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Designing a chemical inhibitor for the AAA protein spastin using active site mutations

Nature Chemical Biology volume 15, pages 444–452 (2019)Cite this article

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Abstract

Spastin is a microtubule-severing AAA (ATPases associated with diverse cellular activities) protein needed for cell division and intracellular vesicle transport. Currently, we lack chemical inhibitors to probe spastin function in such dynamic cellular processes. To design a chemical inhibitor of spastin, we tested selected heterocyclic scaffolds against wild-type protein and constructs with engineered mutations in the nucleotide-binding site that do not substantially disrupt ATPase activity. These data, along with computational docking, guided improvements in compound potency and selectivity and led to spastazoline, a pyrazolyl-pyrrolopyrimidine-based cell-permeable probe for spastin. These studies also identified spastazoline-resistance-conferring point mutations in spastin. Spastazoline, along with the matched inhibitor-sensitive and inhibitor-resistant cell lines we generated, were used in parallel experiments to dissect spastin-specific phenotypes in dividing cells. Together, our findings suggest how chemical probes for AAA proteins, along with inhibitor resistance-conferring mutations, can be designed and used to dissect dynamic cellular processes.

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Fig. 1: Identifying and characterizing ‘variability hot-spot’ mutations in spastin.
Fig. 2: Identifying chemical scaffolds that inhibit Dm-spastin.
Fig. 3: Testing mutant constructs to build a model for the chemical inhibition of spastin.
Fig. 4: Developing a potent and selective inhibitor of human spastin.
Fig. 5: Using spastazoline and a cognate resistance-conferring mutation to probe spastin function in cell division.
Fig. 6: Engineering ‘silent’ mutations to generate models for inhibitor–target interactions and to identify inhibitor resistance-conferring mutations.

Data availability

All data generated or analyzed during this study are included in this published article (and its Supplementary Information files) or are available from the corresponding author on reasonable request.

References

  1. Erzberger, J. P. & Berger, J. M. Evolutionary relationships and structural mechanisms of AAA+ proteins. Annu. Rev. Biophys. Biomol. Struct. 35, 93–114 (2006).

    Article  CAS  Google Scholar 

  2. Lampson, M. A. & Kapoor, T. M. Unraveling cell division mechanisms with small-molecule inhibitors. Nat. Chem. Biol. 2, 19–27 (2006).

    Article  CAS  Google Scholar 

  3. Chou, T. F. et al. Reversible inhibitor of p97, DBeQ, impairs both ubiquitin-dependent and autophagic protein clearance pathways. Proc. Natl Acad. Sci. USA 108, 4834–4839 (2011).

    Article  CAS  Google Scholar 

  4. Firestone, A. J. et al. Small-molecule inhibitors of the AAA+ ATPase motor cytoplasmic dynein. Nature 484, 125–129 (2012).

    Article  CAS  Google Scholar 

  5. Kawashima, S. A. et al. Potent, reversible, and specific chemical inhibitors of eukaryotic ribosome biogenesis. Cell 167, 512–524.e14 (2016).

    Article  CAS  Google Scholar 

  6. Magnaghi, P. et al. Covalent and allosteric inhibitors of the ATPase VCP/p97 induce cancer cell death. Nat. Chem. Biol. 9, 548–556 (2013).

    Article  CAS  Google Scholar 

  7. Roll-Mecak, A. & McNally, F. J. Microtubule-severing enzymes. Curr. Opin. Cell Biol. 22, 96–103 (2010).

    Article  CAS  Google Scholar 

  8. Roll-Mecak, A. & Vale, R. D. The Drosophila homologue of the hereditary spastic paraplegia protein, spastin, severs and disassembles microtubules. Curr. Biol. 15, 650–655 (2005).

    Article  CAS  Google Scholar 

  9. McNally, F. J. & Vale, R. D. Identification of katanin, an ATPase that severs and disassembles stable microtubules. Cell 75, 419–429 (1993).

    Article  CAS  Google Scholar 

  10. Sharp, D. J. & Ross, J. L. Microtubule-severing enzymes at the cutting edge. J. Cell. Sci. 125, 2561–2569 (2012).

    Article  CAS  Google Scholar 

  11. Allison, R. et al. An ESCRT-spastin interaction promotes fission of recycling tubules from the endosome. J. Cell. Biol. 202, 527–543 (2013).

    Article  CAS  Google Scholar 

  12. Allison, R. et al. Defects in ER-endosome contacts impact lysosome function in hereditary spastic paraplegia. J. Cell. Biol. 216, 1337–1355 (2017).

    Article  CAS  Google Scholar 

  13. Vietri, M. et al. Spastin and ESCRT-III coordinate mitotic spindle disassembly and nuclear envelope sealing. Nature 522, 231–235 (2015).

