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We showcase the successful combination of photochemistry and kinetic target-guided synthesis (KTGS) for rapidly pinpointing enzyme inhibitors. KTGS is a fragment-based drug discovery (FBDD) methodology in which the biological target (BT) orchestrates the construction of its own ligand from fragments featuring complementary reactive functionalities. Notably, fragments interacting with the protein binding sites leverage their spatial proximity, facilitating a preferential reaction. Consequently, the resulting bivalent ligand exhibits heightened affinity. Within the realm of KTGS strategies, in situ click chemistry stands out as the most widely used to identify potent protein binders. This approach requires significant protein contributions, such as binding interactions and appropriate orientations of fragments, to overcome high activation barriers. This leads to prolonged incubation times and the potential for generating false negatives, thereby limiting this strategy to proteins that are stable enough in buffer. We herein unveil the possibility to integrate photochemistry into the realm of KTGS, accelerating the ligation reaction between fragments to a time scale of minutes. This approach should significantly expand the narrow reactivity window of traditional KTGS reactions, paving the way for the exploration and development of novel photo-KTGS reactions., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Coumarins still remain one of the most widely explored fluorescent dyes, with a broad spectrum of applications spanning various fields, such as molecular imaging, bioorganic chemistry, materials chemistry, or medical sciences. Their fluorescence is strongly based on a push-pull mechanism involving an electron-donating group (EDG), mainly located at the C7 or C8 positions of the dye core. Unfortunately, up to now, these positions have been very limited to hydroxyl or amino groups. In this study, we present in detail the synthesis of the first series of coumarins bearing a vinyl sulfide as the EDG at the C7 position. These derivatives were prepared by thiol-yne reaction, promoted by ruthenium- or porphyrin-based photoredox catalysis, enabling rapid late-stage diversification. We also functionalized coumarins with short peptides, and BSA protein as a proof-of-concept study, in a single-step process. This strategy, capable of proceeding under aqueous conditions, overcomes the protection/deprotection steps usually required by traditional methods, which also use strong bases and organic solvents. Moreover, the photophysical properties such as absorption and emission of obtained coumarins (for 3-CF 3 , 3-benzothiazole, 6-8-difluoro derivatives), predominantly exhibited large Stokes shifts (up to 204 nm) and maintained intramolecular charge transfer (ICT) characteristics., (© 2024 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

The biocatalyzed oxidative detoxification of the V-series simulant PhX, by mean of the microperoxidase AcMP11, affords the corresponding phosphonothioate as the prominent product instead of the classical P-S and P-O bond cleavage. While PhX is structurally very close to the live agent VX (the methyl group is replaced by a phenyl), assessment with other surrogates missing the nucleophilic amino function displayed more resistance under the same conditions with no phosphonothioate observed. These encouraging results highlight 1) the efficacy of AcMP11 microperoxidase to efficiently detoxify V-series organophosphorus nerve agents (OPNA), and 2) the necessity to use representative alkyl or aryl phosphonothioates simulants such as PhX bearing the appropriate side chain as well as the P-O and P-S cleavable bond to mimic accurately the V-series OPNA to prevent false positive or false negative results., (© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Cross-linking mass spectrometry (XL-MS) has become a very useful tool for studying protein complexes and interactions in living systems. It enables the investigation of many large and dynamic assemblies in their native state, providing an unbiased view of their protein interactions and restraints for integrative modeling. More researchers are turning toward trying XL-MS to probe their complexes of interest, especially in their native environments. However, due to the presence of other potentially higher abundant proteins, sufficient cross-links on a system of interest may not be reached to achieve satisfactory structural and interaction information. There are currently no rules for predicting whether XL-MS experiments are likely to work or not; in other words, if a protein complex of interest will lead to useful XL-MS data. Here, we show that a simple iBAQ (intensity-based absolute quantification) analysis performed from trypsin digest data can provide a good understanding of whether proteins of interest are abundant enough to achieve successful cross-linking data. Comparing our findings to large-scale data on diverse systems from several other groups, we show that proteins of interest should be at least in the top 20% abundance range to expect more than one cross-link found per protein. We foresee that this guideline is a good starting point for researchers who would like to use XL-MS to study their protein of interest and help ensure a successful cross-linking experiment from the beginning. Data are available via ProteomeXchange with identifier PXD045792.

