scholarly journals Permeant Fluorescent Probes Visualize the Activation of SARM1 and Uncover an Anti-neurodegenerative Drug Candidate

2021 ◽  
Author(s):  
Wan Hua Li ◽  
Ke Huang ◽  
Yang Cai ◽  
Qian Wen Wang ◽  
Wen Jie Zhu ◽  
...  
Keyword(s):  
Fluorescent Probes ◽  
Neurodegenerative Disorders ◽  
Drug Target ◽  
Axonal Degeneration ◽  
Live Cells ◽  
Drug Candidate ◽  
Library Screening ◽  
Inactive Conformation ◽  
A Cell ◽  
Covalent Inhibitor

AbstractSARM1 regulates axonal degeneration through its NAD-metabolizing activity and is a drug target for neurodegenerative disorders. We designed and synthesized fluorescent conjugates of styryl derivative with pyridine to serve as substrates of SARM1, which exhibited large red-shifts after conversion. With the conjugates, SARM1 activation was visualized in live cells following elevation of endogenous NMN or treatment with a cell-permeant NMN-analog. In neurons, imaging documented SARM1 activation preceded vincristine-induced axonal degeneration by hours. Library screening identified a derivative of nisoldipine as a covalent inhibitor of SARM1 that reacted with Cys311 in its Armadillo-domain and blocked its NMN-activation, protecting axons from degeneration. CryoEM showed that SARM1 was locked into an inactive conformation by the inhibitor, uncovering an unsuspected neuroprotective mechanism of dihydropyridines.

scholarly journals Permeant fluorescent probes visualize the activation of SARM1 and uncover an anti-neurodegenerative drug candidate

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Wan Hua Li ◽  
Ke Huang ◽  
Yang Cai ◽  
Qian Wen Wang ◽  
Wen Jie Zhu ◽  
...  
Keyword(s):  
Fluorescent Probes ◽  
Neurodegenerative Disorders ◽  
Drug Target ◽  
Axonal Degeneration ◽  
Live Cells ◽  
Drug Candidate ◽  
Library Screening ◽  
Inactive Conformation ◽  
A Cell ◽  
Covalent Inhibitor

SARM1 regulates axonal degeneration through its NAD-metabolizing activity and is a drug target for neurodegenerative disorders. We designed and synthesized fluorescent conjugates of styryl derivative with pyridine to serve as substrates of SARM1, which exhibited large red-shifts after conversion. With the conjugates, SARM1 activation was visualized in live cells following elevation of endogenous NMN or treatment with a cell-permeant NMN-analog. In neurons, imaging documented mouse SARM1 activation preceded vincristine-induced axonal degeneration by hours. Library screening identified a derivative of nisoldipine as a covalent inhibitor of SARM1 that reacted with the cysteines, especially Cys311 in its ARM domain and blocked its NMN-activation, protecting axons from degeneration. The Cryo-EM structure showed that SARM1 was locked into an inactive conformation by the inhibitor, uncovering a potential neuroprotective mechanism of dihydropyridines.

scholarly journals A dual-labeling probe to track functional mitochondria–lysosome interactions in live cells

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Qixin Chen ◽  
Hongbao Fang ◽  
Xintian Shao ◽  
Zhiqi Tian ◽  
Shanshan Geng ◽  
...  
Keyword(s):  
Fluorescence Intensity ◽  
Fluorescent Probes ◽  
Living Cells ◽  
Molecular Probe ◽  
Live Cells ◽  
Blue Fluorescence ◽  
Red Fluorescence ◽  
Dynamic Tracking ◽  
A Cell

AbstractMitochondria–lysosome interactions are essential for maintaining intracellular homeostasis. Although various fluorescent probes have been developed to visualize such interactions, they remain unable to label mitochondria and lysosomes simultaneously and dynamically track their interaction. Here, we introduce a cell-permeable, biocompatible, viscosity-responsive, small organic molecular probe, Coupa, to monitor the interaction of mitochondria and lysosomes in living cells. Through a functional fluorescence conversion, Coupa can simultaneously label mitochondria with blue fluorescence and lysosomes with red fluorescence, and the correlation between the red–blue fluorescence intensity indicates the progress of mitochondria–lysosome interplay during mitophagy. Moreover, because its fluorescence is sensitive to viscosity, Coupa allowed us to precisely localize sites of mitochondria–lysosome contact and reveal increases in local viscosity on mitochondria associated with mitochondria–lysosome contact. Thus, our probe represents an attractive tool for the localization and dynamic tracking of functional mitochondria–lysosome interactions in living cells.

scholarly journals Discovery of catalytic-site-fluorescent probes for tracing phosphodiesterase 5 in living cells

RSC Advances ◽  
2021 ◽  
Vol 11 (51) ◽  
pp. 31967-31971
Author(s):  
Meiying Qiu ◽  
Deyan Wu ◽  
Yi-You Huang ◽  
Yue Huang ◽  
Qian Zhou ◽  
...  
Keyword(s):  
Drug Screening ◽  
Fluorescent Probes ◽  
Drug Target ◽  
Living Cells ◽  
Catalytic Site ◽  
Live Cells ◽  
Tissue Slices ◽  
Phosphodiesterase 5

