Permeant fluorescent probes visualize the activation of SARM1 and uncover an anti-neurodegenerative drug candidate
- State Key Laboratory of Chemical Oncogenomics, Key Laboratory of Chemical Genomics, Peking University Shenzhen Graduate School, China
- Ciechanover Institute of Precision and Regenerative Medicine, School of Life and Health Sciences, The Chinese University of Hong Kong, China
- Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, China
- Department of Biology, Southern University of Science and Technology, China
- Kobilka Institute of Innovative Drug Discovery, School of Life and Health Sciences, The Chinese University of Hong Kong, China
- Shenzhen-Hong Kong Institute of Brain Science-Shenzhen Fundamental Research Institutions, China
Figures
Figure 1 with 13 supplements
Design and characterization of PC probes.
(A) Strategy of fluorescent imaging of the activated SARM1. (B) Designing based on pyridine and styryl derivatives with a donor-π-acceptor framework. (C) Structure of PC6. (D) The kinetics of the …
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Figure 1—source data 1
Source data in excel for Figure 1D-K.
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Figure 1—source data 2
Source data in excel for Figure 1—figure supplements 11C, 12 and Figure 1—figure supplement 13.
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Figure 1—figure supplement 1
Synthetic scheme of PC1–PC9. PC = pyridine conjugate.
Figure 1—figure supplement 2
1H NMR and 13C NMR spectra of PC1 in DMSO-d6.
Figure 1—figure supplement 3
1H NMR and 13C NMR spectra of PC2 in CDCl3.
Figure 1—figure supplement 4
1H NMR and 13C NMR spectra of PC3 in CDCl3.
Figure 1—figure supplement 5
1H NMR and 13C NMR spectra of PC4 in CDCl3.
Figure 1—figure supplement 6
1H NMR and 13C NMR spectra of PC5 in DMSO-d6.
Figure 1—figure supplement 7
1H NMR and 13C NMR spectra of PC6 in CDCl3.
Figure 1—figure supplement 8
1H NMR and 13C NMR spectra of PC7 in CDCl3.
Figure 1—figure supplement 9
1H NMR and 13C NMR spectra of PC8 in CDCl3.
Figure 1—figure supplement 10
1H NMR and 13C NMR spectra of PC9 in CDCl3.
Figure 1—figure supplement 11
Structures of PC1–9 and activity screening.
(A) Chemical structures of PC1–9. (B) Quantification of SARM1-dN. SARM1-dN was pulled down by the BC2 nanobody (Zhao et al., 2019) conjugated beads, which efficiency was close to 100%. The purified …
Figure 1—figure supplement 12
UV-vis absorption spectra scanning of the reactants.
The reactants of 50 μM PCs and 100 μM NAD during the 50 min reactions catalyzed by the activated SARM1.
Figure 1—figure supplement 13
Fluorescence spectra of the reactants.
The reactants of 50 μM PCs and 100 μM NAD during the 50 min reactions catalyzed by the activated SARM1.
Figure 2 with 1 supplement
Live-cell imaging of SARM1 activation.
(A) Western blot of the overexpression of SARM1 and inactive mutant, E642A in HEK293 cells. (B) Confocal fluorescence images of cells in (A) after incubation with PC6 in presence or absence of …
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Figure 2—source data 1
Source data in excel for Figure 2E and G.
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Figure 2—figure supplement 1
Expression level of SARM1 for Figure 2E and the activities of CD38.
(A) Western blots analysis of the expression of SARM1 in the inducible cell lines with or without treatment of 0.5 μg/mL Dox and 100 μM CZ-48 for the indicated time. (B) The HEK293T cells …
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Figure 2—figure supplement 1—source data 1
Source data in excel for Figure 2—figure supplement 1B and C.
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Figure 3 with 1 supplement
SARM1 activation in mouse DRG upon vincristine treatment.
(A, C) Confocal imaging of SARM1 activation in DRG neuronal axons. The neurons were infected with virus expressing TdTomato to provide easy imaging of the axons. Cells were additionally transfected …
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Figure 3—source data 1
Source data in excel for Figure 3B, D-F.
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Figure 3—figure supplement 1
Integrity of axons visualized by the TdTomato fluorescence.
The DRG neurons, on div6, were infected with the lentivirus expressing TdTomato and scramble shRNA (A) or Sarm1-specific shRNA (B) and three days later, incubated with 50 nM VCR or 200 μM CZ-48 with …
Figure 4 with 1 supplement
Identification of dHNN as an inhibitor of SARM1.
(A) Flowchart of the PC6-based high-throughput screening. (B) Inhibitory effects of the 2015 compounds (50 μM) from an approval drug library. The activity of drug-treated SARM1-dN was determined …
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Figure 4—source data 1
Source data in excel for Figure 4B-F.
- https://cdn.elifesciences.org/articles/67381/elife-67381-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
Inhibitory mechanism of dHNN against SARM1.
(A) The inhibition curves of nicotinamide to SARM1-dN measured with PC6-based reaction. (B) HPLC analysis of NSDP after treating by UV at 275 nm for 20 min. Black: standard of NSDP; purple: NSDP …
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Figure 4—figure supplement 1—source data 1
Source data in excel for Figure 4—figure supplement 1A-D.
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Figure 5 with 4 supplements
dHNN reduces AxD by inhibiting SARM1 through covalent modification of the cysteines.
(A) Inhibition of SARM1-dN and SAM-TIR by dHNN in vitro. See 'Materials and methods'. (B) Inhibition of SARM1-dN and SAM-TIR by dHNN in cellulo. See 'Materials and methods'. (C) MS of SARM1-dN …
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Figure 5—source data 1
Source data in excel for Figure 5A, B, D, H and J.
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Figure 5—figure supplement 1
dHNN modifications on the peptides of SARM1 or nonspecific proteins analyzed by LC-MS/MS.
(A-B) Quantification of the intensity of the dHNN-modified peptides covering the indicated cysteine residues of SARM1 by LC-MS/MS following the treatment by 5 μM (A) and 50 μM (B) dHNN, and …
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Figure 5—figure supplement 1—source data 1
Source data in excel of Figure 5—figure supplement 1A–C.
- https://cdn.elifesciences.org/articles/67381/elife-67381-fig5-figsupp1-data1-v2.xlsx
Figure 5—figure supplement 2
Data processing procedure for the SARM1-dHNN structure.
Figure 5—figure supplement 3
Structure of SARM1 was stabled in inactive form after dHNN treatment.
(A-B) Representative 2D class averages of SARM1-dN in the absence (A) or presence (B) of 50 μM dHNN in 100 mM Tris, 150 mM NaCl, and 1 mM EDTA at pH 8.0. (C) Local resolution estimation calculated …
Figure 5—figure supplement 4
dHNN attenuated the axotomy-induced AxD.
DRG neurons were pre-treated with 3 μM dHNN for 0.5 hr and axotomy performed to induce AxD. Images were captured at 0, 24, 48 hr (A) and the AxD index was analyzed by ImageJ (B). AxD = axon …
Author response image 1
Additional files
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Supplementary file 1
Refinement statistics for the SAMR1-dHNN structure.
- https://cdn.elifesciences.org/articles/67381/elife-67381-supp1-v2.xlsx
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Transparent reporting form
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