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

doi:10.1016/j.celrep.2023.113184

Affimer-mediated locking of p21-activated kinase 5 in an intermediate activation state results in kinase inhibition

Cell Rep. 2023 Oct 31;42(10):113184. doi: 10.1016/j.celrep.2023.113184. Epub 2023 Sep 29.

Authors

Heather L Martin¹, Amy L Turner², Julie Higgins³, Anna A Tang², Christian Tiede², Thomas Taylor⁴, Sitthinon Siripanthong², Thomas L Adams², Iain W Manfield², Sandra M Bell⁵, Ewan E Morrison⁵, Jacquelyn Bond⁵, Chi H Trinh², Carolyn D Hurst⁶, Margaret A Knowles⁶, Richard W Bayliss², Darren C Tomlinson⁷

Affiliations

¹ 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.
² 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.
³ BioScreening Technology Group, Leeds Institutes of Molecular Medicine, University of Leeds, Leeds LS9 7TF, UK.
⁴ School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK.
⁵ 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.
⁶ Division of Molecular Medicine, Leeds Institute of Medical Research at St James's University Hospital, University of Leeds, Leeds LS9 7TF, UK.
⁷ 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. Electronic address: d.c.tomlinson@leeds.ac.uk.

PMID: 37776520
DOI: 10.1016/j.celrep.2023.113184

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.

Keywords: Affimer; CP: Cell biology; PAK5; high-content screening; kinase; signaling; structural biology; urothelial cancer.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Humans
Neoplasms*
Phosphorylation
Protein Binding
p21-Activated Kinases* / genetics
p21-Activated Kinases* / metabolism

Substances

p21-Activated Kinases

Abstract

Publication types

MeSH terms

Substances

Grants and funding