Repurposing covalent EGFR/HER2 inhibitors for on-target degradation of human Tribbles 2 (TRIB2) pseudokinase
Authors: Foulkes Daniel M , Byrne Dominic P , Bailey Fiona P , Ferries Samantha , Eyers Claire E , Keeshan Karen , Shrestha Safal , Yeung Wayland , Kannan Natarajan , Wells Carrow , Drewry David H , Zuercher William J , Eyers Patrick A 
Published: April 2018

ONE SENTENCE SUMMARYA Tribbles 2 pseudokinase small molecule screen led to the identification of known EGFR/HER2 inhibitors that alter the stability of TRIB2in vitroand lead to rapid on-target degradation of TRIB2 in human cancer cells.SHORT ABSTRACTTribbles 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 ABSTRACTA 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 TRIB2in 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.ABBREVIATIONSDSFDifferential Scanning FluorimetryEGFREpidermal Growth Factor ReceptorHER2Human Epidermal Growth Factor Receptor 2MSMass spectrometryMSTMicroScale ThermophoresisPKISPublished Kinase Inhibitors SetTRIB2Tribbles 2TSAThermal Stability Assay