Abstract
In recent years, there has been growing interest in SARM1 as a potential breakthrough drug target for treating various pathologies of axon degeneration. SARM1-mediated axon degeneration relies on its TIR domain NADase activity, but recent structural data suggest that the non-catalytic ARM domain could also serve as a pharmacological site as it has an allosteric inhibitory function. Here, we screened for synthetic small molecules that inhibit SARM1, and tested a selected set of these compounds in a DRG axon degeneration assay. Using cryo-EM, we found that one of the newly discovered inhibitors, a calmidazolium designated TK106, not only stabilizes the previously reported inhibited conformation of the octamer, but also a meta-stable structure: a duplex of octamers (16 protomers), which we have now determined to 4.0 Å resolution. In the duplex, each ARM domain protomer is engaged in lateral interactions with neighboring protomers, and is further stabilized by contralateral contacts with the opposing octamer ring. Mutagenesis of the duplex contact sites leads to a moderate increase in SARM1 activation in cultured cells. Based on our data we propose that the duplex assembly constitutes an additional auto-inhibition mechanism that tightly prevents pre-mature activation and axon degeneration.
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Change history
24 February 2023
author name Avraham Yaron was missed during proof stage and now it has been updated
23 February 2023
A Correction to this paper has been published: https://doi.org/10.1007/s00018-023-04701-2
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Acknowledgements
We acknowledge the European Synchrotron Radiation Facility for provision of beam time on CM01 and thank the staff of beamline CM01 of ESRF and members of the Opatowsky lab for technical assistance. We thank Gershon Kunin for IT management. The Israel National Center for Personalized Medicine is supported by a research grant from the Nancy and Stephen Grand. This work was supported by funds from ISF grants no. 1425/15 and 909/19, BSF grant no. 2019150, and ICRF grant 2022-2023 to Y.O. Y.O. is a Katzir Professorial Chair of Biophysics, and A.Y. is an incumbent of the Jack and Simon Djanogly Professorial Chair in Biochemistry.
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This work was supported by funds from ISF grants no. 1425/15 and 909/19 and BSF grant no. 2019150 to Y.O.
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Tami Khazma and Yarden Golan-Vaishenker Co-first authors.
Yarden Opatowsky Lead contact.
- Yarden Opatowsky
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YO: Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing—original draft, Project administration, Writing—review and editing. Lead contact and corresponding author. TK: Data curation, Formal analysis, Investigation, Methodology. First author: YG-V: Formal analysis, Investigation, Methodology. Co-first author: JG-H: Data curation, Formal analysis, Supervision, Investigation, Methodology, Project administration, Writing—review and editing. AY: Contribution Conceptualization, Supervision, Methodology, Writing—review and editing. Co-corresponding author with YO. Atira Grossman: Data curation, Methodology, Investigation, Formal analysis, RS: Methodology. MW: Data curation, Methodology, AP: Data curation, Methodology, RZ: Contribution Data curation, Formal analysis, Methodology. MH: Data curation, Formal analysis, Validation, Methodology, Writing—review and editing. Co-corresponding author with YO.
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18_2022_4641_MOESM1_ESM.docx
Supplementary file1 (DOCX 498 KB) Figure 1 supplement 1. Structure-based sequence alignment of the SARM1 of human, mouse, zebrafish, and the C. elegans ortholog TIR-1. Color-coded highlights and Uniprot protein accession numbers are listed below.
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Supplementary file2 (DOCX 54 KB) Figure 3 supplement 1. A) Chemical structure of 2 additional hSARM1 inhibitory compounds. B) Determination of IC50 values, as in figure 3.
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Supplementary file3 (DOCX 130 KB) Figure 5 supplement 1. A) Representative cryo-EM micrograph of TK106 supplemented hSARM1. B) 2D class averages of the entire dataset. C) Flowchart of the cryo-EM processing steps of the entire dataset.
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Supplementary file4 (DOCX 196 KB) Figure 5 supplement 2. Resolution, angular distribution, and B-factor estimations of the cryo-EM maps of TK106 supplemented hSARM1 duplex and monoplex.
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Supplementary file5 (DOCX 174 KB) Figure 5 supplement 3. Comparison of available cryo-EM map densities of hSARM1, set to similar contour level at the ARM domain’s concave surface [24-27]. Dashed red oval marks the site of interest, highlighting the difference in map density between apo structures (empty site) and NAD+ or TK106 supplemented structures (occupied sites).
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Supplementary file6 (DOCX 177 KB) Figure 6 supplement 1. Extended representation of Fig. 6A. Protective and toxic effects of hSARM1 inhibitors over axon degeneration in mouse DRG explants.
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Khazma, T., Golan-Vaishenker, Y., Guez-Haddad, J. et al. A duplex structure of SARM1 octamers stabilized by a new inhibitor. Cell. Mol. Life Sci. 80, 16 (2023). https://doi.org/10.1007/s00018-022-04641-3
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DOI: https://doi.org/10.1007/s00018-022-04641-3