Mechanism of CFTR correction by type I folding correctors.

Small molecule chaperones have been exploited as therapeutics for the hundreds of diseases caused by protein misfolding. The most successful examples are the CFTR correctors, which transformed cystic fibrosis therapy. These molecules revert folding defects of the ΔF508 mutant and are widely used to treat patients. To investigate the molecular mechanism of their action, we determined cryo-electron microscopy structures of CFTR in complex with the FDA-approved correctors lumacaftor or tezacaftor. Both drugs insert into a hydrophobic pocket in the first transmembrane domain (TMD1), linking together four helices that are thermodynamically unstable. Mutating residues at the binding site rendered ΔF508-CFTR insensitive to lumacaftor and tezacaftor, underscoring the functional significance of the structural discovery. These results support a mechanism in which the correctors stabilize TMD1 at an early stage of biogenesis, prevent its premature degradation, and thereby allosterically rescuing many disease-causing mutations.

Potent, Reversible, and Specific Chemical Inhibitors of Eukaryotic Ribosome Biogenesis.

All cellular proteins are synthesized by ribosomes, whose biogenesis in eukaryotes is a complex multi-step process completed within minutes. Several chemical inhibitors of ribosome function are available and used as tools or drugs. By contrast, we lack potent validated chemical probes to analyze the dynamics of eukaryotic ribosome assembly. Here, we combine chemical and genetic approaches to discover ribozinoindoles (or Rbins), potent and reversible triazinoindole-based inhibitors of eukaryotic ribosome biogenesis. Analyses of Rbin sensitivity and resistance conferring mutations in fission yeast, along with biochemical assays with recombinant proteins, provide evidence that Rbins' physiological target is Midasin, an essential ∼540-kDa AAA+ (ATPases associated with diverse cellular activities) protein. Using Rbins to acutely inhibit or activate Midasin function, in parallel experiments with inhibitor-sensitive or inhibitor-resistant cells, we uncover Midasin's role in assembling Nsa1 particles, nucleolar precursors of the 60S subunit. Together, our findings demonstrate that Rbins are powerful probes for eukaryotic ribosome assembly.

Enantioselective [2+2]-cycloadditions with triplet photoenzymes.

Naturally evolved enzymes, despite their astonishingly large variety and functional diversity, operate predominantly through thermochemical activation. Integrating prominent photocatalysis modes into proteins, such as triplet energy transfer, could create artificial photoenzymes that expand the scope of natural biocatalysis1-3. Here, we exploit genetically reprogrammed, chemically evolved photoenzymes embedded with a synthetic triplet photosensitizer that are capable of excited-state enantio-induction4-6. Structural optimization through four rounds of directed evolution afforded proficient variants for the enantioselective intramolecular [2+2]-photocycloaddition of indole derivatives with good substrate generality and excellent enantioselectivities (up to 99% enantiomeric excess). A crystal structure of the photoenzyme-substrate complex elucidated the non-covalent interactions that mediate the reaction stereochemistry. This study expands the energy transfer reactivity7-10 of artificial triplet photoenzymes in a supramolecular protein cavity and unlocks an integrated approach to valuable enantioselective photochemical synthesis that is not accessible with either the synthetic or the biological world alone.

Superresolution imaging of antibiotic-induced structural disruption of bacteria enabled by photochromic glycomicelles.

Bacterial evolution, particularly in hospital settings, is leading to an increase in multidrug resistance. Understanding the basis for this resistance is critical as it can drive discovery of new antibiotics while allowing the clinical use of known antibiotics to be optimized. Here, we report a photoactive chemical probe for superresolution microscopy that allows for the in situ probing of antibiotic-induced structural disruption of bacteria. Conjugation between a spiropyran (SP) and galactose via click chemistry produces an amphiphilic photochromic glycoprobe, which self-assembles into glycomicelles in water. The hydrophobic inner core of the glycomicelles allows encapsulation of antibiotics. Photoirradiation then serves to convert the SP to the corresponding merocyanine (MR) form. This results in micellar disassembly allowing for release of the antibiotic in an on-demand fashion. The glycomicelles of this study adhere selectively to the surface of a Gram-negative bacterium through multivalent sugar-lectin interaction. Antibiotic release from the glycomicelles then induces membrane collapse. This dynamic process can be imaged in situ by superresolution spectroscopy owing to the "fluorescence blinking" of the SP/MR photochromic pair. This research provides a high-precision imaging tool that may be used to visualize how antibiotics disrupt the structural integrity of bacteria in real time.