The uncharged 3-hydroxy-2-pyridine aldoximes with protonatable tertiary amines are studied as antidotes in toxic organophosphates (OP) poisoning. Due to some of their specific structural features, we hypothesize that these compounds could exert diverse biological activity beyond their main scope of application. To examine this further, we performed an extensive cell-based assessment to determine their effects on human cells (SH-SY5Y, HEK293, HepG2, HK-2, myoblasts and myotubes) and possible mechanism of action. As our results indicated, aldoxime having a piperidine moiety did not induce significant toxicity up to 300 µM within 24 h, while those with a tetrahydroisoquinoline moiety, in the same concentration range, showed time-dependent effects and stimulated mitochondria-mediated activation of the intrinsic apoptosis pathway through ERK1/2 and p38-MAPK signaling and subsequent activation of initiator caspase 9 and executive caspase 3 accompanied with DNA damage as observed already after 4 h exposure. Mitochondria and fatty acid metabolism were also likely targets of 3-hydroxy-2-pyridine aldoximes with tetrahydroisoquinoline moiety, due to increased phosphorylation of acetyl-CoA carboxylase. In silico analysis predicted kinases as their most probable target class, while pharmacophores modeling additionally predicted the inhibition of a cytochrome P450cam. Overall, if the absence of significant toxicity for piperidine bearing aldoxime highlights the potential of its further studies in medical counter-measures, the observed biological activity of aldoximes with tetrahydroisoquinoline moiety could be indicative for future design of compounds either in a negative context in OP antidotes design, or in a positive one for design of compounds for the treatment of other phenomena like cell proliferating malignancies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Proteins are able to irreversibly assemble biologically active ligands from building blocks bearing complementary reactive functions due their spatial proximity, through a kinetic target-guided synthetic process (also named in situ click chemistry). Although linkages thus formed are mostly passive, some of them have shown to significantly contribute to the protein binding through for instance hydrogen bonding and stacking interactions. Biocompatible reactions and click chemistry are a formidable source of inspiration for developing such new protein-directed ligations. This study reports a proximity-induced thiol-yne synthesis of carbonic anhydrase inhibitors. Not only this example widens the arsenal of kinetic target-guided synthesis (KTGS) eligible reactions, but the obtained product displayed unsuspected photophysical properties. The corresponding vinyl sulfide linkage conjugated to a coumarin core proved to be engaged in a monodirectional Z to E photoisomerization process. Further investigations guided by theoretical calculations showed that fine-tuning of the nature of the substituents on the coumarin moiety allows to obtain a bidirectional photochemical process, thus discovering a new photoswitching moiety, displaying moreover fluorescence properties. Due to the spectral tunability of coumarin derivatives, this work should open new opportunities for the design of vinyl sulfide-based photoswitch systems with modular photophysical properties., (© 2022 Wiley-VCH GmbH.)

Recent events demonstrated that organophosphorus nerve agents are a serious threat for civilian and military populations. The current therapy includes a pyridinium aldoxime reactivator to restore the enzymatic activity of acetylcholinesterase located in the central nervous system and neuro-muscular junctions. One major drawback of these charged acetylcholinesterase reactivators is their poor ability to cross the blood-brain barrier. In this study, we propose to evaluate glucoconjugated oximes devoid of permanent charge as potential central nervous system reactivators. We determined their in vitro reactivation efficacy on inhibited human acetylcholinesterase, the crystal structure of two compounds in complex with the enzyme, their protective index on intoxicated mice, and their pharmacokinetics. We then evaluated their endothelial permeability coefficients with a human in vitro model. This study shed light on the structural restrains of new sugar oximes designed to reach the central nervous system through the glucose transporter located at the blood-brain barrier.

Described herein is a quinoxalinone-based photoaffinity probe with caged fluorescence properties. Upon visible blue LED irradiation (λ max 450 nm), this photo-crosslinker is able to covalently capture proteins with concomitant fluorescence labelling. This process enables monitoring applications under "no wash" conditions.

Kinetic target-guided synthesis (KTGS) is a promising tool for the discovery of biologically active compounds. It relies on the identification of potent ligands that are covalently assembled by the biological targets themselves from a pool of reagents. Significant effort is devoted to developing new KTGS strategies; however, only a handful of biocompatible reactions are available, which may be insufficient to meet the specificities (stability, dynamics, active site topology, etc.) of a wide range of biological targets with therapeutic potential. This Topical Review proposes a retrospective analysis of existing KTGS ligation tools, in terms of their kinetics and analogy with other biocompatible reactions, and provides new clues to expand the KTGS toolkit. By way of examples, a nonexhaustive selection of such chemical ligation tools belonging to different classes of reactions as promising candidate reactions for KTGS are suggested.

The combination of the scaffolds of the cholinesterase inhibitor huprine Y and the antioxidant capsaicin results in compounds with nanomolar potencies toward human acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) that retain or improve the antioxidant properties of capsaicin. Crystal structures of their complexes with AChE and BChE revealed the molecular basis for their high potency. Brain penetration was confirmed by biodistribution studies in C57BL6 mice, with one compound ( 5i ) displaying better brain/plasma ratio than donepezil. Chronic treatment of 10 month-old APP/PS1 mice with 5i (2 mg/kg, i.p., 3 times per week, 4 weeks) rescued learning and memory impairments, as measured by three different behavioral tests, delayed the Alzheimer-like pathology progression, as suggested by a significantly reduced Aβ42/Aβ40 ratio in the hippocampus, improved basal synaptic efficacy, and significantly reduced hippocampal oxidative stress and neuroinflammation. Compound 5i emerges as an interesting anti-Alzheimer lead with beneficial effects on cognitive symptoms and on some underlying disease mechanisms.

Specific forms of fatty acids are well known to have beneficial health effects, but their precise mechanism of action remains elusive. Phosphatidic acid (PA) produced by phospholipase D1 (PLD1) regulates the sequential stages underlying secretory granule exocytosis in neuroendocrine chromaffin cells, as revealed by pharmacological approaches and genetic mouse models. Lipidomic analysis shows that secretory granule and plasma membranes display distinct and specific composition in PA. Secretagogue-evoked stimulation triggers the selective production of several PA species at the plasma membrane near the sites of active exocytosis. Rescue experiments in cells depleted of PLD1 activity reveal that mono-unsaturated PA restores the number of exocytotic events, possibly by contributing to granule docking, whereas poly-unsaturated PA regulates fusion pore stability and expansion. Altogether, this work provides insight into the roles that subspecies of the same phospholipid may play based on their fatty acyl chain composition., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Alzheimer's disease (AD) is a neurodegenerative disorder associated with cholinergic dysfunction, provoking memory loss and cognitive dysfunction in elderly patients. The cholinergic hypothesis provided over the years with molecular targets for developing palliative treatments for AD, acting on the cholinergic system, namely, acetylcholinesterase and α7 nicotinic acetylcholine receptor (α7 nAChR). In our synthetic work, we used "click-chemistry" to synthesize two Multi Target Directed Ligands (MTDLs) MB105 and MB118 carrying tacrine and quinuclidine scaffolds which are known for their anticholinesterase and α7 nAChR agonist activities, respectively. Both, MB105 and MB118, inhibit human acetylcholinesterase and human butyrylcholinesterase in the nanomolar range. Electrophysiological recordings on Xenopus laevis oocytes expressing human α7 nAChR showed that MB105 and MB118 acted as partial agonists of the referred nicotinic receptor, albeit, with different potencies despite their similar structure. The different substitution at C-3 on the 2,3-disubstituted quinuclidine scaffold may account for the significantly lower potency of MB118 compared to MB105. Electrophysiological recordings also showed that the tacrine precursor MB320 behaved as a competitive antagonist of human α7 nAChR, in the micromolar range, while the quinuclidine synthetic precursor MB099 acted as a partial agonist. Taken all together, MB105 behaved as a partial agonist of α7 nAChR at concentrations where it completely inhibited human acetylcholinesterase activity paving the way for the design of novel MTDLs for palliative treatment of AD., (Copyright © 2020 Elsevier Inc. All rights reserved.)