To enhance the understanding of PDE5 as the drug target. Herein, we designed catalytic-site-fluorescent probes that can be applied to PDE5 visualization in live cells and tissue slices, implying the potential in diagnosis and drug screening.

scholarly journals Fluorescent Probes for Live Cell Thiol Detection

Molecules ◽  
2021 ◽  
Vol 26 (12) ◽  
pp. 3575
Author(s):  
Shenggang Wang ◽  
Yue Huang ◽  
Xiangming Guan
Keyword(s):  
Fluorescent Probes ◽  
Biological System ◽  
High Sensitivity ◽  
Intracellular Distribution ◽  
Live Cell ◽  
Live Cells ◽  
High Demand ◽  
Non Invasive

Thiols play vital and irreplaceable roles in the biological system. Abnormality of thiol levels has been linked with various diseases and biological disorders. Thiols are known to distribute unevenly and change dynamically in the biological system. Methods that can determine thiols’ concentration and distribution in live cells are in high demand. In the last two decades, fluorescent probes have emerged as a powerful tool for achieving that goal for the simplicity, high sensitivity, and capability of visualizing the analytes in live cells in a non-invasive way. They also enable the determination of intracellular distribution and dynamitic movement of thiols in the intact native environments. This review focuses on some of the major strategies/mechanisms being used for detecting GSH, Cys/Hcy, and other thiols in live cells via fluorescent probes, and how they are applied at the cellular and subcellular levels. The sensing mechanisms (for GSH and Cys/Hcy) and bio-applications of the probes are illustrated followed by a summary of probes for selectively detecting cellular and subcellular thiols.

scholarly journals N-Acetylglucosamine-1-Phosphate Transferase, WecA, as a Validated Drug Target in Mycobacterium tuberculosis

2017 ◽  
Vol 61 (11) ◽  
Author(s):  
Stanislav Huszár ◽  
Vinayak Singh ◽  
Alica Polčicová ◽  
Peter Baráth ◽  
María Belén Barrio ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Target ◽  
Functional Characterization ◽  
Drug Candidate ◽  
Content Type ◽  
Chemical Libraries ◽  
Whole Cells ◽  
Encoding Gene

ABSTRACT The mycobacterial phosphoglycosyltransferase WecA, which initiates arabinogalactan biosynthesis in Mycobacterium tuberculosis, has been proposed as a target of the caprazamycin derivative CPZEN-45, a preclinical drug candidate for the treatment of tuberculosis. In this report, we describe the functional characterization of mycobacterial WecA and confirm the essentiality of its encoding gene in M. tuberculosis by demonstrating that the transcriptional silencing of wecA is bactericidal in vitro and in macrophages. Silencing wecA also conferred hypersensitivity of M. tuberculosis to the drug tunicamycin, confirming its target selectivity for WecA in whole cells. Simple radiometric assays performed with mycobacterial membranes and commercially available substrates allowed chemical validation of other putative WecA inhibitors and resolved their selectivity toward WecA versus another attractive cell wall target, translocase I, which catalyzes the first membrane step in the biosynthesis of peptidoglycan. These assays and the mutant strain described herein will be useful for identifying potential antitubercular leads by screening chemical libraries for novel WecA inhibitors.

scholarly journals Structural basis for SARM1 inhibition and activation under energetic stress

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Michael Sporny ◽  
Julia Guez-Haddad ◽  
Tami Khazma ◽  
Avraham Yaron ◽  
Moshe Dessau ◽  
...  
Keyword(s):  
Cell Death ◽  
Axonal Degeneration ◽  
Structural Basis ◽  
Allosteric Site ◽  
Inactive Conformation ◽  
Catalytic Domains ◽  
Energetic Stress ◽  
Catalytic Sites ◽  
Peripheral Ring ◽  
Cellular Metabolite

SARM1, an executor of axonal degeneration, displays NADase activity that depletes the key cellular metabolite, NAD+, in response to nerve injury. The basis of SARM1 inhibition and its activation under stress conditions are still unknown. Here, we present cryo-EM maps of SARM1 at 2.9 and 2.7 Å resolutions. These indicate that SARM1 homo-octamer avoids premature activation by assuming a packed conformation, with ordered inner and peripheral rings, that prevents dimerization and activation of the catalytic domains. This inactive conformation is stabilized by binding of SARM1’s own substrate NAD+ in an allosteric location, away from the catalytic sites. This model was validated by mutagenesis of the allosteric site, which led to constitutively active SARM1. We propose that the reduction of cellular NAD+ concentration contributes to the disassembly of SARM1's peripheral ring, which allows formation of active NADase domain dimers, thereby further depleting NAD+ to cause an energetic catastrophe and cell death.

