Affimer-mediated locking of p21-activated kinase 5 in an intermediate activation state results in kinase inhibition
Heather L Martin1, Amy L Turner2, Julie Higgins3
1BioScreening Technology Group, Leeds Institutes of Molecular Medicine, University of Leeds, Leeds LS9 7TF, UK; Division of Molecular Medicine, Leeds Institute of Medical Research at St James's University Hospital, University of Leeds, Leeds LS9 7TF, UK; School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK.
Abstract:
Kinases are important therapeutic targets, and their inhibitors are classified according to their mechanism of action, which range from blocking ATP binding to covalent inhibition. Here, a mechanism of inhibition is highlighted by capturing p21-activated kinase 5 (PAK5) in an intermediate state of activation using an Affimer reagent that binds in the P+1 pocket. PAK5 was identified from a non-hypothesis-driven high-content imaging RNAi screen in urothelial cancer cells. Silencing of PAK5 resulted in reduced cell number, G1/S arrest, and enlargement of cells, suggesting it to be important in urothelial cancer cell line survival and proliferation. Affimer reagents were isolated to identify mechanisms of inhibition. The Affimer PAK5-Af17 recapitulated the phenotype seen with siRNA. Co-crystallization revealed that PAK5-Af17 bound in the P+1 pocket of PAK5, locking the kinase into a partial activation state. This mechanism of inhibition indicates that another class of kinase inhibitors is possible.
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