Domino electroreduction of CO2 to methanol on a molecular catalyst.

Electrochemical carbon dioxide (CO2) reduction can in principle convert carbon emissions to fuels and value-added chemicals, such as hydrocarbons and alcohols, using renewable energy, but the efficiency of the process is limited by its sluggish kinetics1,2. Molecular catalysts have well defined active sites and accurately tailorable structures that allow mechanism-based performance optimization, and transition-metal complexes have been extensively explored in this regard. However, these catalysts generally lack the ability to promote CO2 reduction beyond the two-electron process to generate more valuable products1,3. Here we show that when immobilized on carbon nanotubes, cobalt phthalocyanine-used previously to reduce CO2 to primarily CO-catalyses the six-electron reduction of CO2 to methanol with appreciable activity and selectivity. We find that the conversion, which proceeds via a distinct domino process with CO as an intermediate, generates methanol with a Faradaic efficiency higher than 40 per cent and a partial current density greater than 10 milliamperes per square centimetre at -0.94 volts with respect to the reversible hydrogen electrode in a near-neutral electrolyte. The catalytic activity decreases over time owing to the detrimental reduction of the phthalocyanine ligand, which can be suppressed by appending electron-donating amino substituents to the phthalocyanine ring. The improved molecule-based electrocatalyst converts CO2 to methanol with considerable activity and selectivity and with stable performance over at least 12 hours.

Metal-free directed sp2-C-H borylation.

Organoboron reagents are important synthetic intermediates that have a key role in the construction of natural products, pharmaceuticals and organic materials1. The discovery of simpler, milder and more efficient approaches to organoborons can open additional routes to diverse substances2-5. Here we show a general method for the directed C-H borylation of arenes and heteroarenes without the use of metal catalysts. C7- and C4-borylated indoles are produced by a mild approach that is compatible with a broad range of functional groups. The mechanism, which is established by density functional theory calculations, involves BBr3 acting as both a reagent and a catalyst. The potential utility of this strategy is highlighted by the downstream transformation of the formed boron species into natural products and drug scaffolds.

Artificial-Cofactor-Mediated Hydrogen and Electron Transfer Endows AuFe/Polydopamine Superparticles with Enhanced Glucose Oxidase-Like Activity.

Various applications related to glucose catalysis have led to the development of functional nanozymes with glucose oxidase (GOX)-like activity. However, the unsatisfactory catalytic activity of nanozymes is a major challenge for their practical applications due to their inefficient hydrogen and electron transfer. Herein, we present the synthesis of AuFe/polydopamine (PDA) superparticles that exhibit photothermal-enhanced GOX-like activity. Experimental investigations and theoretical calculations reveal that the glucose oxidation process catalyzed by AuFe/PDA follows an artificial-cofactor-mediated hydrogen atom transfer mechanism, which facilitates the generation of carbon-centered radical intermediates. Rather than depending on charged Au surfaces for thermodynamically unstable hydride transfer, Fe(III)-coordinated PDA with abundant amino and phenolic hydroxyl groups serves as cofactor mimics, facilitating both hydrogen atom and electron transfer in the catalytic process. Finally, leveraging the photothermal-enhanced GOX-like and catalase-like activities of AuFe/PDA, we establish a highly sensitive and accurate point-of-care testing blood glucose determination with exceptional anti-jamming capabilities.

Regulating Second Coordination Shell of Ce Atom Site and Reshaping of Carrier Enable Single-Atom Nanozyme to Efficiently Express Oxidase-like Activity.

Single-atom nanozymes (SANs) are considered to be ideal substitutes for natural enzymes due to their high atom utilization. This work reported a strategy to manipulate the second coordination shell of the Ce atom and reshape the carbon carrier to improve the oxidase-like activity of SANs. Internally, S atoms were symmetrically embedded into the second coordination layer to form a Ce-N4S2-C structure, which reduced the energy barrier for O2 reduction, promoted the electron transfer from the Ce atom to O atoms, and enhanced the interaction between the d orbital of the Ce atom and p orbital of O atoms. Externally, in situ polymerization of mussel-inspired polydopamine on the precursor helps capture metal sources and protects the 3D structure of the carrier during pyrolysis. On the other hand, polyethylene glycol (PEG) modulated the interface of the material to enhance water dispersion and mass transfer efficiency. As a proof of concept, the constructed PEG@P@Ce-N/S-C was applied to the multimodal assay of butyrylcholinesterase activity.

