Bioorthogonal chemical labeling of endogenous neurotransmitter receptors in living mouse brains.

Neurotransmitter receptors are essential components of synapses for communication between neurons in the brain. Because the spatiotemporal expression profiles and dynamics of neurotransmitter receptors involved in many functions are delicately governed in the brain, in vivo research tools with high spatiotemporal resolution for receptors in intact brains are highly desirable. Covalent labeling by chemical reaction (chemical labeling) of proteins without genetic manipulation is now a powerful method for analyzing receptors in vitro. However, selective target receptor labeling in the brain has not yet been achieved. This study shows that ligand-directed alkoxyacylimidazole (LDAI) chemistry can be used to selectively tether synthetic probes to target endogenous receptors in living mouse brains. The reactive LDAI reagents with negative charges were found to diffuse well over the whole brain and could selectively label target endogenous receptors, including AMPAR, NMDAR, mGlu1, and GABAAR. This simple and robust labeling protocol was then used for various applications: three-dimensional spatial mapping of endogenous receptors in the brains of healthy and disease-model mice; multi-color receptor imaging; and pulse-chase analysis of the receptor dynamics in postnatal mouse brains. Here, results demonstrated that bioorthogonal receptor modification in living animal brains may provide innovative molecular tools that contribute to the in-depth understanding of complicated brain functions.

Emergence of Dynamic Instability by Hybridizing Synthetic Self-Assembled Dipeptide Fibers with Surfactant Micelles.

Supramolecular chemistry currently faces the challenge of controlling nonequilibrium dynamics such as the dynamic instability of microtubules. In this study, we explored the emergence of dynamic instability through the hybridization of peptide-type supramolecular nanofibers with surfactant micelles. Using real-time confocal imaging, we discovered that the addition of micelles to nanofibers induced the simultaneous but asynchronous growth and shrinkage of nanofibers during which the total number of fibers decreased monotonically. This dynamic phenomenon unexpectedly persisted for 6 days and was driven not by chemical reactions but by noncovalent supramolecular interactions between peptide-type nanofibers and surfactant micelles. This study demonstrates a strategy for inducing autonomous supramolecular dynamics, which will open up possibilities for developing soft materials applicable to biomedicine and soft robotics.

Tyrosinase-Based Proximity Labeling in Living Cells and In Vivo.

Characterizing the protein constituents of a specific organelle and protein neighbors of a protein of interest (POI) is essential for understanding the function and state of the organelle and protein networks associated with the POI. Proximity labeling (PL) has emerged as a promising technology for specific and efficient spatial proteomics. Nevertheless, most enzymes adopted for PL still have limitations: APEX requires cytotoxic H2O2 for activation and thus is poor in biocompatibility for in vivo application, BioID shows insufficient labeling kinetics, and TurboID suffers from high background biotinylation. Here, we introduce a bacterial tyrosinase (BmTyr) as a new PL enzyme suitable for H2O2-free, fast (≤10 min in living cells), and low-background protein tagging. BmTyr is genetically encodable and enables subcellular-resolved PL and proteomics in living cells. We further designed a strategy of ligand-tethered BmTyr for in vivo PL, which unveiled the surrounding proteome of a neurotransmitter receptor (Grm1 and Drd2) in its resident synapse in a live mouse brain. Overall, BmTyr is one promising enzyme that can improve and expand PL-based applications and discoveries.

Chemical, Pharmacological, and Structural Characterization of Novel Acrylamide-Derived Modulators of the GABAA Receptor.

