Repurposing covalent EGFR/HER2 inhibitors for on-target degradation of human Tribbles 2 (TRIB2) pseudokinase



Foulkes, Daniel, Byrne, Dominic, Bailey, Fiona, Ferries, Samantha, Eyers, Claire ORCID: 0000-0002-3223-5926, Keeshan, Karen ORCID: 0000-0001-7266-0890, Shrestha, Safal, Yeung, Wayland, Kannan, Natarajan, Wells, Carrow
et al (show 3 more authors) (2018) Repurposing covalent EGFR/HER2 inhibitors for on-target degradation of human Tribbles 2 (TRIB2) pseudokinase.

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Abstract

ONE SENTENCE SUMMARY A Tribbles 2 pseudokinase small molecule screen led to the identification of known EGFR/HER2 inhibitors that alter the stability of TRIB2 in vitro and lead to rapid on-target degradation of TRIB2 in human cancer cells. SHORT ABSTRACT Tribbles 2 (TRIB2) is a cancer-associated pseudokinase with a diverse interactome, including the AKT signaling module. Substantial evidence demonstrates that TRIB2 dysregulation is important in multiple human tumors. The non-canonical TRIB2 pseudokinase domain contains a unique cysteine rich region and interacts with a peptide motif in its own C-terminal tail. We demonstrate that TRIB2 is a target for previously described small molecule protein kinase ‘inhibitors’, which were originally designed to inhibit the catalytic domain of EGFR/HER2 tyrosine kinases. Using thermal-shift assays and drug repurposing, we classify ligands that stabilize or destabilize the TRIB2 pseudokinase domain. TRIB2 destabilizing agents, including the clinical inhibitor afatinib, lead to rapid and on-target TRIB2 protein degradation in tumor cells, eliciting tractable effects on cell signaling and survival. Our data identifies leads for further development of TRIB2-degrading drugs and highlights compound-induced TRIB2 downregulation, which might be mechanistically relevant for other catalytically-deficient (pseudo)kinases targeted by small molecules. FULL ABSTRACT A major challenge associated with biochemical and cellular analysis of pseudokinases is the lack of target-validated small molecule ligands with which to probe molecular function. Human Tribbles 2 (TRIB2) is a cancer-associated pseudokinase with a diverse interactome, which includes the canonical AKT signaling module. There is substantial evidence that human TRIB2 is a therapeutic target in both solid tumors and blood cancers. The non-canonical TRIB2 pseudokinase domain contains a unique cysteine-rich region and interacts with a peptide motif in its own C-terminal tail, which was previously shown to drive interaction with cellular E3 ubiquitin ligases. In this study we demonstrate that TRIB2 is a target for previously described small molecule protein kinase inhibitors, which were originally designed to inhibit the canonical catalytic domain of the tyrosine kinases EGFR/HER2. Using a thermal-shift assay, we discovered TRIB2 ligands within the Published Kinase Inhibitor Set (PKIS), and employed a drug repurposing approach to classify compounds that either stabilize or destabilize TRIB2 in vitro . Remarkably, TRIB2 destabilizing agents, including the clinical covalent drug afatinib, lead to rapid and on-target TRIB2 degradation in human cells, eliciting tractable effects on signaling and survival. Our data reveal the first drug-leads for development of TRIB2-degrading ligands, which will also be invaluable for unravelling the cellular mechanisms of TRIB2-based signaling. Our study highlights that small molecule-induced protein downregulation through drug ‘off-targets’ might be relevant for other inhibitors that serendipitously target pseudokinases. <jats:def-list> ABBREVIATIONS <jats:def-item> DSF Differential Scanning Fluorimetry <jats:def-item> EGFR Epidermal Growth Factor Receptor <jats:def-item> HER2 Human Epidermal Growth Factor Receptor 2 <jats:def-item> MS Mass spectrometry <jats:def-item> MST MicroScale Thermophoresis <jats:def-item> PKIS Published Kinase Inhibitors Set <jats:def-item> TRIB2 Tribbles 2 <jats:def-item> TSA Thermal Stability Assay

Item Type: Article
Depositing User: Symplectic Admin
Date Deposited: 02 Dec 2019 09:43
Last Modified: 02 May 2022 14:11
DOI: 10.1101/305243
URI: https://livrepository.liverpool.ac.uk/id/eprint/3064397