Metal-Loaded Synthetic Melanin via Oxidative Polymerization of Neurotransmitter Norepinephrine Exhibiting High Photothermal Conversion.

Polydopamine (PDA)-derived melanin-like materials exhibit significant photothermal conversion owing to their broad-spectrum light absorption. However, their low near-infrared (NIR) absorption and inadequate hydrophilicity compromise their utilization of solar energy. Herein, we developed metal-loaded poly(norepinephrine) nanoparticles (PNE NPs) by predoping metal ions (Fe3+, Mn3+, Co2+, Ca2+, Ga3+, and Mg2+) with norepinephrine, a neuron-derived biomimetic molecule, to address the limitations of PDA. The chelation between catechol and metal ions induces a ligand-to-metal charge transfer (LMCT) through the formation of donor-acceptor pairs, modulating the light absorption behavior and reducing the band gap. Under 1 sun illumination, the Fe-loaded PNE coated wood evaporator achieved a high seawater evaporation rate and efficiency of 1.75 kg m-2 h-1 and 92.4%, respectively, owing to the superior hydrophilicity and photothermal performance of PNE. Therefore, this study offers a comprehensive exploration of the role of metal ions in enhancing the photothermal properties of synthetic melanins.

"Cave Effect" Induces Self-Assembled Bimetallic Hollow Structure for Three-in-One Lateral Flow Immunoassay.

Bimetallic hollow structures have attracted much attention due to their unique properties, but they still face the problems of nonuniform alloys and excessive etching leading to structural collapse. Here, uniform bimetallic hollow nanospheres are constructed by pore engineering and then highly loaded with hemin (Hemin@MOF). Interestingly, in the presence of polydopamine (PDA), the competitive coordination between anionic polymer (γ-PGA) and dimethylimidazole does not lead to the collapse of the external framework but self-assembly into a hollow structure. By constructing the Hemin@MOF immune platform and using E. coli O157:H7 as the detection object, we find that the visual detection limits can reach 10, 3, and 3 CFU/mL in colorimetric, photothermal, and catalytic modes, which is 4 orders of magnitude lower than the traditional gold standard. This study provides a new idea for the morphological modification of the metal-organic skeleton and multifunctional immunochromatography detection.

Physiological Microenvironment Dependent Self-Cross-Linking of Multifunctional Nanohybrid for Prolonged Antibacterial Therapy via Synergistic Chemodynamic-Photothermal-Biological Processes.

Herein, a multifunctional nanohybrid (PL@HPFTM nanoparticles) was fabricated to perform the integration of chemodynamic therapy, photothermal therapy, and biological therapy over the long term at a designed location for continuous antibacterial applications. The PL@HPFTM nanoparticles consisted of a polydopamine/hemoglobin/Fe2+ nanocomplex with comodification of tetrazole/alkene groups on the surface as well as coloading of antimicrobial peptides and luminol in the core. During therapy, the PL@HPFTM nanoparticles would selectively cross-link to surrounding bacteria via tetrazole/alkene cycloaddition under chemiluminescence produced by the reaction between luminol and overexpressed H2O2 at the infected area. The resulting PL@HPFTM network not only significantly damaged bacteria by Fe2+-catalyzed ROS production, effective photothermal conversion, and sustained release of antimicrobial peptides but dramatically enhanced the retention time of these therapeutic agents for prolonged antibacterial therapy. Both in vitro and in vivo results have shown that our PL@HPFTM nanoparticles have much higher bactericidal efficiency and remarkably longer periods of validity than free antibacterial nanoparticles.

LRG1-Targeted Nintedanib Delivery for Enhanced Renal Fibrosis Mitigation.