Acrylamide-derived compounds have been previously shown to act as modulators of members of the Cys-loop transmitter-gated ion channel family, including the mammalian GABAA receptor. Here we have synthesized and functionally characterized the GABAergic effects of a series of novel compounds (termed "DM compounds") derived from the previously characterized GABAA and the nicotinic α7 receptor modulator (E)-3-furan-2-yl-N-p-tolyl-acrylamide (PAM-2). Fluorescence imaging studies indicated that the DM compounds increase apparent affinity to the transmitter by up to 80-fold in the ternary αßγ GABAA receptor. Using electrophysiology, we show that the DM compounds, and the structurally related (E)-3-furan-2-yl-N-phenylacrylamide (PAM-4), have concurrent potentiating and inhibitory effects that can be isolated and observed under appropriate recording conditions. The potentiating efficacies of the DM compounds are similar to those of neurosteroids and benzodiazepines (ΔG ∼ -1.5 kcal/mol). Molecular docking, functionally confirmed by site-directed mutagenesis experiments, indicate that receptor potentiation is mediated by interactions with the classic anesthetic binding sites located in the transmembrane domain of the intersubunit interfaces. Inhibition by the DM compounds and PAM-4 was abolished in the receptor containing the α1(V256S) mutation, suggestive of similarities in the mechanism of action with that of inhibitory neurosteroids. Functional competition and mutagenesis experiments, however, indicate that the sites mediating inhibition by the DM compounds and PAM-4 differ from those mediating the action of the inhibitory steroid pregnenolone sulfate. SIGNIFICANCE STATEMENT: We have synthesized and characterized the actions of novel acrylamide-derived compounds on the mammalian GABAA receptor. We show that the compounds have concurrent potentiating effects mediated by the classic anesthetic binding sites, and inhibitory actions that bear mechanistic resemblance to but do not share binding sites with, the inhibitory steroid pregnenolone sulfate.

Visualizing Formation and Dynamics of a Three-Dimensional Sponge-like Network of a Coacervate in Real Time.

Coacervates, which are formed by liquid-liquid phase separation, have been extensively explored as models for synthetic cells and membraneless organelles, so their in-depth structural analysis is crucial. However, both the inner structure dynamics and formation mechanism of coacervates remain elusive. Herein, we demonstrate real-time confocal observation of a three-dimensional sponge-like network in a dipeptide-based coacervate. In situ generation of the dipeptide allowed us to capture the emergence of the sponge-like network via unprecedented membrane folding of vesicle-shaped intermediates. We also visualized dynamic fluctuation of the network, including reversible engagement/disengagement of cross-links and a stochastic network kissing event. Photoinduced transient formation of a multiphase coacervate was achieved with a thermally responsive phase transition. Our findings expand the fundamental understanding of synthetic coacervates and provide opportunities to manipulate their physicochemical properties by engineering the inner network for potential applications in development of artificial cells and life-like material fabrication.

Lysine-Reactive N-Acyl-N-aryl Sulfonamide Warheads: Improved Reaction Properties and Application in the Covalent Inhibition of an Ibrutinib-Resistant BTK Mutant.

The covalent inhibition of a target protein has gained widespread attention in the field of drug discovery. Most of the current covalent drugs utilize the high reactivity of cysteines toward modest electrophiles. However, there is a growing need for warheads that can target lysine residues to expand the range of covalently druggable proteins and to deal with emerging proteins with mutations resistant to cysteine-targeted covalent drugs. We have recently developed an N-acyl-N-alkyl sulfonamide (NASA) as a lysine-targeted electrophile. Despite its successful application, this NASA warhead suffered from instability in physiological environments, such as serum-containing medium, because of its high intrinsic reactivity. In this study, we sought to modify the structure of the NASA warhead and found that N-acyl-N-aryl sulfonamides (ArNASAs) are promising electrophiles for use in a lysine-targeted covalent inhibition strategy. We prepared a focused library of ArNASA derivatives with diverse structures and reactivity and identified several warhead candidates with suppressed hydrolysis-mediated inactivation and reduced nonspecific reactions with off-target proteins, without sacrificing the reactivity toward the target. These reaction properties enabled the improved covalent inhibition of intracellular heat shock protein 90 (HSP90) in the presence of serum and the development of the first irreversible inhibitor for ibrutinib-resistant Bruton's tyrosine kinase (BTK) bearing the C481S mutation. This study clearly demonstrated the use of a set of ArNASA warheads to create highly potent covalent drugs and highlighted the importance of enriching the current arsenal of lysine-reactive warheads.