    Article  CAS  Google Scholar 

  14. Kuo, T. C. et al. Purine-type compounds induce microtubule fragmentation and lung cancer cell death through interaction with katanin. J. Med. Chem. 59, 8521–8534 (2016).

    Article  CAS  Google Scholar 

  15. Banerjee, S. et al. 2.3 Å resolution cryo-EM structure of human p97 and mechanism of allosteric inhibition. Science 351, 871–875 (2016).

    Article  CAS  Google Scholar 

  16. Pöhler, R. et al. A non-competitive inhibitor of VCP/p97 and VPS4 reveals conserved allosteric circuits in type I and II AAA ATPases. Angew. Chem. Int. Ed. Engl. 57, 1576–1580 (2018).

    Article  Google Scholar 

  17. Beyer, A. Sequence analysis of the AAA protein family. Protein Sci. 6, 2043–2058 (1997).

    Article  CAS  Google Scholar 

  18. Puchades, C. et al. Structure of the mitochondrial inner membrane AAA+ protease YME1 gives insight into substrate processing. Science 358, eaao0464 (2017).

    Article  Google Scholar 

  19. Davis, A. M., Teague, S. J. & Kleywegt, G. J. Application and limitations of X-ray crystallographic data in structure-based ligand and drug design. Angew. Chem. Int. Ed. Engl. 42, 2718–2736 (2003).

    Article  CAS  Google Scholar 

  20. Han, T. et al. Anticancer sulfonamides target splicing by inducing RBM39 degradation via recruitment to DCAF15. Science 356, eaal3755 (2017).

    Article  Google Scholar 

  21. Wacker, S. A., Houghtaling, B. R., Elemento, O. & Kapoor, T. M. Using transcriptome sequencing to identify mechanisms of drug action and resistance. Nat. Chem. Biol. 8, 235–237 (2012).

    Article  CAS  Google Scholar 

  22. Kapoor, T. M. & Miller, R. M. Leveraging chemotype-specific resistance for drug target identification and chemical biology. Trends Pharmacol. Sci. 38, 1100–1109 (2017).

    Article  CAS  Google Scholar 

  23. Anderson, D. J. et al. Targeting the AAA ATPase p97 as an approach to treat cancer through disruption of protein homeostasis. Cancer Cell 28, 653–665 (2015).

    Article  CAS  Google Scholar 

  24. Roll-Mecak, A. & Vale, R. D. Structural basis of microtubule severing by the hereditary spastic paraplegia protein spastin. Nature 451, 363–367 (2008).

    Article  CAS  Google Scholar 

  25. Rouiller, I. et al. Conformational changes of the multifunction p97 AAA ATPase during its ATPase cycle. Nat. Struct. Biol. 9, 950–957 (2002).

    Article  CAS  Google Scholar 

  26. Ye, Q. et al. TRIP13 is a protein-remodeling AAA+ ATPase that catalyzes MAD2 conformation switching. eLife 4, e07367 (2015).

    Article  Google Scholar 

  27. Loughlin, R., Wilbur, J. D., McNally, F. J., Nédélec, F. J. & Heald, R. Katanin contributes to interspecies spindle length scaling in Xenopus. Cell 147, 1397–1407 (2011).

    Article  CAS  Google Scholar 

  28. Karlberg, T. et al. Crystal structure of human fidgetin-like protein 1 in complex with ADP. https://doi.org/10.2210/pdb3D8B/pdb (2008).

  29. Whitehead, E., Heald, R. & Wilbur, J.D. N-terminal phosphorylation of p60 katanin directly regulates microtubule severing. J. Mol. Biol. 425, 214–221 (2013).

    Article  CAS  Google Scholar 

  30. Taylor, J. L., White, S. R., Lauring, B. & Kull, F. J. Crystal structure of the human spastin AAA domain. J. Struct. Biol. 179, 133–137 (2012).

    Article  CAS  Google Scholar 

  31. Amaro, R. E. et al. Ensemble docking in drug discovery. Biophys. J. 114, 2271–2278 (2018).

    Article  CAS  Google Scholar 

  32. Statsuk, A. V. et al. Tuning a three-component reaction for trapping kinase substrate complexes. J. Am. Chem. Soc. 130, 17568–17574 (2008).

    Article  CAS  Google Scholar 

  33. Claudiani, P., Riano, E., Errico, A., Andolfi, G. & Rugarli, E. I. Spastin subcellular localization is regulated through usage of different translation start sites and active export from the nucleus. Exp. Cell Res. 309, 358–369 (2005).

    Article  CAS  Google Scholar 

  34. Connell, J. W., Lindon, C., Luzio, J. P. & Reid, E. Spastin couples microtubule severing to membrane traffic in completion of cytokinesis and secretion. Traffic 10, 42–56 (2009).

