Potential path for countering oxidative stress in a range of diseases

Scientists at Scripps Research have made a surprising discovery in their mission to understand how cells stay healthy, uncovering an important connection between a cell’s sugar metabolism and its antioxidant response, one of the cell’s key mechanisms to protect itself from oxidative stress and other damaging agents.

Targeting this link, the researchers identified a small molecule capable of activating the antioxidant response, a potential therapy for a range of diseases involving oxidative stress such as chronic kidney disease, neurodegeneration, and autoimmune disease. They reported their discovery recently in Nature.

“This study demonstrates that there’s clear cross-talk between central carbon metabolism and the antioxidant response and opens the door to develop these findings to target a range of diseases,” says Luke Lairson, PhD, an assistant professor at Scripps Research and co-senior author of the study. “The small molecule we developed targets the glycolysis pathway in a reversible way to activate the antioxidant response, which could represent a new strategy for developing drugs to turn on this protective pathway.”

The key player in this cross-talk is a protein called KEAP1, which acts as a sensor for potentially damaging reactive molecules in cells. When these reactive molecules build up, KEAP1 triggers the antioxidant stress response to clean them up.

Because reactive molecules are linked to the development of so many diseases — from cancers to autoimmune and neurological disorders — scientists are searching for a way to target KEAP1 and take control of the cell stress response. There has been good progress in finding a “covalent” mechanism to directly target KEAP1, but this approach has the downside of potentially affecting other proteins and causing side effects.

To discover alternative mechanisms to activate this pathway, the Scripps Research scientists decided to employ a chemical genetics-based approach to find non-covalent activators of KEAP1. “We identified a completely new mechanism by first searching for molecules that activate this pathway in an unbiased cell-based screen — and then retroactively looking for their targets and mechanisms of action,” says Michael Bollong, PhD, a Scripps Fellow and first author of the new study.

This approach led the scientists to a molecule called CBR-470-1, which was then optimized by researchers at Calibr, a nonprofit drug discovery division of Scripps Research.

“This is a great example of how screening for active molecules at a cellular level that interrogates all molecules in cells in an unbiased way, rather than targeting a specific protein, can both provide new biological insights as well as lead to potential new drug development opportunities,” says Peter Schultz, PhD, president of Scripps Research and president of Calibr, who serves as co-senior author of the study.

Experiments with CBR-470-1 and its analogue, CBR-470-2, led to the surprising conclusion that KEAP1 is modified through a mechanism that is part of a cell’s response to glucose, called glycolysis, which breaks sugar molecules into energy.

“You could say these two pathways are communicating with each other,” says Bollong. “That had never been shown before.”

This understanding of the molecule’s mechanism of action came from study collaborators at the University of Chicago, led by Raymond Moellering, PhD, who found that KEAP1 activation occurred through a previously unknown post-translational modification, a change made to a cellular protein after its initial production that changes its function

Molecules in the CBR-470 series inhibit a central carbon metabolism enzyme. Inhibiting that enzyme leads to the accumulation of an endogenous metabolite that modifies KEAP1.

The researchers next tested an analogue of the compound in a mouse model of UV damage, a source of cell stress. The analogue CBR-470-2 had the advantage of being more bioavailable than the first two molecules in the series. The researchers found that the mechanism was relevant in the adult mouse, and it was not an artifact of tissue culture.

“The mechanism has the ability to beneficially impact disease progression following systemic delivery of the compound at well tolerated doses,” says Lairson.

Currently, molecules in the CBR-470 series serve to show the “proof of concept” that KEAP1 can be activated by non-covalent drug-like molecules. Going forward, the Bollong lab is looking for new mechanisms and molecules targeting the antioxidant response that may serve as promising drug candidates.

“We’re also testing this mechanism in other disease models to see where activating this response might be useful,” says Bollong.

The study, “A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signalling,” was supported by a Kwanjeong Educational Fellowship; the National Institutes of Health (grants R00CA175399, R01CA211916 and DP2GM128199; an NIH MSTP Training Grant (grant T32GM007281); the V Foundation for Cancer Research (grant V2015-020); the Damon Runyon Cancer Research Foundation (grant DFS08-14); The Skaggs Institute for Chemical Biology, and The University of Chicago.

The paper was co-authored by Michael J. Bollong, Gihoon Lee, John S. Coukos, Hwayoung Yun, Claudio Zambaldo, Jae Won Chang, Emily N. Chin, Insha Ahmad, Arnab K. Chatterjee, Luke L. Lairson, Peter G. Schultz and Raymond E. Moellering.

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