Microscopic Imaging Techniques for Molecular Assemblies: Electron, Atomic Force, and Confocal Microscopies.

Self-assembly is promising for construction of a wide variety of supramolecular assemblies, whose 1D/2D/3D structures are typically relevant to their functions. In-depth understanding of their structure-function relationships is essential for rational design and development of functional molecular assemblies. Microscopic imaging has been used as a powerful tool to elucidate structures of individual molecular assemblies with subnanometer to millimeter resolution, which is complementary to conventional spectroscopic techniques that provide the ensemble structural information. In this review, we highlight the representative examples of visualization of molecular assemblies by use of electron microscopy, atomic force microscopy, confocal microscopy, and super-resolution microscopy. This review comprehensively describes imaging of supramolecular nanofibers/gels, micelles/vesicles, coacervate droplets, polymer assemblies, and protein/DNA assemblies. Advanced imaging techniques that can address key challenges, like evaluation of dynamics of molecular assemblies, multicomponent self-assembly, and self-assembly/disassembly in complex cellular milieu, are also discussed. We believe this review would provide guidelines for deeper structural analyses of molecular assemblies to develop the next-generation materials.

Temporal Stimulus Patterns Drive Differentiation of a Synthetic Dipeptide-Based Coacervate.

The fate of living cells often depends on their processing of temporally modulated information, such as the frequency and duration of various signals. Synthetic stimulus-responsive systems have been intensely studied for >50 years, but it is still challenging for chemists to create artificial systems that can decode dynamically oscillating stimuli and alter the systems' properties/functions because of the lack of sophisticated reaction networks that are comparable with biological signal transduction. Here, we report morphological differentiation of synthetic dipeptide-based coacervates in response to temporally distinct patterns of the light pulse. We designed a simple cationic diphenylalanine peptide derivative to enable the formation of coacervates. The coacervates concentrated an anionic methacrylate monomer and a photoinitiator, which provided a unique reaction environment and facilitated light-triggered radical polymerization─even in air. Pulsed light irradiation at 9.0 Hz (but not at 0.5 Hz) afforded anionic polymers. This dependence on the light pulse patterns is attributable to the competition of reactive radical intermediates between the methacrylate monomer and molecular oxygen. The temporal pulse pattern-dependent polymer formation enabled the coacervates to differentiate in terms of morphology and internal viscosity, with an ultrasensitive switch-like mode. Our achievements will facilitate the rational design of smart supramolecular soft materials and are insightful regarding the synthesis of sophisticated chemical cells.

Organelle membrane-specific chemical labeling and dynamic imaging in living cells.

Lipids play crucial roles as structural elements, signaling molecules and material transporters in cells. However, the functions and dynamics of lipids within cells remain unclear because of a lack of methods to selectively label lipids in specific organelles and trace their movement by live-cell imaging. We describe here a technology for the selective labeling and fluorescence imaging (microscopic or nanoscopic) of phosphatidylcholine in target organelles. This approach involves the metabolic incorporation of azido-choline, followed by a spatially limited bioorthogonal reaction that enables the visualization and quantitative analysis of interorganelle lipid transport in live cells. More importantly, with live-cell imaging, we obtained direct evidence that the autophagosomal membrane originates from the endoplasmic reticulum. This method is simple and robust and is thus powerful for real-time tracing of interorganelle lipid trafficking.

Fluorescence identification of arthropathic calcium pyrophosphate single crystals using alizarin red S and a xanthene dipicolylamine ZnII complex.

Calcium pyrophosphate deposition disease, previously known as pseudogout, is a type of chronic and painful joint arthropathy. Accurate identification of calcium pyrophosphate dihydrate (CPPD) single crystals is crucial for determining the best course of treatment. In this study, a two-step method involving alizarin red S (ARS) and a xanthene dipicolylamine ZnII (XDZ) complex was employed for the identification of CPPD single crystals in both triclinic and monoclinic forms using a fluorescence microscope and a microplate reader. The accurate identification method proposed in this study has the potential to advance the diagnosis and treatment of patients suffering from painful gouty arthritis.

