# Control of Executioner Caspases with an allosteric switch - Supplement

> **NIH NIH R01** · UNIVERSITY OF MASSACHUSETTS AMHERST · 2020 · $124,131

## Abstract

PROJECT SUMMARY
 Caspases are cysteine proteases that control apoptotic cell death. If caspases are activated, cancer
cells die; conversely, inhibiting caspases can prevent cell death in diseases such as heart attack and stroke.
Thus, there has been significant interest in caspases as drug targets. This interest heightened further when
caspase-6 was discovered to play a central role in neurodegeneration. Unfortunately, to date, no caspase-
directed therapies are on the market, primarily because work has focused on the active site, which is the
most overlapping and conserved region of the family. It is becoming increasingly clear that each caspase is
regulated in a unique and nuanced manner, so the only hope for achieving caspase-specific inhibition is by
harnessing their regulation, which is usually mediated at allosteric sites and exosites. In order to target a
specific caspase, group of caspases, or subset of caspase substrates, it is essential to understand the dif-
ferences between individual caspases and the similarities within caspase subgroups. Thus, our long-term
project goal has been to define and exploit unique regulatory features for each of the apoptotic caspases.
 By identifying allosteric sites and exosites, we have observed and described four major mechanistic
classes of exosite and allosteric regulation. The first, shared by many disparate regulators, is the indirect
disruption of the loops that cooperatively form the substrate-binging groove and impact catalysis. Based on
our studies of caspase regulation via loop disruption, we developed an allosteric inhibitor that is more potent
than any reported and is also by far the most selective, preferring caspase-6 by 500-fold over all other
caspases. This selectivity is achievable because this new allosteric site is present exclusively in caspase-6.
Given this success, we aim to investigate the remaining three classes of allosteric regulation. In Aim 1, we
focus on class II, identifying exosites on caspase-6 and its substrates. This concerted analysis is possible
for the first time due to our development of a hybrid caspase with the active site specificity of caspase-6 but
the exosites of caspase-7. We aim to block particular exosites and explore the impact on a proteome-wide
basis. We anticipate that this approach will enable the development of new inhibitors that block cleavage of
disease-causing caspase-6 substrates like DJ-1,Tau or huntingtin in Alzheimer and Huntington but not other
substrates. In Aim 2, we focus on class III, native small molecule binding. Our recent discovery that ATP
binds to an orphan allosteric cavity and new methods will allow us to identify both covalent and non-
covalent native ligands that regulate caspase-6 from this site. This goal is significant as it will provide need-
ed insights into the intersection between caspases and metabolism. In Aim 3, we focus on class IV, which
impact the folded state. We interrogate a caspase-9 site that when phosphorylate...

## Key facts

- **NIH application ID:** 10134722
- **Project number:** 3R01GM080532-11S1
- **Recipient organization:** UNIVERSITY OF MASSACHUSETTS AMHERST
- **Principal Investigator:** Jeanne Ann Hardy
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $124,131
- **Award type:** 3
- **Project period:** 2008-07-01 → 2022-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10134722

## Citation

> US National Institutes of Health, RePORTER application 10134722, Control of Executioner Caspases with an allosteric switch - Supplement (3R01GM080532-11S1). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10134722. Licensed CC0.

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