Drug upgrade: A complete methodology from old drug to new chemical entities using Nematic Protein Organization Technique.

Drug repurposing is used to propose new therapeutic perspectives. Here, we introduce "Drug Upgrade", that is, characterizing the mode of action of an old drug to generate new chemical entities and new therapeutics. We proposed a novel methodology covering target identification to pharmacology validation. As an old drug, we chose hydroxychloroquine (HCQ) for its well-documented clinical efficacy in lupus and its side effect, retinal toxicity. Using the Nematic Protein Organization Technique (NPOT®) followed by liquid chromatography-tandem mass spectrometry analyses, we identified myeloperoxidase (MPO) and alpha-crystallin ß chain (CRYAB) as primary and secondary targets to HCQ from lupus patients' peripheral blood mononuclear cells (PBMCs) and isolated human retinas. Surface plasmon resonance (SPR) and enzymatic assays confirmed the interaction of HCQ with MPO and CRYAB. We synthesized INS-072 a novel analog of HCQ that increased affinity for MPO and decreased binding to CRYAB compared to HCQ. INS-072 delayed cutaneous eruption significantly compared to HCQ in the murine MRL/lpr model of spontaneous lupus and prevents immune complex vasculitis in mice. In addition, long-term HCQ treatment caused retinal toxicity in mice, unlike INS-072. Our study illustrates a method of drug development, where new applications or improvements can be explored by fully characterizing the drug's mode of action.

Targeted delivery of immune-stimulating bispecific RNA, inducing apoptosis and anti-tumor immunity in cancer cells.

Double-stranded RNAs (dsRNA)-based strategies appeared as promising therapies to induce an inflammation in the tumor microenvironment. However, currently described systems generally lack active targeting of tissues, and their clinical translation is thus limited to intratumoral injection. Herein, we developed an antibody-siRNA-5'triphosphate conjugate with multiple modes of action, combining cell surface EphA2-specific internalization, leading to a simultaneous gene silencing and activation of the receptor retinoic acid-inducible gene I (RIG-I). Recognition of cytosolic siRNA-5'triphosphate by RIG-I triggers the expression of interferons and pro-inflammatory cytokines, inducing an inflammation of the tumor environment and activating neighboring immune cells. In addition, these RIG-I-specific effects synergized with siRNA-mediated PLK1 silencing to promote cancer cell death by apoptosis. Altogether, such immune-stimulating antibody-RNA conjugate opens a novel modality to overcome some limitations encountered by dsRNA molecules currently in clinical trials.

Site-Selective Protein Conjugation by a Multicomponent Ugi Reaction.

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.

Reinvestigation of the Automated Synthesis of Stoichiometrically Conjugated Antibodies to Access High Molecular Weight Payloads and Multiplexed Conjugation via an In-Solution Trans-Tagging Process.

Protein conjugates have found applications in a wide variety of fields, ranging from therapeutics to imaging and detection. However, robust control over the parameters of the conjugation process (such as sites and degree of conjugation) remains challenging. Previously, our group introduced Equimolar NAtive Chemical Tagging (ENACT), a method which allows for the monofunctionalization of proteins by combining an iterative low-conversion bioconjugation, an automated process, and a bioorthogonal trans-tagging reaction. However, while the automated ENACT was dimensioned to achieve monoconjugation at the mg scale, in early stage research, because of the rarity and cost of the starting materials, it is often necessary to prepare conjugates at the lower, µg, scale. Here, we introduce modified ENACT protocols, as well as a new ENACT conjugation reagent, which allow for the monofunctionalization of proteins on the micrograms scale, using minimal quantities of payload.

Microfluidic Droplet Stabilization via SPAAC Promoted Antibody Conjugation at the Water/Oil Interface.

Droplet-based microfluidics is leading the development of miniaturized, rapid, and sensitive version of enzyme-linked immunosorbent assays (ELISAs), a central method for protein detection. These assays involve the use of a functionalized surface able to selectively capture the desired analyte. Using the droplet's oil water interface as a capture surface requires designing custom-perfluorinated fluorosurfactants bearing azide-containing polar groups, which spontaneously react when forming the droplet with strain-alkyne-functionalized antibodies solubilized in the aqueous phase. In this article, we present our research on the influence of the structure of surfactant's hydrophilic heads on the efficiency of SPAAC functionalization and on the effect of this antibody grafting process on droplet stability. We have shown that while short linkers lead to high grafting efficiency, long linkers lead to high stability, and that an intermediate size is required to balance both parameters. In the described family of surfactants, the optimal structure proved to be a PEG4 linker connecting a polar di-azide head and a per-fluoropolyether tail (Krytox). We also found that grafting an increasing amount of antibody, thus increasing interface coverage, increases droplet stability. It thus appears that such a bi-partite system with a reactive fluoro-surfactant in the oil phase and reactive antibody counterpart in the aqueous phase gives access in situ to novel surfactant construct providing unexplored interface structures and droplet functionality.