Phototriggered Spatially Controlled Out-of-Equilibrium Patterns of Peptide Nanofibers in a Self-Sorting Double Network Hydrogel.

Out-of-equilibrium patterns arising from diffusion processes are ubiquitous in nature, although they have not been fully exploited for the design of artificial materials. Here, we describe the formation of phototriggered out-of-equilibrium patterns using photoresponsive peptide-based nanofibers in a self-sorting double network hydrogel. Light irradiation using a photomask followed by thermal incubation induced the spatially controlled condensation of peptide nanofibers. According to confocal images and spectroscopic analyses, metastable nanofibers photodecomposed in the irradiated areas, where thermodynamically stable nanofibers reconstituted and condensed with a supply of monomers from the nonirradiated areas. These supramolecular events were regulated by light and diffusion to facilitate the creation of unique out-of-equilibrium patterns, including two lines from a one-line photomask and a line pattern of a protein immobilized in the hydrogel.

Enhanced Suppression of a Protein-Protein Interaction in Cells Using Small-Molecule Covalent Inhibitors Based on an N-Acyl-N-alkyl Sulfonamide Warhead.

Protein-protein interactions (PPIs) intimately govern various biological processes and disease states and therefore have been identified as attractive therapeutic targets for small-molecule drug discovery. However, the development of highly potent inhibitors for PPIs has proven to be extremely challenging with limited clinical success stories. Herein, we report irreversible inhibitors of the human double minute 2 (HDM2)/p53 PPI, which employ a reactive N-acyl-N-alkyl sulfonamide (NASA) group as a warhead. Mass-based analysis successfully revealed the kinetics of covalent inhibition and the modification sites on HDM2 to be the N-terminal α-amine and Tyr67, both rarely seen in traditional covalent inhibitors. Finally, we demonstrated prolonged p53-pathway activation and more effective induction of the p53-mediated cell death in comparison to a noncovalent inhibitor. This study highlights the potential of the NASA warhead as a versatile electrophile for the covalent inhibition of PPIs and opens new avenues for the rational design of potent covalent PPI inhibitors.

Selective and reversible modification of kinase cysteines with chlorofluoroacetamides.

Irreversible inhibition of disease-associated proteins with small molecules is a powerful approach for achieving increased and sustained pharmacological potency. Here, we introduce α-chlorofluoroacetamide (CFA) as a novel warhead of targeted covalent inhibitor (TCI). Despite weak intrinsic reactivity, CFA-appended quinazoline showed high reactivity toward Cys797 of epidermal growth factor receptor (EGFR). In cells, CFA-quinazoline showed higher target specificity for EGFR than the corresponding Michael acceptors in a wide concentration range (0.1-10 µM). The cysteine adduct of the CFA derivative was susceptible to hydrolysis and reversibly yielded intact thiol but was stable in solvent-sequestered ATP-binding pocket of EGFR. This environment-dependent hydrolysis can potentially reduce off-target protein modification by CFA-based drugs. Oral administration of CFA quinazoline NS-062 significantly suppressed tumor growth in a mouse xenograft model. Further, CFA-appended pyrazolopyrimidine irreversibly inhibited Bruton's tyrosine kinase with higher target specificity. These results demonstrate the utility of CFA as a new class warheads for TCI.

Site-specific covalent labeling of His-tag fused proteins with N-acyl-N-alkyl sulfonamide reagent.