A brief literature survey reveals that metal-free ligation such as the maleimide-based cycloaddition with electron-rich (hetero)dienes is a widespread tool for the assembly of (bio)molecular systems with applications in biotechnology, materials science, polymers and bio-organic chemistry. Despite their everyday use, only scattered data about their kinetics as well as the stabilities of corresponding products under physiological conditions, are accessible. These key parameters are yet, of paramount importance to ensure the rapid and effective preparation of stable compounds. Herein is reported a systematic study regarding the different classes of dienes used in chemoselective ligation, including their accessibility and stability, as well as comparative kinetic experiments and products stability assays. We took advantage of these data to develop a double labeling strategy from the combined use of cyclopentadiene and oxazole dienes.

Chromogranin A (CgA) is a key luminal actor of secretory granule biogenesis at the trans-Golgi network (TGN) level but the molecular mechanisms involved remain obscure. Here, we investigated the possibility that CgA acts synergistically with specific membrane lipids to trigger secretory granule formation. We show that CgA preferentially interacts with the anionic glycerophospholipid phosphatidic acid (PA). In accordance, bioinformatic analysis predicted a PA-binding domain (PABD) in CgA sequence that effectively bound PA (36:1) or PA (40:6) in membrane models. We identified PA (36:1) and PA (40:6) as predominant species in Golgi and granule membranes of secretory cells, and we found that CgA interaction with these PA species promotes artificial membrane deformation and remodeling. Furthermore, we demonstrated that disruption of either CgA PABD or phospholipase D (PLD) activity significantly alters secretory granule formation in secretory cells. Our findings show for the first time the ability of CgA to interact with PLD-generated PA, which allows membrane remodeling and curvature, key processes necessary to initiate secretory granule budding., (© 2020 Federation of American Societies for Experimental Biology.)

The copper-catalyzed alkyne-azide cycloaddition (CuAAC) is one of the most powerful chemical strategies for selective fluorescent labeling of biomolecules in in vitro or biological systems. In order to accelerate the ligation process and ensure efficient formation of conjugates under diluted conditions, external copper(I) ligands or sophisticated copper(I)-chelating azides are used. This latter strategy, however, increases the bulkiness of the triazole linkage, thus perturbing the biological function or dynamic behavior of the conjugates. In a proof-of-concept study, we investigated the use of an extremely compact fluorophore-based copper(I) chelating azide in order to accelerate the CuAAC with concomitant fluorescence labeling; in our strategy, the fluorophore is able to complex copper(I) species while retaining its photophysical properties. It is believed that this unprecedented approach which was applied for the labeling of a short peptide molecule and the fluorescent labeling of live cells, could be extended to other families of nitrogen-based fluorophores in order to tune both the reaction rate and photophysical characteristics.

Nerve agents, the deadliest chemical warfare agents, are potent inhibitors of acetylcholinesterase (AChE) and cause rapid cholinergic crisis with serious symptoms of poisoning. Oxime reactivators of AChE are used in medical practice in the treatment of nerve agent poisoning, but the search for novel improved reactivators with central activity is an ongoing pursuit. For numerous oximes synthesized, in vitro reactivation is a standard approach in biological evaluation with little attention given to the pharmacokinetic properties of the compounds. This study reports a comprehensive physicochemical, pharmacokinetic, and safety profiling of five lipophilic 3-hydroxy-2-pyridine aldoximes, which were recently shown to be potent AChE reactivators with a potential to be centrally active. The oxime JR595 was singled out as highly metabolically stable in human liver microsomes, noncytotoxic oxime for SH-SY5Y neuroblastoma and 1321N1 astrocytoma cell lines, and its pharmacokinetic profile was determined after intramuscular administration in mice. JR595 was rapidly absorbed into blood after 15 min with simultaneous distribution to the brain at up to about 40% of its blood concentration; however, it was eliminated from both the brain and blood within an hour. In addition, the MDCKII-MDR1 cell line assay showed that oxime JR595 was not a P-glycoprotein efflux pump substrate. Finally, the preliminary antidotal study against multiple LD 50 doses of VX and sarin in mice showed the potential of JR595 to provide desirable therapeutic outcomes with future improvements in its circulation time.

Fluorescein isothiocyanate (FITC) is one of the most extensively used fluorescent probes for the labeling of biomolecules. The isothiocyanate function reacts with lysine residues of proteins to provide a chemically stable thiourea linkage without releasing any byproduct. However, diversification of isothiocyanate-based reagents is still hampered by the lack of mild conditions to generate isothiocyanate chemical functions, as well as by their poor stability and limited solutions available to increase water solubility, restricting the use of isothiocyanate labeling to highly water-soluble fluorophores. Inspired by plant biological processes, we report a safe and biocompatible myrosinase-assisted in situ formation of isothiocyanate conjugates from a highly water-soluble and stable glucosinolate precursor. This method was applied for the fluorescence labeling of a plasmatic protein and fluorescence imaging of living cells.

