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

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.

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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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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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