Studying electrophile signaling is marred by difficulties in parsing changes in pathway flux attributable to on-target, vis-à-vis off-target, modifications. By combining bolus dosing, knockdown, and Z-REX-a tool investigating on-target/on-pathway electrophile signaling, we document that electrophile labeling of one zebrafish-Keap1-paralog (zKeap1b) stimulates Nrf2- driven antioxidant response (AR) signaling (like the human-ortholog). Conversely, zKeap1a is a dominant-negative regulator of electrophile-promoted Nrf2-signaling, and itself is nonpermissive for electrophile-induced Nrf2-upregulation. This behavior is recapitulated in human cells, wherein following electrophile treatment: (1) zKeap1b-transfected cells are permissive for augmented AR-signaling through reduced zKeap1b-Nrf2 binding; (2) zKeap1a-transfected cells are non-permissive for AR-upregulation, as zKeap1a-Nrf2 binding capacity remains unaltered; (3) 1:1 ZKeap1a:zKeap1b-transfected cells show no Nrf2-release from the Keap1-complex, rendering these cells unable to upregulate AR. We identified a zKeap1a-specific point-mutation (C273I) responsible for zKeap1a's behavior. Human-Keap1(C273I), of known diminished Nrf2-regulatory capacity, dominantly muted electrophile-induced Nrf2-signaling. These studies highlight divergent and interdependent electrophile signaling behaviors, despite conserved electrophile sensing.
The data generated in this study using these materials are provided in Main Figure 1-8, accompanied by 17 associated Figure Supplements, and the Source Data Files associated with Main Figure 1-8 and 17 associated Figure Supplements.
- Yimon Aye
- Yimon Aye
- Yimon Aye
- Jesse R Poganik
- Jesse R Poganik
- Saba Parvez
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Animal experimentation: All procedures performed at Cornell (2017-2018) and EPFL (2018-present) conform to the animal care, maintenance, and experimentation procedures followed by Cornell University's and EPFL's Institutional Animal Care and Use Committee (IACUC) guidelines and approved by the respective institutional committees. All experiments with zebrafish performed at EPFL (2018-present) have been performed in accordance with the Swiss regulations on Animal Experimentation (Animal Welfare Act SR 455 and Animal Welfare Ordinance SR 455.1), in the EPFL zebrafish unit, cantonal veterinary authorization VD-H23).
- Jonathan A Cooper, Fred Hutchinson Cancer Research Center, United States
- Received: September 9, 2022
- Preprint posted: October 12, 2022 (view preprint)
- Accepted: October 12, 2022
- Accepted Manuscript published: October 27, 2022 (version 1)
- Accepted Manuscript updated: October 31, 2022 (version 2)
- Version of Record published: December 15, 2022 (version 3)
- Version of Record updated: June 1, 2023 (version 4)
© 2022, Van Hall-Beauvais et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Kinase inhibitors are successful therapeutics in the treatment of cancers and autoimmune diseases and are useful tools in biomedical research. However, the high sequence and structural conservation of the catalytic kinase domain complicates the development of selective kinase inhibitors. Inhibition of off-target kinases makes it difficult to study the mechanism of inhibitors in biological systems. Current efforts focus on the development of inhibitors with improved selectivity. Here, we present an alternative solution to this problem by combining inhibitors with divergent off-target effects. We develop a multicompound-multitarget scoring (MMS) method that combines inhibitors to maximize target inhibition and to minimize off-target inhibition. Additionally, this framework enables optimization of inhibitor combinations for multiple on-targets. Using MMS with published kinase inhibitor datasets we determine potent inhibitor combinations for target kinases with better selectivity than the most selective single inhibitor and validate the predicted effect and selectivity of inhibitor combinations using in vitro and in cellulo techniques. MMS greatly enhances selectivity in rational multitargeting applications. The MMS framework is generalizable to other non-kinase biological targets where compound selectivity is a challenge and diverse compound libraries are available.
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