Both cholinesterases (AChE and BChE) and kinases, such as GSK-3α/β, are associated with the pathology of Alzheimer's disease. Two scaffolds, targeting AChE (tacrine) and GSK-3α/β (valmerin) simultaneously, were assembled, using copper(I)-catalysed azide alkyne cycloaddition (CuAAC), to generate a new series of multifunctional ligands. A series of eight multi-target directed ligands (MTDLs) was synthesized and evaluated in vitro and in cell cultures. Molecular docking studies, together with the crystal structures of three MTDL/TcAChE complexes, with three tacrine-valmerin hybrids allowed designing an appropriate linker containing a 1,2,3-triazole moiety whose incorporation preserved, and even increased, the original inhibitory potencies of the two selected pharmacophores toward the two targets. Most of the new derivatives exhibited nanomolar affinity for both targets, and the most potent compound of the series displayed inhibitory potencies of 9.5 nM for human acetylcholinesterase (hAChE) and 7 nM for GSK-3α/β. These novel dual MTDLs may serve as suitable leads for further development, since, in the micromolar range, they exhibited low cytotoxicity on a panel of representative human cell lines including the human neuroblastoma cell line SH-SY5Y. Moreover, these tacrine-valmerin hybrids displayed a good ability to penetrate the blood-brain barrier (BBB) without interacting with efflux pumps such as P-gp., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Background: EGFR mutations are routinely explored in lung adenocarcinoma by sequencing tumoral DNA. The aim of this study was to evaluate a fluorescent-labelled erlotinib based theranostic agent for the molecular imaging of mutated EGFR tumours in vitro and ex vivo using a mice xenograft model and fibred confocal fluorescence microscopy (FCFM)., Methods: The fluorescent tracer was synthesized in our laboratory by addition of fluorescein to an erlotinib molecule. Three human adenocarcinoma cell lines with mutated EGFR (HCC827, H1975 and H1650) and one with wild-type EGFR (A549) were xenografted on 35 Nude mice. MTT viability assay was performed after exposure to our tracer. In vitro imaging was performed at 1 μM tracer solution, and ex vivo imaging was performed on fresh tumours excised from mice and exposed to a 1 μM tracer solution in PBS for 1 h. Real-time molecular imaging was performed using FCFM and median fluorescence intensity (MFI) was recorded for each experiment., Results: MTT viability assay confirmed that addition of fluorescein to erlotinib did not suppress the cytotoxic of erlotinib on tumoral cells. In vitro FCFM imaging showed that our tracer was able to distinguish cell lines with mutated EGFR from those lines with wild-type EGFR (p < 0.001). Ex vivo FCFM imaging of xenografts with mutated EGFR had a significantly higher MFI than wild-type (p < 0.001). At a cut-off value of 354 Arbitrary Units, MFI of our tracer had a sensitivity of 100% and a specificity of 96.3% for identifying mutated EGFR tumours., Conclusion: Real time molecular imaging using fluorescent erlotinib is able to identify ex vivo tumours with EGFR mutations.

Chemoselective, biocompatible ligation reactions are the key components for efficient and modular access to biomolecular scaffolds. Tetrazine ligation leads to the formation of a mixture of isomers, which makes reaction monitoring, purification and characterization of conjugates difficult. We report herein a modified tetrazine ligation strategy based on the use of a pyrazolone coupling partner, which provides a single molecule conjugate.

A novel approach for brain protection against poisoning by organophosphorus agents is developed based on the combination treatment of dual delivery of two oximes. Pralidoxime chloride (2-PAM) and a novel reactivator, 6-(5-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)pentyl)-3-hydroxy picolinaldehyde oxime (3-HPA), have been loaded in solid-lipid nanoparticles (SLNs) to offer distinct release profile and systemic half-life for both oximes. To increase the therapeutic time window of both oximes, SLNs with two different compartments were designed to load each respective drug. Oxime-loaded SLNs of hydrodynamic diameter between 100 and 160 nm and negative zeta potential (-30 to -25 mV) were stable for a period of 10 months at 4 °C. SLNs displayed longer circulation time in the bloodstream compared to free 3-HPA and free 2-PAM. Oxime-loaded SLNs were suitable for intravenous (iv) administration. Paraoxon-poisoned rats (0.8 × LD 50 ) were treated with 3-HPA-loaded SLNs and 2-PAM+3-HPA-loaded SLNs at the dose of 3-HPA and 2-PAM of 5 mg/kg. Brain AChE reactivation up to 30% was slowly achieved in 5 h after administration of 3-HPA-SLNs. For combination therapy with two oximes, a time-dependent additivity and increased reactivation up to 35% were observed., (Copyright © 2018 Elsevier B.V. All rights reserved.)

We report the use of air-stable Cu(I)-NHC complex 4a as a catalyst for the efficient microwave-assisted synthesis of peptidotriazoles on solid phase. Compared with the usual conditions (CuI or CuSO 4 /NaAsc), catalyst 4a allowed the preparation of a series of peptidomimetic compounds containing a 1,2,3-triazole ring in their backbone without the oxidation of common side-chains. Overall, the peptidotriazoles were obtained in good yields (61-87%), in excellent purity (higher than 94%) and with low copper contamination.