Renal fibrosis lacks effective nephroprotective drugs in clinical settings due to poor accumulation of therapeutic agents in damaged kidneys, underscoring the urgent need for advanced renal-targeted delivery systems. Herein, we exploited the significantly increased expression of the leucine-rich α-2 glycoprotein 1 (LRG1) protein during renal fibrosis to develop a novel drug delivery system. Our engineered nanocarrier, DENNM, preferentially targets fibrotic kidneys via the decorated ET peptide's high affinity for LRG1. Once internalized by damaged renal cells, DENNM releases its encapsulated nintedanib, triggered by the active caspase-3 protease, disrupting the nanomedicine's structural integrity. The released nintedanib effectively reduces the level of expression of the extracellular matrix and impedes the progression of renal fibrosis by inhibiting the transforming growth factor-ß (TGF-ß)-Smad2/3 pathway. Our comprehensive in vitro and in vivo studies validate DENNM's antifibrotic efficacy, emphasizing LRG1's potential in renal targeted drug delivery and introducing an innovative approach to nanomedicine for treating renal fibrosis.

Polydopamine Synergizes with Quercetin Nanosystem to Reshape the Perifollicular Microenvironment for Accelerating Hair Regrowth in Androgenetic Alopecia.

Accumulated reactive oxygen species (ROS) and their resultant vascular dysfunction in androgenic alopecia (AGA) hinder hair follicle survival and cause permanent hair loss. However, safe and effective strategies to rescue hair follicle viability to enhance AGA therapeutic efficiency remain challenging. Herein, we fabricated a quercetin-encapsulated (Que) and polydopamine-integrated (PDA@QLipo) nanosystem that can reshape the perifollicular microenvironment to initial hair follicle regeneration for AGA treatment. Both the ROS scavenging and angiogenesis promotion abilities of PDA@QLipo were demonstrated. In vivo assays revealed that PDA@QLipo administrated with roller-microneedles successfully rejuvenated the "poor" perifollicular microenvironment, thereby promoting cell proliferation, accelerating hair follicle renewal, and facilitating hair follicle recovery. Moreover, PDA@QLipo achieved a higher hair regeneration coverage of 92.5% in the AGA mouse model than minoxidil (87.8%), even when dosed less frequently. The nanosystem creates a regenerative microenvironment by scavenging ROS and augmenting neovascularity for hair regrowth, presenting a promising approach for AGA clinical treatment.

DON/DRP-104 as potent serine protease inhibitors implicated in SARS-CoV-2 infection: Comparative binding modes with human TMPRSS2 and novel therapeutic approach.

Human transmembrane serine protease 2 (TMPRSS2) is an important member of the type 2 transmembrane serine protease (TTSP) family with significant therapeutic markings. The search for potent TMPRSS2 inhibitors against severe acute respiratory syndrome coronavirus 2 infection with favorable tissue specificity and off-site toxicity profiles remains limited. Therefore, probing the anti-TMPRSS2 potential of enhanced drug delivery systems, such as nanotechnology and prodrug systems, has become compelling. We report the first in silico study of TMPRSS2 against a prodrug, [isopropyl(S)-2-((S)-2-acetamido-3-(1H-indol-3-yl)-propanamido)-6-diazo-5-oxo-hexanoate] also known as DRP-104 synthesized from 6-Diazo-5-oxo-l-norleucine (DON). We performed comparative studies on DON and DRP-104 against a clinically potent TMPRSS2 inhibitor, nafamostat, and a standard serine protease inhibitor, 4-(2-Aminoethyl) benzenesulfonyl fluoride (AEBSF) against TMPRSS2 and found improved TMPRSS2 inhibition through synergistic binding of the S1/S1' subdomains. Both DON and DRP-104 had better thermodynamic profiles than AEBSF and nafamostat. DON was found to confer structural stability with strong positive correlated inter-residue motions, whereas DRP-104 was found to confer kinetic stability with restricted residue displacements and reduced loop flexibility. Interestingly, the Scavenger Receptor Cysteine-Rich (SRCR) domain of TMPRSS2 may be involved in its inhibition mechanics. Two previously unidentified loops, designated X (270-275) and Y (293-296) underwent minimal and major structural transitions, respectively. In addition, residues 273-277 consistently transitioned to a turn conformation in all ligated systems, whereas unique transitions were identified for other transitioning residue groups in each TMPRSS2-inhibitor complex. Intriguingly, while both DON and DRP-104 showed similar loop transition patterns, DRP-104 preserved loop structural integrity. As evident from our systematic comparative study using experimentally/clinically validated inhibitors, DRP-104 may serve as a potent and novel TMPRSS2 inhibitor and warrants further clinical investigation.