The ability to incorporate a desired functionality into proteins of interest in a site-specific manner can provide powerful tools for investigating biological systems and creating therapeutic conjugates. However, there are not any universal methods that can be applied to all proteins, and it is thus important to explore the chemical strategy for protein modification. In this paper, we developed a new reactive peptide tag/probe pair system for site-specific covalent protein labeling. This method relies on the recognition-driven reaction of a peptide tag and a molecular probe, which comprises the lysine-containing short histidine tag (KH6 or H6K) and a binuclear nickel (II)- nitrilotriacetic acid (Ni2+-NTA) complex probe containing a lysine-reactive N-acyl-N-alkyl sulfonamide (NASA) group. The selective interaction of the His-tag and Ni2+-NTA propeles a rapid nucleophilic reaction between a lysine residue of the tag and the electrophilic NASA group of the probe by the proximity effect, resulting in the tag-site-specific functionalization of proteins. We characterized the reactive profile and site-specificity of this method using model peptides and proteins in vitro, and demonstrated the general utility for production of a nanobody-chemical probe conjugate without compromising its binding ability.

Recent applications of N-acyl imidazole chemistry in chemical biology.

N-Acyl imidazoles are unique electrophiles that exhibit moderate reactivity, relatively long-half life, and high solubility in water. Thanks to their tunable reactivity and chemical selectivity, the application of N-acyl imidazole derivatives has launched to a number of chemical biology researches, which include chemical synthesis of peptide/protein, chemical labeling of native proteins of interest (POIs), and structural analysis and functional manipulation of RNAs. Since proteins and RNAs play pivotal roles in numerous biological events in all living organisms, the methods that enable the chemical modification of endogenously existing POIs and RNAs in live cells may offer a variety of opportunities not only for fundamental scientific study but also for biotechnology and drug development. In this review, we discuss the recent progress of N-acyl imidazole chemistry that contributes to the chemical labeling and functional control of endogenous proteins and RNAs under multimolecularly crowded biological conditions of live cells.

Development of a Cell-Based Ligand-Screening System for Identifying Hsp90 Inhibitors.

Because of its critical roles in regulating cellular signal transduction, the molecular chaperone heat-shock protein 90 (Hsp90) has become a novel therapeutic target for various diseases, including cancer, inflammation, and neurological diseases. However, the lack of methods that allow us to directly evaluate the binding of small molecule ligands to intracellular Hsp90 makes the inhibitor development more difficult. Here, we report a simple cell-based assay system for the Hsp90 inhibitor in live-cell environments. In this strategy, the binding activity of ligands of interest is evaluated by competitive inhibition of ligand-directed N-acyl-N-alkyl sulfonamide (LDNASA) chemistry-mediated Hsp90 labeling. Using several known Hsp90 inhibitors, we demonstrated that our method could easily detect the ligand-binding event of Hsp90 in live cells. Our system is applicable to high-throughput ligand screening, and we discovered a new small molecule candidate that binds to the N-terminal ATP binding domain of Hsp90. These results demonstrate the use of the competitive LDNASA-based approach to directly evaluate ligand activity in live cells and identify potent drug candidates from chemical libraries.

Imaging and Profiling of Proteins under Oxidative Conditions in Cells and Tissues by Hydrogen-Peroxide-Responsive Labeling.

Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) can inflict damage to biomolecules under oxidative stress and also act as signaling molecules at physiological levels. Here we developed a unique chemical tool to elucidate the biological roles of ROS using both fluorescence imaging and conditional proteomics. H2O2-responsive protein labeling reagents (Hyp-L) were designed to selectively tag proteins under the oxidative conditions in living cells and tissues. The Hyp-L signal remained even after sample fixation, which was compatible with conventional immunostaining. Moreover, Hyp-L allowed proteomic profiling of the labeled proteins using a conditional proteomics workflow. The integrative analysis enabled the identification of ROS generation and/or accumulation sites with a subcellular resolution. For the first time, we characterized that autophagosomes were enriched with H2O2 in activated macrophages. Hyp-L was further applied to mouse brain tissues and clearly revealed oxidative stress within mitochondria by the conditional proteomics.

Activity-Based Sensing with a Metal-Directed Acyl Imidazole Strategy Reveals Cell Type-Dependent Pools of Labile Brain Copper.