Acetylcholinesterase (AChE), a key enzyme in the central and peripheral nervous systems, is the principal target of organophosphorus nerve agents. Quaternary oximes can regenerate AChE activity by displacing the phosphyl group of the nerve agent from the active site, but they are poorly distributed in the central nervous system. A promising reactivator based on tetrahydroacridine linked to a nonquaternary oxime is also an undesired submicromolar reversible inhibitor of AChE. X-ray structures and molecular docking indicate that structural modification of the tetrahydroacridine might decrease inhibition without affecting reactivation. The chlorinated derivative was synthesized and, in line with the prediction, displayed a 10-fold decrease in inhibition but no significant decrease in reactivation efficiency. X-ray structures with the derivative rationalize this outcome. We thus show that rational design based on structural studies permits the refinement of new-generation pyridine aldoxime reactivators that may be more effective in the treatment of nerve agent intoxication.

Since their first use in bioconjugation more than 50 years ago, maleimides have become privileged chemical partners for the site-selective modification of proteins via thio-Michael addition of biothiols and, to a lesser extent, via Diels-Alder (DA) reactions with biocompatible dienes. Prominent examples include immunotoxins and marketed maleimide-based antibody-drug conjugates (ADCs) such as Adcetris, which are used in cancer therapies. Among the key factors in the success of these groups is the availability of several maleimides that can be N-functionalized by fluorophores, affinity tags, spin labels, and pharmacophores, as well as their unique reactivities in terms of selectivity and kinetics. However, maleimide conjugate reactions have long been considered irreversible, and only recently have systematic studies regarding their reversibility and stability toward hydrolysis been reported. This review provides an overview of the diverse applications for maleimides in bioconjugation, highlighting their strengths and weaknesses, which are being overcome by recent strategies. Finally, the fluorescence quenching ability of maleimides was leveraged for the preparation of fluorogenic probes, which are mainly used for the specific detection of thiol analytes. A summary of the reported structures, their photophysical features, and their relative efficiencies is discussed in the last part of the review.

A new series of 3-hydroxy-2-pyridine aldoxime compounds have been designed, synthesised and tested in vitro, in silico, and ex vivo as reactivators of human acetylcholinesterase (hAChE) and butyrylcholinesterase (hBChE) inhibited by organophosphates (OPs), for example, VX, sarin, cyclosarin, tabun, and paraoxon. The reactivation rates of three oximes (16-18) were determined to be greater than that of 2-PAM and comparable to that of HI-6, two pyridinium aldoximes currently used by the armies of several countries. The interactions important for a productive orientation of the oxime group within the OP-inhibited enzyme have been clarified by molecular-modelling studies, and by the resolution of the crystal structure of the complex of oxime 17 with Torpedo californica AChE. Blood-brain barrier penetration was predicted for oximes 15-18 based on their physicochemical properties and an in vitro brain membrane permeation assay. Among the evaluated compounds, two morpholine-3-hydroxypyridine aldoxime conjugates proved to be promising reactivators of OP-inhibited cholinesterases. Moreover, efficient ex vivo reactivation of phosphylated native cholinesterases by selected oximes enabled significant hydrolysis of VX, sarin, paraoxon, and cyclosarin in whole human blood, which indicates that the oximes have scavenging potential., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Glucosinolates are sulfur-containing secondary metabolites found in plants of the Brassicale order. They are precursors of isothiocyanate species, resulting from C-S hydrolysis catalysed by the thioglucohydrolase myrosinase. We describe the synthesis of bifunctional glucosinolate-mannoside glycoconjugates combining both the structural features of a substrate of myrosinase and a ligand of the lectin FimH. We show that these glycoconjugates serve as enzyme substrates and that myrosinase can indeed hydrolyze the glucosinolate moiety with affinities (KM, Vmax) comparable to the natural substrates glucomoringin and sinigrin. This enzymatic hydrolysis of the thioglycosidic bond led to the efficient formation of an isothiocyanate which was assessed by the formation of the corresponding dithiocarbamate derivatives. Finally, we show that our synthetic bifunctional glycoconjugates also serve as FimH ligands where the glucosinolate moiety does not hamper the interaction with the lectin. Our findings set the stage for an original bioconjugation tool, allowing for myrosinase-triggered specific labelling of lectins using glucosinolate glycoconjugates as non-toxic, water soluble isothiocyanate precursors.

Symptomatic treatment of myasthenia gravis is based on the use of peripherally-acting acetylcholinesterase (AChE) inhibitors that, in some cases, must be discontinued due to the occurrence of a number of side-effects. Thus, new AChE inhibitors are being developed and investigated for their potential use against this disease. Here, we have explored two alternative approaches to get access to peripherally-acting AChE inhibitors as new agents against myasthenia gravis, by structural modification of the brain permeable anti-Alzheimer AChE inhibitors tacrine, 6-chlorotacrine, and huprine Y. Both quaternization upon methylation of the quinoline nitrogen atom, and tethering of a triazole ring, with, in some cases, the additional incorporation of a polyphenol-like moiety, result in more polar compounds with higher inhibitory activity against human AChE (up to 190-fold) and butyrylcholinesterase (up to 40-fold) than pyridostigmine, the standard drug for symptomatic treatment of myasthenia gravis. The novel compounds are furthermore devoid of brain permeability, thereby emerging as interesting leads against myasthenia gravis., Competing Interests: The authors declare no conflict of interest.