Targeted Anticancer Agent with Original Mode of Action Prepared by Supramolecular Assembly of Antibody Oligonucleotide Conjugates and Cationic Nanoparticles.

Despite their clinical success, Antibody-Drug Conjugates (ADCs) are still limited to the delivery of a handful of cytotoxic small-molecule payloads. Adaptation of this successful format to the delivery of alternative types of cytotoxic payloads is of high interest in the search for novel anticancer treatments. Herein, we considered that the inherent toxicity of cationic nanoparticles (cNP), which limits their use as oligonucleotide delivery systems, could be turned into an opportunity to access a new family of toxic payloads. We complexed anti-HER2 antibody-oligonucleotide conjugates (AOC) with cytotoxic cationic polydiacetylenic micelles to obtain Antibody-Toxic-Nanoparticles Conjugates (ATNPs) and studied their physicochemical properties, as well as their bioactivity in both in vitro and in vivo HER2 models. After optimising their AOC/cNP ratio, the small (73 nm) HER2-targeting ATNPs were found to selectively kill antigen-positive SKBR-2 cells over antigen-negative MDA-MB-231 cells in serum-containing medium. Further in vivo anti-cancer activity was demonstrated in an SKBR-3 tumour xenograft model in BALB/c mice in which stable 60% tumour regression could be observed just after two injections of 45 pmol of ATNP. These results open interesting prospects in the use of such cationic nanoparticles as payloads for ADC-like strategies.

Droplet Surface Immunoassay by Relocation (D-SIRe) for High-Throughput Analysis of Cytosolic Proteins at the Single-Cell Level.

Enzyme-linked immunosorbent assay (ELISA) is a central analytic method in biological science for the detection of proteins. Introduction of droplet-based microfluidics allowed the development of miniaturized, less-consuming, and more sensitive ELISA assays by coencapsulating the biological sample and antibody-functionalized particles. We report herein an alternative in-droplet immunoassay format, which avoids the use of particles. It exploits the oil/aqueous-phase interface as a protein capture and detection surface. This is achieved using tailored perfluorinated surfactants bearing azide-functionalized PEG-based polar headgroups, which spontaneously react when meeting at the droplet formation site, with strained alkyne-functionalized antibodies solubilized in the water phase. The resulting antibody-functionalized inner surface can then be used to capture a target protein. This surface capture process leads to concomitant relocation at the surface of a labeled detection antibody and in turn to a drastic change in the shape of the fluorescence signal from a convex shape (not captured) to a characteristic concave shape (captured). This novel droplet surface immunoassay by fluorescence relocation (D-SIRe) proved to be fast and sensitive at 2.3 attomoles of analyte per droplet. It was further demonstrated to allow detection of cytosolic proteins at the single bacteria level.

A Novel Family of Acid-Cleavable Linker Based on Cyclic Acetal Motifs for the Production of Antibody-Drug Conjugates with High Potency and Selectivity.

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.

An overview of chemo- and site-selectivity aspects in the chemical conjugation of proteins.

The bioconjugation of proteins-that is, the creation of a covalent link between a protein and any other molecule-has been studied for decades, partly because of the numerous applications of protein conjugates, but also due to the technical challenge it represents. Indeed, proteins possess inner physico-chemical properties-they are sensitive and polynucleophilic macromolecules-that make them complex substrates in conjugation reactions. This complexity arises from the mild conditions imposed by their sensitivity but also from selectivity issues, viz the precise control of the conjugation site on the protein. After decades of research, strategies and reagents have been developed to address two aspects of this selectivity: chemoselectivity-harnessing the reacting chemical functionality-and site-selectivity-controlling the reacting amino acid residue-most notably thanks to the participation of synthetic chemistry in this effort. This review offers an overview of these chemical bioconjugation strategies, insisting on those employing native proteins as substrates, and shows that the field is active and exciting, especially for synthetic chemists seeking new challenges.

Plasma induced acceleration and selectivity in strain-promoted azide-alkyne cycloadditions.

Strain-promoted azide-alkyne cycloaddition (SPAAC) is an important member of the bioorthogonal reaction family. Over the past decade, much work has been dedicated to the generation of new strained alkynes with improved reactivity. While kinetics studies of SPAAC are often conducted in organic solvents, buffered solutions or mixtures, these media do not reflect the complexity of in vivo systems. In this work, we show that performing SPAAC in human plasma leads to intriguing kinetics and selectivity effects. In particular, we observed that reactions in plasma could be accelerated up to 70-fold compared to those in methanol, and that selective couplings between a pair of reagents could be possible in competition experiments. These findings highlight the value of evaluating bioorthogonal reactions in such a complex medium, especially when in vivo applications are planned, as unsuspected behaviour can be observed, disrupting the usual rules governing the reactivity in simple solvent systems.

