Affimer-mediated locking of p21-activated kinase 5 in an intermediate activation state results in kinase inhibition
Heather L. Martin,
Amy L. Turner,
Julie Higgins,
Anna A. Tang,
Christian Tiede,
Thomas Taylor,
Sitthinon Siripanthong,
Thomas L. Adams,
Iain W. Manfield,
Sandra M. Bell,
Ewan E. Morrison,
Jacquelyn Bond,
Chi H. Trinh,
Carolyn D. Hurst,
Margaret A. Knowles,
Richard W. Bayliss,
Darren C. Tomlinson
Affiliations
Heather L. Martin
BioScreening 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
Amy L. Turner
School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK; Astbury Centre for Structural and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK
Julie Higgins
BioScreening Technology Group, Leeds Institutes of Molecular Medicine, University of Leeds, Leeds LS9 7TF, UK
Anna A. Tang
School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK; Astbury Centre for Structural and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK
Christian Tiede
School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK; Astbury Centre for Structural and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK
Thomas Taylor
School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK
Sitthinon Siripanthong
School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK; Astbury Centre for Structural and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK
Thomas L. Adams
School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK; Astbury Centre for Structural and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK
Iain W. Manfield
School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK; Astbury Centre for Structural and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK
Sandra M. Bell
BioScreening 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
Ewan E. Morrison
BioScreening 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
Jacquelyn Bond
BioScreening 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
Chi H. Trinh
School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK; Astbury Centre for Structural and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK
Carolyn D. Hurst
Division of Molecular Medicine, Leeds Institute of Medical Research at St James’s University Hospital, University of Leeds, Leeds LS9 7TF, UK
Margaret A. Knowles
Division of Molecular Medicine, Leeds Institute of Medical Research at St James’s University Hospital, University of Leeds, Leeds LS9 7TF, UK
Richard W. Bayliss
School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK; Astbury Centre for Structural and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK
Darren C. Tomlinson
BioScreening Technology Group, Leeds Institutes of Molecular Medicine, University of Leeds, Leeds LS9 7TF, UK; School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK; Astbury Centre for Structural and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK; Corresponding author
Summary: 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.
