.54±0.18×103 â M-1 s-1 ) under dilute aqueous conditions, enabling selectivity over other nucleophilic amino acids. Additionally, the versatility of these new bioconjugation handles was demonstrated in the cross-linking of in-chain or C-terminal cysteines with nearby lysine residues. NHS-activated acrylamides are compatible with the use of other cysteine selective reagents, allowing for orthogonal dual-modifications. This strategy was successfully applied to the late-stage functionalization of peptides and proteins with a PEG unit, fluorescent probe, and cytotoxic agent. The level of molecular control offered by NHS-activated acrylamides is expected to promote amino-sulfhydryl stapling technology as a powerful strategy to design functional bioconjugates.
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The development of metal-based anticancer drugs has been hampered, among other reasons, by their lack of selectivity for cancer cells. In a recent article, Zou and co-workers presented the successful intracellular activation of organogold(I) complexes for potential cancer treatment through Pd(II)-mediated transmetallation, overcoming some off-target activity of novel gold-based drugs. This unique strategy builds the perfect bridge between metallodrug usage and bioorthogonal intracellular catalysis for more advanced and selective therapies. Such an approach will hopefully pave the way for forthcoming studies in medicinal inorganic chemistry.