Hydroxamic acids are outstanding zinc chelating groups that can be used to design potent and selective metalloenzyme inhibitors in various therapeutic areas. Some hydroxamic acids display a high plasma clearance resulting in poor in vivo activity, though they may be very potent compounds in vitro. We designed a 57-member library of hydroxamic acids to explore the structure-plasma stability relationships in these series and to identify which enzyme(s) and which pharmacophores are critical for plasma stability. Arylesterases and carboxylesterases were identified as the main metabolic enzymes for hydroxamic acids. Finally, we suggest structural features to be introduced or removed to improve stability. This work thus provides the first medicinal chemistry toolbox (experimental procedures and structural guidance) to assess and control the plasma stability of hydroxamic acids and realize their full potential as in vivo pharmacological probes and therapeutic agents. This study is particularly relevant to preclinical development as it allows obtaining compounds equally stable in human and rodent models.

Prolyl-peptidyl isomerases (PPIases) are enzymes that are found in all living organisms. They form an essential part of the cellular protein folding homeostasis machinery. PPIases are associated with many important human diseases, e.g. cardiovascular disease, cancer and Alzheimer's. The development of novel PPIase inhibitors has been limited by the lack of a rapid, laboratory-based assay for these enzymes, as their substrates and products are challenging to distinguish. A well described continuous assay, coupled with the hydrolysis of a peptide by chymotrypsin is highly effective, but comparatively slow. To address this, we developed an improved version of the traditional assay using a temperature controlled plate reader. This assay allows semi-automated medium throughput assays in an academic laboratory for 84 samples per day. The assay shows lower errors, with an average Z' of 0.72. We further developed the assay using a fluorogenic peptide-based FRET probe. This provides an extremely sensitive PPIase assay using substrate at 200 nM, which approaches single turnover conditions. The fluorescent probe achieves an excellent quenching efficiency of 98.6%, and initial experiments showed acceptable Z' of 0.31 and 0.30 for cyclophilin A and hFKBP12 respectively. The assays provide an improved toolset for the quantitative, biochemical analysis of PPIases., (Crown Copyright © 2017. Published by Elsevier Inc. All rights reserved.)

Correction for 'Metal-free oxidative ring contraction of benzodiazepinones: an entry to quinoxalinones' by Hasan Mtiraoui, et al., Org. Biomol. Chem., 2017, 15, 3060-3068.

The use of nonfluorescent azo dyes as dark quenchers in activatable optical bioprobes based on the Förster resonance energy transfer (FRET) mechanism and designed to target a wide range of enzymes has been established for over two decades. The key value of the azo moiety (-N=N-) to act as an efficient "ON-OFF" switch of fluorescence once introduced within the core structure of conventional organic-based fluorophores (mainly fluorescent aniline derivatives) has recently been exploited in the development of alternative reaction-based small-molecule probes based on the "profluorescence" concept. These unprecedented "azobenzene-caged" fluorophores are valuable tools for the detection of a wide range of reactive (bio)analytes. This review highlights the most recent and relevant advances made in the design and biosensing/bioimaging applications of azo-based fluorogenic probes. Emphasis is also placed on relevant achievements in the synthesis of bioconjugatable/biocompatible azo dyes used as starting building blocks in the rational and rapid construction of these fluorescent chemodosimeters. Finally, a brief glimpse of possible future biomedical applications (theranostics) of these "smart" azobenzene-based molecular systems is presented., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A novel and practical synthesis of 3-benzoylquinoxalin-2(1H)-ones from benzodiazepin-2-ones in two steps from commercially available starting materials is reported. The reaction was achieved in the presence of N-bromosuccinimide in DMSO which served both as a solvent and an oxidant. Significantly, the yet unknown ketone to alcohol fluorescence turn-on of benzoylquinoxalinones was unveiled through the preparation of a fluorescently labelled cholesterol conjugate.

Acetylcholinesterase (AChE), an enzyme of the serine hydrolase superfamily, is a mediator of signal transmission at cholinergic synapses by catalyzing acetylcholine cleavage into acetate and choline. This enzyme is vulnerable to covalent inhibition by organophosphate compounds (like VX). Covalent inhibition of AChE does not revert spontaneously. Known reactivator compounds have limited action in restoring catalytic activity. QM/MM simulations of VX-inhibited AChE reactivation by pralidoxime (2-PAM), a classical reactivator, were performed. These afforded a broad view of the effect of protonation states of active-site residues, and provide evidence for the role of Glu202, which needs to be protonated for reactivation to occur. In situ deprotonation of 2-PAM for both protonation states of Glu202 showed that His447 is able to deprotonate 2-PAM with the assistance of Glu202. Because the active site of serine hydrolases is highly conserved, this work provides new insights on the interplay between the catalytic triad residues and this glutamate, newly identified as protonatable., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Organophosphorus nerve agents, like VX, are highly toxic due to their strong inhibition potency against acetylcholinesterase (AChE). AChE inhibited by VX can be reactivated using powerful nucleophilic molecules, most commonly oximes, which are one major component of the emergency treatment in case of nerve agent intoxication. We present here a comparative in vivo study on Swiss mice of four reactivators: HI-6, pralidoxime and two uncharged derivatives of 3-hydroxy-2-pyridinaldoxime that should more easily cross the blood-brain barrier and display a significant central nervous system activity. The reactivability kinetic profile of the oximes is established following intraperitoneal injection in healthy mice, using an original and fast enzymatic method based on the reactivation potential of oxime-containing plasma samples. HI-6 displays the highest reactivation potential whatever the conditions, followed by pralidoxime and the two non quaternary reactivators at the dose of 50 mg/kg bw. But these three last reactivators display equivalent reactivation potential at the same dose of 100 μmol/kg bw. Maximal reactivation potential closely correlates to surviving test results of VX intoxicated mice., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Fluorogenic reactions are largely underrepresented in the toolbox of chemoselective ligations despite their tremendous potential, particularly in chemical biology and biochemistry. In this respect, we have investigated in full detail the fluorescence behaviour of the azaphthalamide, a scaffold which is generated through a hetero-Diels-Alder reaction of 5-alkoxyoxazole and maleimide derivatives under mild conditions that are compatible with, among others, peptide chemistry. The scope and limitations of such a fluorogenic labelling strategy were examined through four distinct applications, which target enzymatic activities or bioorthogonal reactions., (© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Two near infra-red (NIR) fluorescent probes HupNIR1 and HupNIR2 based on the huprine scaffold and cyanine 5.0 dye have been synthesised and evaluated in situ for the detection of acetylcholinesterases in different tissues. As anticipated by the initial properties of huprine, both probes displayed a high affinity and selectivity for AChE toward BChE, with IC50 values in the nanomolar range and without any non-specific binding in the tissues. HupNIR2 appears the best probe for AChE with a great selectivity and sensitivity for AChE even in the brain region displaying a low AChE concentration as striatum. Moreover, the binding of HupNIR2 is affected when AChE is inhibited with toxic molecules such as organophosphates. This work provides a new tool to visualize active AChE in biological applications.

