Modulating pseudokinase conformation from the ATP-binding site.

Pseudokinase domains (PsKDs) mediate key signaling processes involved in human disease. Despite transformative success in targeting catalytically active protein kinases, drug development for pseudokinases has lagged behind. Although they lack catalytic activity, pseudokinases are conformationally dynamic and are thought to function by switching between conformations that bind different signaling partners. This conformational switching model suggests the possibility of modulating pseudokinase signaling using small molecules that alter or bias conformational transitions. Focusing on the three PsKDs in receptor tyrosine kinases (RTKs) that retain nucleotide binding (ErbB3, EphB6, and EphA10), we used hydrogen-deuterium exchange mass spectrometry (HDX-MS) to ask whether different small molecules can promote distinct changes in conformation or dynamics. We detected distinct conformational effects in different locations within the PsKDs when different small molecules bind their respective ATP-binding sites. Our results suggest that similar small molecules will be useful probes for understanding pseudokinase function and ultimately for developing possible therapeutics.