Using Small Molecule Tools to Study ATPase Mechanoenzymes

<p>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. For the small molecule activator part of my thesis work, from a screen optimized to identify compounds that stimulate VCP ATPase activity as little as 20%, I discovered activators that represent five chemotypes. Minimal modification of the most potent hit, an isoindoline-based compound, resulted in VCP Activator 1 (VA1), a compound that dose-dependently stimulates VCP ATPase activity up to ~3-fold. Cryo-EM studies resulted instructures (~2.9-3.5 Å-resolution) of VCP in apo and ADP-bound states, and reveal VA1 binding an allosteric pocket near the C-terminus in both states. Finally, I engineered mutations in the VA1 binding site that confer resistance to VA1, and furthermore, modulate VCP ATPase activity to a similar level as VA1-mediated activation. Together, these findings suggest a chemical genetics approach to decipher AAA protein cellular functions and uncover a druggable allosteric site that can also be occupied by VCP's C-terminal tail to control activity.</p>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Thesis

Year dc:date.available

2024

Author dc:creator

• Jones, Natalie

Contributors dc:contributor

• Tarun Kapoor

• AAA proteins
• katanin
• ASPIRe-1
• VCP
• allosteric activation
• chemical genetics
• Life Sciences

Repository record dc:identifier

https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/760

OAI identifier oai:identifier

oai:digitalcommons.rockefeller.edu:student_theses_and_dissertations-1764