Non-specific interactions of antibody-oligonucleotide conjugates with living cells.

Antibody-Oligonucleotide Conjugates (AOCs) represent an emerging class of functionalized antibodies that have already been used in a wide variety of applications. While the impact of dye and drug conjugation on antibodies' ability to bind their target has been extensively studied, little is known about the effect caused by the conjugation of hydrophilic and charged payloads such as oligonucleotides on the functions of an antibody. Previous observations of non-specific interactions of nucleic acids with untargeted cells prompted us to further investigate their impact on AOC binding abilities and cell selectivity. We synthesized a series of single- and double-stranded AOCs, as well as a human serum albumin-oligonucleotide conjugate, and studied their interactions with both targeted and non-targeted living cells using a time-resolved analysis of ligand binding assay. Our results indicate that conjugation of single strand oligonucleotides to proteins induce consistent non-specific interactions with cell surfaces while double strand oligonucleotides have little or no effect, depending on the preparation method.

Bicyclo[6.1.0]nonyne carboxylic acid for the production of stable molecular probes.

Bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN alcohol) is the most prominent strained-alkyne scaffold in chemical biology. Described herein is the synthesis of an oxidized analogue - BCN acid - whose facile functionalization via amide bond formation yields more stable derivatives than the classically encountered carbamates.

Manniindole, an indole derivative from the roots of Anonidium mannii and combined antischistosomal and enzymatic activities.

A new alkaloid, manniindole 1, together with four known compounds: aristolactam AII 2, aristolactam BII 3, piperolactam D 4 and polycarpol 5 were isolated from the crude extract EtOH-H2O (8:2) of the roots of Anonidium mannii by chromatographic separation. The structure elucidation was performed on the basis of a spectroscopic analysis (IR, HRESI MS, 1D and 2D NMR) as well as a comparison of their spectral data with those reported in the literature. For the first time, the crude extract and those isolated compounds were evaluated for their anti-schistosomal activity against Schistosoma mansoni and for cytotoxicity activity against Huh7 and A549 cells. Furthermore, they were also tested in vitro on the recent characterized Schistosoma mansoni NAD+ catabolizing enzyme (SmNACE) for their impact on this enzyme which is localized on the outer surface of the adult parasite. Compound 2 displayed quite good worm killing capability, while 4 showed significant inhibition of SmNACE.

Investigating Ugi/Passerini Multicomponent Reactions for the Site-Selective Conjugation of Native Trastuzumab*.

Site-selective modification of proteins has been the object of intense studies over the past decades, especially in the therapeutic field. Prominent results have been obtained with recombinant proteins, for which site-specific conjugation is made possible by the incorporation of particular amino acid residues or peptide sequences. In parallel, methods for the site-selective and site-specific conjugation of native and natural proteins are starting to thrive, allowing the controlled functionalization of various types of amino acid residues. Pursuing the efforts in this field, we planned to develop a new type of site-selective method, aiming at the simultaneous conjugation of two amino acid residues. We reasoned that this should give higher chances of developing a site-selective strategy compared to the great majority of existing methods that solely target a single residue. We opted for the Ugi four-centre three-component reaction to implement this idea, with the aim of conjugating the side-chain amine and carboxylate groups of two neighbouring lysine and aspartate/glutamate. Herein, we show that this strategy can give access to valuable antibody conjugates bearing several different payloads; furthermore, the approach limits the potential conjugation sites to only six on the model antibody trastuzumab.

On the use of DNA as a linker in antibody-drug conjugates: synthesis, stability and in vitro potency.

Here we present the synthesis and evaluation of antibody-drug conjugates (ADCs), for which antibody and drug are non-covalently connected using complementary DNA linkers. These ADCs are composed of trastuzumab, an antibody targeting HER2 receptors overexpressed on breast cancer cells, and monomethyl auristatin E (MMAE) as a drug payload. In this new ADC format, trastuzumab conjugated to a 37-mer oligonucleotide (ON) was prepared and hybridized with its complementary ON modified at 5-end with MMAE (cON-MMAE) in order to obtain trastuzumab-DNA-MMAE. As an advantage, the cON-MMAE was completely soluble in water, which decreases overall hydrophobicity of toxic payload, an important characteristic of ADCs. The stability in the human plasma of these non-engineered ON-based linkers was investigated and showed a satisfactory half-life of 5.8 days for the trastuzumab-DNA format. Finally, we investigated the in vitro cytotoxicity profile of both the DNA-linked ADC and the ON-drug conjugates and compared them with classical covalently linked ADC. Interestingly, we found increased cytotoxicity for MMAE compared to cON-MMAE and an EC50 in the nanomolar range for trastuzumab-DNA-MMAE on HER2-positive cells. Although this proved to be less potent than classically linked ADC with picomolar range EC50, the difference in cytotoxicity between naked payload and conjugated payload was significant when an ON linker was used. We also observed an interesting increase in cytotoxicity of trastuzumab-DNA-MMAE on HER2-negative cells. This was attributed to enhanced non-specific interaction triggered by the DNA strand as it could be confirmed using ligand tracer assay.