Chemogenetic Evolution of Diversified Photoenzymes for Enantioselective [2 + 2] Cycloadditions in Whole Cells.

Artificial photoenzymes with novel catalytic modes not found in nature are in high demand; yet, they also present significant challenges in the field of biocatalysis. In this study, a chemogenetic modification strategy is developed to facilitate the rapid diversification of photoenzymes. This strategy integrates site-specific chemical conjugation of various artificial photosensitizers into natural protein cavities and the iterative mutagenesis in cell lysates. Through rounds of directed evolution, prominent visible-light-activatable photoenzyme variants were developed, featuring a thioxanthone chromophore. They successfully enabled the enantioselective [2 + 2] photocycloaddition of 2-carboxamide indoles, a class of UV-sensitive substrates that are traditionally challenging for known photoenzymes. Furthermore, the versatility of this photoenzyme is demonstrated in enantioselective whole-cell photobiocatalysis, enabling the efficient synthesis of enantioenriched cyclobutane-fused indoline tetracycles. These findings significantly expand the photophysical properties of artificial photoenzymes, a critical factor in enhancing their potential for harnessing excited-state reactivity in stereoselective transformations.

Discovery of new acetamide derivatives of 5-indole-1,3,4-oxadiazol-2-thiol as inhibitors of HIV-1 Tat-mediated viral transcription.

Human immunodeficiency virus-1 (HIV-1) encodes a transcriptional factor called Tat, which is critical for viral transcription. Tat-mediated transcription is highly ordered apart from the cellular manner; therefore, it is considered a target for developing anti-HIV-1 drugs. However, drugs targeting Tat-mediated viral transcription are not yet available. Our high-throughput screen of a compound library employing a dual-reporter assay identified a 1,3,4-oxadiazole scaffold against Tat and HIV-1 infection. Furthermore, a serial structure-activity relation (SAR) study performed with biological assays found 1,3,4-oxadiazole derivatives (9 and 13) containing indole and acetamide that exhibited potent inhibitory effects on HIV-1 infectivity, with half-maximal effective concentrations (EC50) of 0.17 (9) and 0.24 µM (13), respectively. The prominent derivatives specifically interfered with the viral transcriptional step without targeting other infection step(s) and efficiently inhibited the HIV-1 replication cycle in the T cell lines and peripheral blood mononuclear cells infected with HIV-1. Additionally, compared to the wild type, the compounds exhibited similar potency against anti-retroviral drug-resistant HIV-1 strains. In a series of mode-of-action studies, the compounds inhibited the ejection of histone H3 for facilitating viral transcription on the long-terminal repeat (LTR) promoter. Furthermore, SAHA (a histone deacetylase inhibitor) treatment abolished the compound potency, revealing that the compounds can possibly target Tat-regulated epigenetic modulation of LTR to inhibit viral transcription. Overall, our screening identified novel 1,3,4-oxadiazole compounds that inhibited HIV-1 Tat, and subsequent SAR-based optimization led to the derivatives 9 and 13 development that could be a promising scaffold for developing a new class of therapeutic agents for HIV-1 infection.

Fluorine-19 labeling of the tryptophan residues in the G protein-coupled receptor NK1R using the 5-fluoroindole precursor in Pichia pastoris expression.

In NMR spectroscopy of biomolecular systems, the use of fluorine-19 probes benefits from a clean background and high sensitivity. Therefore, 19F-labeling procedures are of wide-spread interest. Here, we use 5-fluoroindole as a precursor for cost-effective residue-specific introduction of 5-fluorotryptophan (5F-Trp) into G protein-coupled receptors (GPCRs) expressed in Pichia pastoris. The method was successfully implemented with the neurokinin 1 receptor (NK1R). The 19F-NMR spectra of 5F-Trp-labeled NK1R showed one well-separated high field-shifted resonance, which was assigned by mutational studies to the "toggle switch tryptophan". Residue-selective labeling thus enables site-specific investigations of this functionally important residue. The method described here is inexpensive, requires minimal genetic manipulation and can be expected to be applicable for yeast expression of GPCRs at large.

