Covalent targeting of the vacuolar H+-ATPase activates autophagy via mTORC1 inhibition.

Autophagy is a lysosomal degradation pathway that eliminates aggregated proteins and damaged organelles to maintain cellular homeostasis. A major route for activating autophagy involves inhibition of the mTORC1 kinase, but current mTORC1-targeting compounds do not allow complete and selective mTORC1 blockade. Here, we have coupled screening of a covalent ligand library with activity-based protein profiling to discover EN6, a small-molecule in vivo activator of autophagy that covalently targets cysteine 277 in the ATP6V1A subunit of the lysosomal v-ATPase, which activates mTORC1 via the Rag guanosine triphosphatases. EN6-mediated ATP6V1A modification decouples the v-ATPase from the Rags, leading to inhibition of mTORC1 signaling, increased lysosomal acidification and activation of autophagy. Consistently, EN6 clears TDP-43 aggregates, a causative agent in frontotemporal dementia, in a lysosome-dependent manner. Our results provide insight into how the v-ATPase regulates mTORC1, and reveal a unique approach for enhancing cellular clearance based on covalent inhibition of lysosomal mTORC1 signaling

Design, synthesis, photophysics and self-assembly study of platinum (II) terpyridine complexes and their utilization as stimuli-responsive smart materials and probes for molecules and macromolecules of biological interest

A series of water-soluble platinum(II) terpyridine complexes with functionalized alkynyl ligands and a boronic acid-containing polymer, PAAPBA, have been synthesized and characterized. The photophysical and electrochemical properties of all the platinum(II) complexes have been studied. Some of the complexes have been demonstrated to show ground-state aggregation in organic solvents and aqueous solutions at high concentrations, leading to Pt…Pt and/or π–π interactions and hence the emergence of metal-metal-to-ligand charge transfer (MMLCT) transitions in both the UV−visible and emission spectra. The induced self-assembly of [Pt(tpy)(C≡CC6H4−CH2NMe3-4)](OTf)2 by PAAPBA has been explored for the development of glucose sensing protocols and α-glucosidase assay by monitoring the triplet metal-metal-to-ligand charge transfer (3MMLCT) emission in the near-infrared (NIR) region. [Pt(tpy){C≡CC6H4− {NHC(=NH2+)(NH2)}-4}](OTf)2 has been observed to undergo induced aggregation in the presence of citrate, with good selectivity over other mono- and dicarboxylates in the tricarboxylic acid (TCA) cycle. Enzymatic activity of citrate lyase has also been probed by the emission spectral changes of the complex in the NIR region. A series of water-soluble alkynylplatinum(II) terpyridine complexes and water-soluble conjugated polyelectrolytes (CPEs) have been synthesized and characterized. The UV–vis absorption and emission properties of the platinum(II) complexes and CPEs have been investigated in organic solvents and/or aqueous buffer solutions. The electrochemical properties and ground-state aggregation at high concentrations of the platinum(II) complexes have also been examined. Two-component ensembles containing selected platinum(II) complexes and PPE-SO3− have been studied, and Förster resonance energy transfer (FRET) has been demonstrated from the PPE-SO3− donor to the aggregated complexes as acceptors. The ensemble containing PPE-SO3− and [Pt(tpy)(C≡CC6H4CH2NMe3-4)](OTf)2 has been employed for a “proof-of-principle” label-free detection of human serum albumin (HSA) in pH 3 buffer solutions with high selectivity and sensitivity, while another ensemble containing PPE-SO3− and [Pt{tpy(C6H4CH2NMe3-4)-4’}(C≡CC6H5)](OTf)2 has been utilized for selective label-free detection of G-quadruplex structure of the human telomeric DNA in physiological buffer solutions. A series of water-soluble platinum(II) terpyridine complexes with stimuli-responsive alkynyl ligands and a series of water-soluble platinum(II) metallosupramolecular triblock copolymers have been synthesized and characterized. The photophysical and electrochemical properties as well as the ground-state aggregation of the complexes have been investigated. Some of them have been found to show different electronic absorption and emission properties in aqueous solution at different pHs due to aggregation/deaggregation of the complexes. One of the complexes has been employed for live-cell imaging experiments to locate acidic organelles, such as lysosomes, in MDCK cells. The water-soluble platinum(II) metallosupramolecular triblock copolymers have been found to show an increase in 3MMLCT emission intensity in the red-NIR region with temperature, which has been attributed to the formation of spherical polymeric micelles. The platinum(II) triblock copolymer with pH-responsive –CH2NMe2 moieties has been demonstrated as a NIR-emitting dual sensor for pH and temperature through the changes in hydrophilicity and hence the emission properties with pH and temperature simultaneously.published_or_final_versionChemistryDoctoralDoctor of Philosoph

N-Acryloylindole-alkyne (NAIA) enables profiling new ligandable hotspots in chemoproteomics experiments and imaging thiol oxidation