Copper is a required nutrient for life and particularly important to the brain and central nervous system. Indeed, copper redox activity is essential to maintaining normal physiological responses spanning neural signaling to metabolism, but at the same time copper misregulation is associated with inflammation and neurodegeneration. As such, chemical probes that can track dynamic changes in copper with spatial resolution, especially in loosely bound, labile forms, are valuable tools to identify and characterize its contributions to healthy and disease states. In this report, we present an activity-based sensing (ABS) strategy for copper detection in live cells that preserves spatial information by a copper-dependent bioconjugation reaction. Specifically, we designed copper-directed acyl imidazole dyes that operate through copper-mediated activation of acyl imidazole electrophiles for subsequent labeling of proximal proteins at sites of elevated labile copper to provide a permanent stain that resists washing and fixation. To showcase the utility of this new ABS platform, we sought to characterize labile copper pools in the three main cell types in the brain: neurons, astrocytes, and microglia. Exposure of each of these cell types to physiologically relevant stimuli shows distinct changes in labile copper pools. Neurons display translocation of labile copper from somatic cell bodies to peripheral processes upon activation, whereas astrocytes and microglia exhibit global decreases and increases in intracellular labile copper pools, respectively, after exposure to inflammatory stimuli. This work provides foundational information on cell type-dependent homeostasis of copper, an essential metal in the brain, as well as a starting point for the design of new activity-based probes for metals and other dynamic signaling and stress analytes in biology.

Screening of protein-ligand interactions under crude conditions by native mass spectrometry.

A convenient analytical system for protein-ligand interactions under crude conditions was developed using native mass spectrometry (MS). As a model protein, Escherichia coli (E. coli) dihydrofolate reductase (DHFR) with and without a histidine tag was used for the study. First, overexpressed DHFR with a His-tag was roughly purified with a Ni-sepharose resin and subjected to native mass spectrometry with or without incubation with an inhibitor, Methotrexate (MTX). Even only with the minimum cleanup by the Ni-sepharose resin, intact ions of DHFR-nicotinamide adenine dinucleotide phosphate (NADPH) and DHFR-NADPH-ligand complexes were successfully observed. By optimizing the preparation procedures of the crude sample for native MS, e.g., avoiding sonication for cell lysis, we successfully observed intact ions of the specific DHFR-NADPH-MTX ternary complex starting with cultivation of E. coli in ≤ 25 mL medium. When the crude DHFR sample was mixed with two, four, or eight candidate compounds, only ions of the specific protein-ligand complex were observed. This indicates that the present system can be used as a rapid and convenient method for the rough determination of binding of specific ligands to the target protein without the time-consuming purification of protein samples. Moreover, it is important to rapidly determine specific interactions with target proteins under conditions similar to those in "real" biological systems. Graphical abstract.

Fluorescence Differentiation of ATP-related Multiple Enzymatic Activities in Synovial Fluid as a Marker of Calcium Pyrophosphate Deposition Disease using Kyoto Green.

Calcium pyrophosphate deposition disease (CPPD) is a crystal induced inflammation in joints, and causes severe pain in elderly people. The accumulation of pyrophosphate (PPi) in synovial fluid (SF) results from several enzymatic reactions, especially the highly activated e-NPPs, which catalyze the conversion of ATP to PPi. This study demonstrates the detection of relative catalytic activity of 3 enzymes-ecto-nucleotide pyrophosphatase/phosphodiesterases (e-NPPs), tissue nonspecific alkaline phosphatase (TNAP), and ecto-nucleoside triphosphate diphosphohydrolases (e-NTPDases)-using a single molecular sensor called Kyoto Green. Kyoto Green exhibits excellent performance in sensing the catalytic activity of the commercial representatives of the e-NPPs, TNAP, and e-NTPDases, which are ENPP1, PPase, and apyrase, respectively, in both single-enzyme and multi-enzyme assays. Analysis of SF enzymes in 19 SF samples from human and swine revealed moderate activity of e-NPPs, high activity of e-NTPDases, and low activity of TNAP. Our newly developed method for analysis of multiple enzymatic activities using Kyoto Green in biological SF will assist improvement in accuracy of the CPPD prognosis/diagnosis, which will minimize unnecessary medical procedures.