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

Nat Chem Biol. 2019 May;15(5):444-452. doi: 10.1038/s41589-019-0225-6. Epub 2019 Feb 18.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Catalytic Domain / drug effects
  • Catalytic Domain / genetics
  • Drug Design*
  • Enzyme Inhibitors / chemical synthesis
  • Enzyme Inhibitors / chemistry
  • Enzyme Inhibitors / pharmacology*
  • Heterocyclic Compounds / chemical synthesis
  • Heterocyclic Compounds / chemistry
  • Heterocyclic Compounds / pharmacology*
  • Models, Molecular
  • Molecular Structure
  • Mutation*
  • Spastin / antagonists & inhibitors*
  • Spastin / genetics*
  • Spastin / metabolism

Substances

  • Enzyme Inhibitors
  • Heterocyclic Compounds
  • Spastin
  • SPAST protein, human