BASHY Dyes Are Highly Efficient Lipid Droplet-Targeting Photosensitizers that Induce Ferroptosis through Lipid Peroxidation.

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).

Polyoxazoline-Based Nanovaccine Synergizes with Tumor-Associated Macrophage Targeting and Anti-PD-1 Immunotherapy against Solid Tumors.

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.

N-Terminal Cysteine Bioconjugation with (2-Cyanamidophenyl)boronic Acids Enables the Direct Formation of Benzodiazaborines on Peptides.

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.

Rabbit derived VL single-domains as promising scaffolds to generate antibody-drug conjugates.

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.

Towards Selective Delivery of a Ruthenium(II) Polypyridyl Complex-Containing Bombesin Conjugate into Cancer Cells.

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.

Interactive Bioconjugation at N-Terminal Cysteines by Using O-Salicylaldehyde Esters towards Dual Site-Selective Functionalization.

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.

Cysteine-Assisted Click-Chemistry for Proximity-Driven, Site-Specific Acetylation of Histones.

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.

Multivalent NHS-activated acrylates for orthogonal site-selective functionalisation of peptides at cysteine residues.

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.

The BASHY Platform Enables the Assembly of a Fluorescent Bortezomib-GV1001 Conjugate.

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).

BASHY Dye Platform Enables the Fluorescence Bioimaging of Myelin Debris Phagocytosis by Microglia during Demyelination.

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.

Chemoselective cysteine or disulfide modification via single atom substitution in chloromethyl acryl reagents.

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.

Diazaborines Are a Versatile Platform to Develop ROS-Responsive Antibody Drug Conjugates*.

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 54.1 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=-74.3 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.

Dual Stimuli-Responsive Dynamic Covalent Peptide Tags: Toward Sequence-Controlled Release in Tumor-like Microenvironments.

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.

A 2-formylphenylboronic acid (2FPBA)-maleimide crosslinker: a versatile platform for Cys-peptide-hydrazine conjugation and interplay.

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.

Unveiling the Potential of Transition Metal Complexes for Medicine: Translational in Situ Activation of Metal-Based Drugs from Bench to in Vivo Applications.

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.

Efficient Amino-Sulfhydryl Stapling on Peptides and Proteins Using Bifunctional NHS-Activated Acrylamides.

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  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.

Engineering Boron Hot Spots for the Site-Selective Installation of Iminoboronates on Peptide Chains.

Boronic acids (BAs) are a promising bioconjugation function to design dynamic materials as they can establish reversible covalent bonds with oxygen/nitrogen nucleophiles that respond to different pH, ROS, carbohydrates and glutathione levels. However, the dynamic nature of these bonds also limits the control over the stability and site-selectivity of the bioconjugation, which ultimately leads to heterogeneous conjugates with poor stability under physiological conditions. Here we disclose a new strategy to install BAs on peptide chains. In this study, a "boron hot spot" based on the 3-hydroxyquinolin-2(1H)-one scaffold was developed and upon installation on a peptide N-terminal cysteine, enables the site-selective formation of iminoboronates with 2-formyl-phenyl boronic acids (Ka of 58128±2 m-1 ). The reaction is selective in the presence of competing lysine ϵ-amino groups, and the resulting iminoboronates, displayed improved stability in buffers solutions and a cleavable profile in the presence of glutathione. Once developed, the methodology was used to prepare cleavable fluorescent conjugates with a laminin fragment, which enabled the validation of the 67LR receptor as a target to deliver cargo to cancer HT29 cells.

Immunization with mannosylated nanovaccines and inhibition of the immune-suppressing microenvironment sensitizes melanoma to immune checkpoint modulators.

A low response rate, acquired resistance and severe side effects have limited the clinical outcomes of immune checkpoint therapy. Here, we show that combining cancer nanovaccines with an anti-PD-1 antibody (αPD-1) for immunosuppression blockade and an anti-OX40 antibody (αOX40) for effector T-cell stimulation, expansion and survival can potentiate the efficacy of melanoma therapy. Prophylactic and therapeutic combination regimens of dendritic cell-targeted mannosylated nanovaccines with αPD-1/αOX40 demonstrate a synergism that stimulates T-cell infiltration into tumours at early treatment stages. However, this treatment at the therapeutic regimen does not result in an enhanced inhibition of tumour growth compared to αPD-1/αOX40 alone and is accompanied by an increased infiltration of myeloid-derived suppressor cells in tumours. Combining the double therapy with ibrutinib, a myeloid-derived suppressor cell inhibitor, leads to a remarkable tumour remission and prolonged survival in melanoma-bearing mice. The synergy between the mannosylated nanovaccines, ibrutinib and αPD-1/αOX40 provides essential insights to devise alternative regimens to improve the efficacy of immune checkpoint modulators in solid tumours by regulating the endogenous immune response.

Bioconjugation with Maleimides: A Useful Tool for Chemical Biology.

Maleimide chemistry stands out in the bioconjugation toolbox by virtue of its synthetic accessibility, excellent reactivity, and practicability. The second-generation of clinically approved antibody-drug conjugates (ADC) and much of the current ADC pipeline in clinical trials contain the maleimide linkage. However, thiosuccinimide linkages are now known to be less robust than once thought, and ergo, are correlated with suboptimal pharmacodynamics, pharmacokinetics, and safety profiles in some ADC constructs. Rational design of novel generations of maleimides and maleimide-type reagents have been reported to address the shortcomings of classical maleimides, allowing for the formation of robust bioconjugate linkages. This review highlights the main strategies for rational reagent design that have allowed irreversible bioconjugations in cysteines, reversible labelling strategies and disulfide re-bridging.

N,O-Iminoboronates: Reversible Iminoboronates with Improved Stability for Cancer Cells Targeted Delivery.

Herein a new class of iminoboronates obtained from 2-acetylbenzene boronic acids and aminophenols is presented. The N,O-ligand topology enabled the formation of an additional B-O bond that locks the boron center in a tetrahedral geometry. This molecular arrangement decisively contributes to improve the construct's stability in biocompatible conditions and retaining the iminoboronate reversibility in more acidic environments. 2-Acetylbenzene boronic acid was reacted with a fluorescent amino-coumarin to yield a stable and non-fluorescent N,O-iminoboronate. This mechanism was further used to assemble a folate receptor targeting conjugate that selectively delivered the fluorescent amino-coumarin to MDA-MB-231 human breast cancer cells.