Using Small Molecule Tools to Study ATPase Mechanoenzymes

Author

Natalie Jones

Date of Award

2024

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Kapoor Laboratory

Abstract

The dysregulation of AAA (ATPases associated with diverse cellular activities) mechanoenzymes has been linked to diseases, and chemical inhibitors and activators can be powerful tools to probe mechanisms and test therapeutic hypotheses. However,the structural conservation across the AAA protein family makes designing selective chemical inhibitors challenging. Additionally, unlike chemical inhibitors that can stabilizea single conformational state of an enzyme, activator binding must be permissive to different conformational states needed for enzyme function, and we do not know how AAA proteins can be activated by small molecules. My thesis work covers the development of a chemical genetics approach to inhibit AAA proteins, starting from atriazolopyridine-based fragment that binds the AAA domain of the microtubule severing protein katanin, and the identification of a druggable site for chemical activators in valosin-containing protein (VCP)/p97, a AAA unfoldase whose loss of function has been linked to protein aggregation-based disorders.For the chemical genetics approach, we designed ASPIRe-1 (Allele-Specific, Proximity-Induced Reactivity-based inhibitor-1), a cell-permeable compound that selectively inhibits katanin with an engineered cysteine mutation. Only in cells expressing mutant katanin,ASPIRe-1 treatment increases the accumulation of CAMSAP2 at microtubule minus-ends, confirming specific on-target cellular activity. Importantly, ASPIRe-1 also selectively targets engineered cysteine mutants of VPS4B, FIGL1, and VCP.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

Recommended Citation

Jones, Natalie, "Using Small Molecule Tools to Study ATPase Mechanoenzymes" (2024). Student Theses and Dissertations. 760.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/760