Ethynylation of Cysteine Residues: From Peptides to Proteins in Vitro and in Living Cells.

Current approaches to introduce terminal alkynes for bioorthogonal reactions into biomolecules still present limitations in terms of either reactivity, selectivity, or adduct stability. We present a method for the ethynylation of cysteine residues based on the use of ethynylbenziodoxolone (EBX) reagents. The acetylene group is directly introduced onto the thiol group of cysteine and can be used for copper-catalyzed alkyne-azide cycloaddition (CuAAC) without further processing. Labeling proceeded with reaction rates comparable to or higher than the most often used iodoacetamide on peptides or maleimide on the antibody trastuzumab, and high cysteine selectivity was observed. The reagents were also used in living cells for cysteine proteomic profiling and displayed improved coverage of the cysteinome compared to previously reported iodoacetamide or hypervalent iodine reagents. Fine-tuning of the EBX reagents allows optimization of their reactivity and physical properties.

Automated linkage of proteins and payloads producing monodisperse conjugates.

Controlled protein functionalization holds great promise for a wide variety of applications. However, despite intensive research, the stoichiometry of the functionalization reaction remains difficult to control due to the inherent stochasticity of the conjugation process. Classical approaches that exploit peculiar structural features of specific protein substrates, or introduce reactive handles via mutagenesis, are by essence limited in scope or require substantial protein reengineering. We herein present equimolar native chemical tagging (ENACT), which precisely controls the stoichiometry of inherently random conjugation reactions by combining iterative low-conversion chemical modification, process automation, and bioorthogonal trans-tagging. We discuss the broad applicability of this conjugation process to a variety of protein substrates and payloads.

Antibody-Oligonucleotide Conjugates as Therapeutic, Imaging, and Detection Agents.

Antibody-oligonucleotide conjugates (AOCs) are a novel class of synthetic chimeric biomolecules that has been continually gaining traction in different fields of modern biotechnology. This is mainly due to the unique combination of the properties of their two constituents, exceptional targeting abilities and antibody biodistribution profiles, in addition to an extensive scope of oligonucleotide functional and structural roles. Combining these two classes of biomolecules in one chimeric construct has therefore become an important milestone in the development of numerous biotechnological applications, including imaging (DNA-PAINT), detection (PLA, PEA), and therapeutics (targeted siRNA/antisense delivery). Numerous synthetic approaches have been developed to access AOCs ranging from stochastic chemical bioconjugation to site-specific conjugation with reactive handles, introduced into antibody sequences through protein engineering. This Review gives a general overview of the current status of AOC applications with a specific emphasis on the synthetic methods used for their preparation. The reported synthetic techniques are discussed in terms of their practical aspects and limitations. The importance of the development of novel methods for the facile generation of AOCs possessing a defined constitution is highlighted as a priority in AOC research to ensure the advance of their new applications.

Design and evaluation of ionizable peptide amphiphiles for siRNA delivery.

Small interfering RNAs (siRNAs) can down-regulate the expression of a target mRNA molecule in a sequence-specific manner, making them an attractive new class of drugs with broad potential for the treatment of diverse human diseases. Here, we report the synthesis of a series of cationic amphiphiles which were obtained by the coupling of amino acids and dipeptides onto a lipidic double chain. The new amphiphiles presenting a peptidic motif on a short hydrophilic spacer group were evaluated for selective gene silencing through RNA interference. Our results show that tryptophan residues boost siRNA delivery in an unexpected manner. The silencing experiments performed with very low concentrations of siRNA showed that the best formulations could induce significant death of tumor cells after silencing of polo-like kinase 1 which is implicated in cell cycle progression. In addition, these Trp containing peptide amphiphiles were highly efficient siRNA delivery vectors even in presence of competing serum proteins.

Sydnone-based turn-on fluorogenic probes for no-wash protein labeling and in-cell imaging.

We report the synthesis and use of sydnone-based profluorophores as tools for imaging applications. These new probes display exquisite reactivity towards strain promoted cycloaddition reactions with cycloalkynes allowing fast, efficient and selective labeling in biological media. Styryl-pyridinium sydnone probes were found particularly interesting for click reactions to proceed selectively inside cells.