Fluorescent coumarins are an important class of small-molecule organic fluorophores ubiquitous in different well-established and emerging fields of research including, among others, biochemistry and chemical biology. The present work aims at covering the poor detectability of coumarin-based conjugates by mass spectrometry while keeping important photophysical properties of the coumarin core. In this context, the synthesis of readily functionalizable phosphonium-tagged coumarin derivatives enabling a dual mass-tag and fluorescence labelling of analytes or (bio)molecules of interest through a single-step protocol, is reported. The utility of these coumarins is illustrated through the preparation of fluorogenic substrates that facilitated identification of the peptide fragment released by specific proteolytic cleavages.

Photoluminescent materials, that are now ubiquitous in our everyday life, have particularly attracted the attention of the scientific community these past few years due to potential important applications such as in bioimaging, sensing, or optoelectronics. In this context, relatively few different families of molecules have been reported to exhibit fluorescence in the aggregated or solid-state through the excited-state intramolecular proton transfer (ESIPT) photochemical process. The preparation and subsequent determination of photochemical properties of an underexplored family of 1,5-benzodiazepin-2-one derivatives are reported. From these data and X-ray diffraction analysis study, it emerged that photoluminescence (in the range 520-655 nm) was mostly attributed to ESIPT. The photoluminescent potential of 1,5-benzodiazepin-2-ones, their facile access, and functionalization were demonstrated through the preparation of two fluorogenic probes for the selective detection of biothiols.

Members of a series of boron difluoride complexes with 3-(heteroaryl)-2-iminocoumarin ligands bearing both a phenolic hydroxyl group (acting as a fluorogenic center) and an N-aryl substituent (acting as a stabilizing moiety) have been synthesized in good yields by applying a straightforward two-step method. These novel fluorogenic dyes belong to the family of "Boricos" (D. Frath et al., Chem. Commun.- 2013, 49, 4908-4910) and are the first examples of phenol-based fluorophores of which the photophysical properties in the green-yellow spectral range are dramatically improved by N,N-chelation of a boron atom. Modulation of their fluorescence properties through reversible chemical modification of their phenol moieties has been demonstrated by the preparation of the corresponding 2,4-dinitrophenyl (DNP) ethers, which led to a dramatic "OFF-ON" fluorescence response upon reaction with thiols. Additionally, to expand the scope of these "7-hydroxy-Borico" derivatives, particularly in biolabeling, amine or carboxylic acid functionalities amenable to (bio)conjugation have been introduced within their scaffold. Their utility has been demonstrated in the preparation of fluorescent bovine serum albumin (BSA) conjugates and "Borico"-DOTA-like scaffolds in an effort to design novel monomolecular multimodal fluorescence- radioisotope imaging agents., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

New sulforhodamine-based fluorescent 'turn-on' probes have been developed for the direct imaging of cellular hypoxia. Rapid access to this novel class of water-soluble 'azobenzene-caged' fluorophores was made possible through an easily-implementable azo-coupling reaction between a fluorescent primary arylamine derived from a sulforhodamine 101 scaffold (named SR101-NaphtNH 2 ) and a tertiary aniline whose N-substituents are neutral, cationic, or zwitterionic. The detection mechanism is based on the bioreductive cleavage of the azo bond that restores strong far-red fluorescence (emission maximum at 625 nm) by regenerating the original sulforhodamine SR101-NaphtNH 2 . This valuable fluorogenic response was obtained for the three 'smart' probes studied in this work, as shown by an in vitro assay using rat liver microsomes placed under aerobic and then under hypoxic conditions. Most importantly, the probe namely SR101-NaphtNH 2 -Hyp-diMe was successfully applied for imaging the hypoxic status of tumor cells (A549 cells).

The kinetic target-guided synthesis (KTGS) strategy is an unconventional discovery approach that takes advantage of the presence of the biological target itself in order to irreversibly assemble the best inhibitors from an array of building blocks. This strategy has grown over the last two decades notably after the introduction of the in situ click chemistry concept by Sharpless and colleagues in the early 2000s based on the use of the Huisgen cycloaddition between terminal alkynes and azides. KTGS is a captivating area of research offering an unprecedented and powerful strategy to probe the macromolecular complexity and dynamics of biological targets. After a brief introduction listing all chemical ligation reactions reported to date in KTGS, this review focuses on the last five years' progress to expand the repertoire of the click or "click-like" tool box targeting proteins, as well as to overcome limitations arising in particular from false negatives, i.e. potent ligands that are not formed, or formed in undetectable trace amounts. Furthermore, we wish to analyze the new twists and novelties described in some of these applications in order to better understand the conditions that govern this strategy and the extent to which it can be developed and generalized for a more efficient process.