scholarly journals Fluorescent Probes with Unnatural Amino Acids to Monitor Proteasome Activity in Real-Time

2020 ◽  
Author(s):  
Breanna L. Zerfas ◽  
Rachel A. Coleman ◽  
Andres Salazar Chaparro ◽  
Nathaniel J. Macatangay ◽  
Darci Trader
Keyword(s):  
Amino Acids ◽  
Small Molecule ◽  
Fluorescent Probes ◽  
Unnatural Amino Acids ◽  
Cell Types ◽  
Proteasome Activity ◽  
Live Cells ◽  
Fluorescent Substrate ◽  
The Individual ◽  
Small Molecule Modulators

<div> <div> <div> <p>The proteasome is an essential protein complex that, when dysregulated, can result in various diseases in eukaryotic cells. As such, understanding the enzymatic activity of the proteasome and what can alter it is crucial to elucidating its roles in these diseases. This can be done effectively by using activity-based fluorescent substrate probes, of which there are many commercially available that target the individual protease-like subunits in the 20S CP of the proteasome. Unfortunately, these probes have not displayed appropriate characteristics for their use in live cell-based assays. In the work presented here, we have developed a set of probes which have shown improved fluorescence properties and selectivity towards the proteasome compared to other cellular proteases. By including unnatural amino acids, we have found probes which can be utilized in various applications, including monitoring the effects of small molecule stimulators of the proteasome in live cells and comparing the relative proteasome activity across different cancer cell types. In future studies, we expect the fluorescent probes presented here will serve as tools to support the discovery and characterization of small molecule modulators of proteasome activity. </p> </div> </div> </div>

scholarly journals Nanoscale Chromatin Imaging and Analysis (nano-ChIA) platform bridges 4-D chromatin organization with molecular function

2020 ◽  
Author(s):  
Yue Li ◽  
Adam Eshein ◽  
Ranya K.A. Virk ◽  
Aya Eid ◽  
Wenli Wu ◽  
...  
Keyword(s):  
Chromatin Structure ◽  
Electron Tomography ◽  
Population Level ◽  
Average Diameter ◽  
Chromatin Organization ◽  
Live Cells ◽  
Molecular Function ◽  
Label Free ◽  
A Cell ◽  
Chromatin Packing

AbstractIn eukaryotic cells, chromatin structure is linked to transcription processes through the regulation of genome organization. Extending across multiple length-scales - from the nucleosome to higher-order three-dimensional structures - chromatin is a dynamic system which evolves throughout the lifetime of a cell. However, no individual technique can fully elucidate the behavior of chromatin organization and its relation to molecular function at all length- and timescales at both a single-cell and a cell population level. Herein, we present a multi-technique nanoscale Chromatin Imaging and Analysis (nano-ChIA) platform that bridges electron tomography and optical superresolution imaging of chromatin conformation and transcriptional processes, with resolution down to the level of individual nucleosomes, with high-throughput, label-free analysis of chromatin packing and its dynamics in live cells. Utilizing nano-ChIA, we observed that chromatin is localized into spatially separable packing domains, with an average diameter of around 200 nm, sub-Mb genomic size, and an internal fractal structure. The chromatin packing behavior of these domains is directly influenced by active gene transcription. Furthermore, we demonstrated that the chromatin packing domain structure is correlated among progenitor cells and all their progeny, indicating that the organization of chromatin into fractal packing domains is heritable across cell division. Further studies employing the nano-ChIA platform have the potential to provide a more coherent picture of chromatin structure and its relation to molecular function.

scholarly journals Bifunctional fluorescent probes for detection of amyloid aggregates and reactive oxygen species

2018 ◽  
Vol 5 (2) ◽  
pp. 171399 ◽  
Author(s):  
Lisa-Maria Needham ◽  
Judith Weber ◽  
James W. B. Fyfe ◽  
Omaru M. Kabia ◽  
Dung T. Do ◽  
...  
Keyword(s):  
Fluorescent Probes ◽  
Neurodegenerative Disorders ◽  
Oxygen Species ◽  
Fluorescence Measurements ◽  
Amyloid Aggregate ◽  
Amyloid Deposits ◽  
Amyloid Aggregates ◽  
Highly Sensitive ◽  
Key Features ◽  
And Oxidative Stress

Protein aggregation into amyloid deposits and oxidative stress are key features of many neurodegenerative disorders including Parkinson's and Alzheimer's disease. We report here the creation of four highly sensitive bifunctional fluorescent probes, capable of H 2 O 2 and/or amyloid aggregate detection. These bifunctional sensors use a benzothiazole core for amyloid localization and boronic ester oxidation to specifically detect H 2 O 2 . We characterized the optical properties of these probes using both bulk fluorescence measurements and single-aggregate fluorescence imaging, and quantify changes in their fluorescence properties upon addition of amyloid aggregates of α-synuclein and pathophysiological H 2 O 2 concentrations. Our results indicate these new probes will be useful to detect and monitor neurodegenerative disease.

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