We report a new class of compounds, N-acryloylindole-alkynes (NAIAs), as promising cysteine-reactive probes for proteome-wide cysteine profiling and imaging of thiol oxidative modifications. NAIAs showed superior cysteine reactivity owing to delocalization of π electrons of the acrylamide warhead over the whole indole scaffold, resulting in its activation for faster reaction with cysteines. This allows NAIAs to ligand functional cysteines more effectively than IAA, as well as to image oxidized thiols in cells facing oxidative stress by confocal fluorescence microscopy. In MS-based ABPP experiments, NAIAs successfully captured a new pool of ligandable cysteines and proteins even compared to the current state-of-the-art cysteine profiling data. Competitive ABPP experiments further demonstrate the ability of NAIA to discover hit compounds targeting these new cysteines and proteins. This work should initiate development of new cysteine-reactive probes, particularly those with activated acrylamide, for advancing cysteine imaging and profiling, and covalent ligand screening for drug research

A puromycin-dependent activity-based sensing probe for histochemical staining of hydrogen peroxide in cells and animal tissues

Hydrogen peroxide (H2O2) is a key member of the reactive oxygen species family of transient small molecules that has broad contributions to oxidative stress and redox signaling. The development of selective and sensitive chemical probes can enable the study of H2O2 biology in cell, tissue and animal models. Peroxymycin-1 is a histochemical activity-based sensing probe that responds to H2O2 via chemoselective boronate oxidation to release puromycin, which is then covalently incorporated into nascent proteins by the ribosome and can be detected by antibody staining. Here, we describe an optimized two-step, one-pot protocol for synthesizing Peroxymycin-1 with improved yields over our originally reported procedure. We also present detailed procedures for applying Peroxymycin-1 to a broad range of biological samples spanning cells to animal tissues for profiling H2O2 levels through histochemical detection by using commercially available anti-puromycin antibodies. The preparation of Peroxymycin-1 takes 9 h, the confocal imaging experiments of endogenous H2O2 levels across different cancer cell lines take 1 d, the dot blot analysis of mouse liver tissues takes 1 d and the confocal imaging of mouse liver tissues takes 3-4 d

Versatile Histochemical Approach to Detection of Hydrogen Peroxide in Cells and Tissues Based on Puromycin Staining

Hydrogen peroxide (H₂O₂) is a central reactive oxygen species (ROS) that contributes to diseases from obesity to cancer to neurodegeneration but is also emerging as an important signaling molecule. We now report a versatile histochemical approach for detection of H₂O₂ that can be employed across a broad range of cell and tissue specimens in both healthy and disease states. We have developed a first-generation H₂O₂-responsive analogue named Peroxymycin-1, which is based on the classic cell-staining molecule puromycin and enables covalent staining of biological samples and retains its signal after fixation. H₂O₂-mediated boronate cleavage uncages the puromycin aminonucleoside, which leaves a permanent and dose-dependent mark on treated biological specimens that can be detected with high sensitivity and precision through a standard immunofluorescence assay. Peroxymycin-1 is selective and sensitive enough to image both exogenous and endogenous changes in cellular H₂O₂ levels and can be exploited to profile resting H₂O₂ levels across a panel of cell lines to distinguish metastatic, invasive cancer cells from less invasive cancer and nontumorigenic counterparts, based on correlations with ROS status. Moreover, we establish that Peroxymycin-1 is an effective histochemical probe for in vivo H₂O₂ analysis, as shown through identification of aberrant elevations in H₂O₂ levels in liver tissues in a murine model of nonalcoholic fatty liver disease, thus demonstrating the potential of this approach for studying disease states and progression associated with H₂O₂. This work provides design principles that should enable development of a broader range of histochemical probes for biological use that operate via activity-based sensing

Induced self-assembly and disassembly of water-soluble alkynylplatinum( ii ) terpyridyl complexes with switchable near-infrared (NIR) emission modulated by metalmetal interactions over physiological pH: demonstration of pH-responsive NIR luminescent probes in cell-imaging studies

Water-soluble alkynylplatinum( ii ) terpyridine complexes, [Pt{tpy(C 6 H 4 CH 2 NMe 3 -4)-4}(CCAr)](OTf) 2 [Ar = C 6 H 3 (OH) 2 -3,5 ( 1 ), C 6 H 4 OH-4 ( 2 ), C 6 H 3 (OMe) 2 -3,5 ( 3 )], have been synthesized and characterized. The photophysical and electrochemical properties of the complexes have been studied. Complex 1 has been found to undergo aggregation at low pHs, leading to metalmetal and/or interactions and the emergence of a triplet metal-metal-to-ligand charge transfer ( 3 MMLCT) emission in the near-infrared (NIR) region, the intensity of which has been enhanced 1350-fold over that at physiological pH. Such switchable NIR emission of complex 1 was employed in cell-imaging experiments. The pH response of the 3 MMLCT emission of complex 1 in cellular compartments has been studied using experiments with fixed MadinDarby canine kidney (MDCK) cells, while live cell-imaging experiments revealed that complex 1 could function as a NIR luminescent probe for the tracking of the location of acidic organelles such as lysosomes