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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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.
Erzberger, J. P. & Berger, J. M. Evolutionary relationships and structural mechanisms of AAA+ proteins. Annu. Rev. Biophys. Biomol. Struct. 35, 93–114 (2006).
Lampson, M. A. & Kapoor, T. M. Unraveling cell division mechanisms with small-molecule inhibitors. Nat. Chem. Biol. 2, 19–27 (2006).
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).
Firestone, A. J. et al. Small-molecule inhibitors of the AAA+ ATPase motor cytoplasmic dynein. Nature 484, 125–129 (2012).
Kawashima, S. A. et al. Potent, reversible, and specific chemical inhibitors of eukaryotic ribosome biogenesis. Cell 167, 512–524.e14 (2016).
Magnaghi, P. et al. Covalent and allosteric inhibitors of the ATPase VCP/p97 induce cancer cell death. Nat. Chem. Biol. 9, 548–556 (2013).
Roll-Mecak, A. & McNally, F. J. Microtubule-severing enzymes. Curr. Opin. Cell Biol. 22, 96–103 (2010).
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).
McNally, F. J. & Vale, R. D. Identification of katanin, an ATPase that severs and disassembles stable microtubules. Cell 75, 419–429 (1993).
Sharp, D. J. & Ross, J. L. Microtubule-severing enzymes at the cutting edge. J. Cell. Sci. 125, 2561–2569 (2012).
Allison, R. et al. An ESCRT-spastin interaction promotes fission of recycling tubules from the endosome. J. Cell. Biol. 202, 527–543 (2013).
Allison, R. et al. Defects in ER-endosome contacts impact lysosome function in hereditary spastic paraplegia. J. Cell. Biol. 216, 1337–1355 (2017).
Vietri, M. et al. Spastin and ESCRT-III coordinate mitotic spindle disassembly and nuclear envelope sealing. Nature 522, 231–235 (2015).
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).
Banerjee, S. et al. 2.3 Å resolution cryo-EM structure of human p97 and mechanism of allosteric inhibition. Science 351, 871–875 (2016).
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).
Beyer, A. Sequence analysis of the AAA protein family. Protein Sci. 6, 2043–2058 (1997).
Puchades, C. et al. Structure of the mitochondrial inner membrane AAA+ protease YME1 gives insight into substrate processing. Science 358, eaao0464 (2017).
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).
Han, T. et al. Anticancer sulfonamides target splicing by inducing RBM39 degradation via recruitment to DCAF15. Science 356, eaal3755 (2017).
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).
Kapoor, T. M. & Miller, R. M. Leveraging chemotype-specific resistance for drug target identification and chemical biology. Trends Pharmacol. Sci. 38, 1100–1109 (2017).
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).
Roll-Mecak, A. & Vale, R. D. Structural basis of microtubule severing by the hereditary spastic paraplegia protein spastin. Nature 451, 363–367 (2008).
Rouiller, I. et al. Conformational changes of the multifunction p97 AAA ATPase during its ATPase cycle. Nat. Struct. Biol. 9, 950–957 (2002).
Ye, Q. et al. TRIP13 is a protein-remodeling AAA+ ATPase that catalyzes MAD2 conformation switching. eLife 4, e07367 (2015).
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).
Karlberg, T. et al. Crystal structure of human fidgetin-like protein 1 in complex with ADP. https://doi.org/10.2210/pdb3D8B/pdb (2008).
Whitehead, E., Heald, R. & Wilbur, J.D. N-terminal phosphorylation of p60 katanin directly regulates microtubule severing. J. Mol. Biol. 425, 214–221 (2013).
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).
Amaro, R. E. et al. Ensemble docking in drug discovery. Biophys. J. 114, 2271–2278 (2018).
Statsuk, A. V. et al. Tuning a three-component reaction for trapping kinase substrate complexes. J. Am. Chem. Soc. 130, 17568–17574 (2008).
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).
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).
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).
Schellhaus, A. K., De Magistris, P. & Antonin, W. Nuclear reformation at the end of mitosis. J. Mol. Biol. 428, 1962–1985 (2016).
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).
Olmos, Y., Hodgson, L., Mantell, J., Verkade, P. & Carlton, J. G. ESCRT-III controls nuclear envelope reformation. Nature 522, 236–239 (2015).
Blackstone, C., O’Kane, C. J. & Reid, E. Hereditary spastic paraplegias: membrane traffic and the motor pathway. Nat. Rev. Neurosci. 12, 31–42 (2011).
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).
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).
Uphoff, C. C. & Drexler, H. G. Detection of mycoplasma contaminations. Methods Mol. Biol. 946, 1–13 (2013).
Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).
Feng, B. Y. & Shoichet, B. K. A detergent-based assay for the detection of promiscuous inhibitors. Nat. Protoc. 1, 550–553 (2006).
Ziółkowska, N. E. & Roll-Mecak, A. In vitro microtubule severing assays. Methods Mol. Biol. 1046, 323–334 (2013).
Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).
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).
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.
The authors declare no competing interests.
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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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