    Article  CAS  Google Scholar 

  35. Mierzwa, B. E. et al. Dynamic subunit turnover in ESCRT-III assemblies is regulated by Vps4 to mediate membrane remodelling during cytokinesis. Nat. Cell Biol. 19, 787–798 (2017).

    Article  CAS  Google Scholar 

  36. Schellhaus, A. K., De Magistris, P. & Antonin, W. Nuclear reformation at the end of mitosis. J. Mol. Biol. 428, 1962–1985 (2016).

    Article  CAS  Google Scholar 

  37. Yang, D. et al. Structural basis for midbody targeting of spastin by the ESCRT-III protein CHMP1B. Nat. Struct. Mol. Biol. 15, 1278–1286 (2008).

    Article  CAS  Google Scholar 

  38. Olmos, Y., Hodgson, L., Mantell, J., Verkade, P. & Carlton, J. G. ESCRT-III controls nuclear envelope reformation. Nature 522, 236–239 (2015).

    Article  CAS  Google Scholar 

  39. Blackstone, C., O’Kane, C. J. & Reid, E. Hereditary spastic paraplegias: membrane traffic and the motor pathway. Nat. Rev. Neurosci. 12, 31–42 (2011).

    Article  CAS  Google Scholar 

  40. Zempel, H. et al. Amyloid-β oligomers induce synaptic damage via Tau-dependent microtubule severing by TTLL6 and spastin. EMBO J. 32, 2920–2937 (2013).

    Article  CAS  Google Scholar 

  41. Mancuso, G. & Rugarli, E. I. A cryptic promoter in the first exon of the SPG4 gene directs the synthesis of the 60-kDa spastin isoform. BMC Biol. 6, 31 (2008).

    Article  Google Scholar 

  42. Uphoff, C. C. & Drexler, H. G. Detection of mycoplasma contaminations. Methods Mol. Biol. 946, 1–13 (2013).

    Article  CAS  Google Scholar 

  43. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

    Article  CAS  Google Scholar 

  44. Feng, B. Y. & Shoichet, B. K. A detergent-based assay for the detection of promiscuous inhibitors. Nat. Protoc. 1, 550–553 (2006).

    Article  CAS  Google Scholar 

  45. Ziółkowska, N. E. & Roll-Mecak, A. In vitro microtubule severing assays. Methods Mol. Biol. 1046, 323–334 (2013).

    Article  Google Scholar 

  46. Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  Google Scholar 

  47. Friesner, R. A. et al. Extra precision glide: docking and scoring incorporating a model of hydrophobic enclosure for protein-ligand complexes. J. Med. Chem. 49, 6177–6196 (2006).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank J. Steinman and P. Verma for help with AAA protein purification, and M. Grasso for the purification of human VPS4B. We thank F. Glickman and C. Adura of the Rockefeller University High-Throughput and Spectroscopy Resource Center for assistance with assay development and A. North of the Rockefeller Bioimaging Resource Center. We are especially grateful to L. Kapitein, K. Jansen and W. Nijenhuis (Utrecht University) for testing our spastin inhibitors in cellular assays. We are also grateful to R. Heald (University of California, Berkeley) and C. Campsteijn (Oslo University Hospital) for plasmids and to A. Roll-Mecak (NIH) for a plasmid and a protein sample for the initial assay validation. T.C. was supported by the EMBO Long-Term Fellowship for post-doctoral studies and by the Kestenbaum award for research in neurodegenerative diseases. M.E.K. acknowledges support from an NIH training grant (GM066699). T.M.K. is grateful to the NIH (GM98578) for supporting this research.

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Authors and Affiliations

  1. Laboratory of Chemistry and Cell Biology, The Rockefeller University, New York, NY, USA

    Tommaso Cupido, Rudolf Pisa, Megan E. Kelley & Tarun M. Kapoor

  2. Tri-Institutional PhD program in Chemical Biology, The Rockefeller University, New York, NY, USA

    Rudolf Pisa

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  1. Tommaso Cupido
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Contributions

T.C., R.P., and T.M.K. conceived the project and designed experiments. T.C. and R.P. synthesized compounds, performed assays, and analyzed data. M.E.K. performed western blot analyses and assisted with cell imaging experiments and data analysis. T.M.K. supervised the research. T.C. and T.M.K. wrote the manuscript with input from all authors.

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Correspondence to Tarun M. Kapoor.

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Cupido, T., Pisa, R., Kelley, M.E. et al. Designing a chemical inhibitor for the AAA protein spastin using active site mutations. Nat Chem Biol 15, 444–452 (2019). https://doi.org/10.1038/s41589-019-0225-6

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