Cell Reports (Oct 2023)

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

Journal volume & issue
Vol. 42, no. 10
p. 113184

Abstract

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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.

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