Indole-3-pyruvate: Analysis and Control of Tautomerism and Reactivity.

It is well-known in biochemistry that structure confers function, meaning that chemical structural elucidation is critical to truly understanding the function of a given metabolite. Indole-3-pyruvate (IPyA) exists in an equilibrium between the keto and enol tautomeric forms. IPyA is suggested to play a role in immune function; however, determining whether the tautomeric forms function differently can only be studied if an analytical method is capable of distinguishing between the two forms. Herein, we describe the use of UHPLC-HRMS to gain insight into the physical variables that govern IPyA tautomer equilibrium, reactivity, and detection limit. We use hydrogen-deuterium exchange (HDX) to identify enol and keto peaks, and we show that tautomers exhibit a valley of fronting followed by a tailing peak shape (though separation is still attainable) and identical MS/MS spectra. We observed drastically different ratios of keto and enol forms in different solvents, which is an important consideration for in vitro studies. IPyA was found to be highly unstable with accelerated reactivity in peroxides. Through in vitro reactivity studies, IPyA produced a myriad of known and unknown metabolites via nonenzymatic processes, many of which were mapped in vivo via the analysis of human plasma. Finally, we show that vitamin C (ascorbic acid) can slow this reactivity and enable sensitive detection in whole blood.

Divergent Metabolic Fates of Aromatic Amino Acid-Derived Isomers: Insights from Ex Vivo Metabolomics and HDX-HRMS/MS-Based Resolution of Tautomers.

Tautomers are one of the many types of isomers, and differences in tautomeric structures confer altered chemical and biological properties. Using ultrahigh-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) ex vivo metabolomics, we investigate, in whole blood, the divergent metabolism of enol and keto forms of indole-3-pyruvate (IPyA), a tautomeric product of aromatic amino acid metabolism. Two new compounds resulting from IPyA metabolism were discovered, 3-(1H-indol-3-yl)-2,3-dioxopropanoic acid or "indole-3-oxopyruvic acid" and glutathionyl-indole pyruvate (GSHIPyA), which were characterized via ultraviolet photodissociation (UVPD) and higher-energy collisional dissociation (HCD). Computational calculations support the hypothesis that GSHIPyA forms specifically through the enol form of IPyA. GSHIPyA is also hypothesized to be tautomeric, and a hydrogen-deuterium exchange-high-resolution tandem mass spectrometry (HDX-HRMS/MS) approach is developed to prove the presence of an enol and keto tautomer. HDX of GSHIPyA labels the keto form with an additional deuterium, relative to the enol form. HRMS/MS of the labeled isomers is employed to leverage the relationship of resolving power scaling inversely with the square root of m/z, for Orbitrap mass analyzers. HRMS/MS yields a smaller-molecular-weight deuterated tautomeric product ion, reducing the analyte ion m/z and thus lowering the resolving power necessary to separate the deuterated keto tautomer product ion from the [13]C product ion.

Near-Infrared Spontaneously Blinking Fluorophores for Live Cell Super-Resolution Imaging with Minimized Phototoxicity.

Single-molecule localization microscopy (SMLM) requires high-intensity laser irradiation, typically exceeding kW/cm2, to yield a sufficient photon count. However, this intense visible light exposure incurs substantial cellular toxicity, hindering its use in living cells. Here, we developed a class of near-infrared (NIR) spontaneously blinking fluorophores for SMLM. These NIR fluorophores are a combination of rhodamine spirolactams and merocyanine derivatives, where the rhodamine spirolactam component converts between a bright and dark state based on pH-dependent spirocyclization and merocyanine derivatives shift the excitation wavelength into the infrared. Single-molecule characterizations demonstrated their potential for SMLM. At a moderate power density of 3.93 kW/cm2, these probes exhibit duty cycle as low as 0.18% and an emission rate as high as 26,700 photons/s. Phototoxicity assessment under single-molecule imaging conditions reveals that NIR illumination (721 nm) minimizes harm to living cells. Employing these NIR fluorophores, we successfully captured time-lapse super-resolution tracking of mitochondria at a Fourier ring correlation (FRC) resolution of 69.4 nm and reconstructed the ultrastructures of endoplasmic reticulum (ER) in living cells.