The variety of protein cross-linkers developed in recent years illustrates the current requirement for efficient reagents optimized for mass spectrometry (MS) analysis. To date, the most widely used strategy relies on commercial cross-linkers that bear an isotopically labeled tag and N-hydroxysuccinimid-ester (NHS-ester) moieties. Moreover, an enrichment step using liquid chromatography is usually performed after enzymatic digestion of the cross-linked proteins. Unfortunately, this approach suffers from several limitations. First, it requires large amounts of proteins. Second, NHS-ester cross-linkers are poorly efficient because of their fast hydrolysis in water. Finally, data analysis is complicated because of uneven fragmentation of complex isotopic cross-linked peptide mixtures. We therefore synthesized a new type of trifunctional cross-linker to overrule these limitations. This reagent, named NNP9, comprises a rigid core and bears two activated carbamate moieties and an azido group. NNP9 was used to establish intra- and intermolecular cross-links within creatine kinase, then to map the interaction surfaces between α-Synuclein (α-Syn), the aggregation of which leads to Parkinson's disease, and the molecular chaperone Hsc70. We show that NNP9 cross-linking efficiency is significantly higher than that of NHS-ester commercial cross-linkers. The number of cross-linked peptides identified was increased, and a high quality of MS/MS spectra leading to high sequence coverage was observed. Our data demonstrate the potential of NNP9 for an efficient and straightforward characterization of protein-protein interfaces and illustrate the power of using different cross-linkers to map thoroughly the surface interfaces within protein complexes.

The two main pathological hallmarks of Alzheimer's disease (AD) in the brain are senile plaques (SPs) composed of beta-amyloid (Aβ) peptides and neurofibrillary tangles (NFTs) of hyperphosphorylated tau protein. These hallmarks are associated with a cholinergic deficit. While the process leading to the development of AD is complex and multifactorial, and the etiology of AD is not completely known, it is nowadays clear that AD is a multifaceted illness requiring the combination of synergetic treatment strategies. Because definite diagnosis is achieved by postmortem examination of the brain, new noninvasive diagnostic imaging modalities for AD are in high demand, both to detect and monitor the evolution of this sickness, and evaluate the efficacy of treatments. Positron Emission Tomography (PET) is a nuclear molecular imaging technique that uses radiopharmaceuticals labeled with a positron-emitting isotope (carbon-11, fluorine-18, copper-64, gallium- 68…), to visualize in vivo cellular metabolism with high-spatial resolution and unique sensitivity, while Single-Photon Emission Computed Tomography (SPECT) using radioisotopes such as technetium-99m or iodine-123. Besides being a powerful tool for diagnosis (mostly in oncology with [(18)F]-FDG), PET experiments can provide information about biochemical mechanisms in living tissues or interactions between neurotransmitter and brain receptors. For the past two decades, numerous radiopharmaceuticals have been developed for imaging the lesions observed in AD patients. Tau aggregates and Aβ plaques can also be visualized and quantified by mean of specific radioligands. The latter has been the focus of intense research efforts lately, leading to new FDA approved radiopharmaceuticals. This paper aimed at summarizing the recent advances in PET and SPECT imaging of AD pathophysiology.

Diversification of existing chemoselective ligations is required to efficiently access complex and well-defined biomolecular assemblies with unique and valuable properties. The development and bioconjugation applications of a novel Diels-Alder-based irreversible site-specific ligation are reported. The strategy is based on a Kondrat'eva cycloaddition between bioinert and readily functionalizable 5-alkoxyoxazoles and maleimides that readily react together under mild and easily tunable reaction conditions to afford a fully stable pyridine scaffold. The potential of this novel bioconjugation is demonstrated through the preparation of fluorescent conjugates of biomolecules and a novel Förster resonance energy transfer (FRET)-based probe suitable for the in vivo detection and imaging of urokinase-like plasminogen activator (uPA), which is a key protease involved in cancer invasion and metastasis.

Apoptosis is a regulated process, leading to cell death, which is involved in several pathologies including neurodegenerative diseases and stroke. Caspase-3 is a key enzyme of the apoptotic pathway and is considered as a major target for the treatment of abnormal cell death. Sensitive and non-invasive methods to monitor caspase-3 activity in cells and in the brain of living animals are needed to test the efficiency of novel therapeutic strategies. In the present study, we have biochemically characterized a caspase-3 far-red fluorescent probe, QCASP3.2, that can be used to detect apoptosis in vivo. The specificity of cleavage of QCASP3.2 was demonstrated using recombinant caspases and protease inhibitors. The functionality of the probe was also established in cerebellar neurons cultured in apoptotic conditions. QCASP3.2 did not exhibit any toxicity and appeared to accurately reflect the induction and inhibition of caspase activity by H2O2 and PACAP, respectively, both in cell lysates and in cultured neurons. Finally, intravenous injection of the probe after cerebral ischemia revealed activation of caspase-3 in the infarcted hemisphere. Thus, the present study demonstrates that QCASP3.2 is a suitable probe to monitor apoptosis both in vitro and in vivo and illustrates some of the possible applications of this caspase-3 fluorescent probe.

The bioactive metabolite sphingosine-1-phosphate (S1P), a product of sphingosine kinases (SphKs), mediates diverse biological processes such as cell differentiation, proliferation, survival and angiogenesis. A fluorinated analogue of S1P receptor agonist has been synthesized by utilizing a ring opening reaction of oxacycles by a lithiated difluoromethylphosphonate anion as the key reaction. In vitro activity of this S1P analogue is also reported., (Copyright © 2014 Elsevier Ltd. All rights reserved.)