Ligand discrimination between active and inactive activation loop conformations of Aurora-A kinase is unmodified by phosphorylation

James Gilburt, Paul Girvan, Julian Blagg, Liming Ying, Charlotte Dodson

Research output: Contribution to journalArticle

1 Citation (Scopus)
16 Downloads (Pure)

Abstract

Structure-based drug design is commonly used to guide the development of potent and specific enzyme inhibitors. Many enzymes – such as protein kinases – adopt multiple conformations, and conformational interconversion is expected to impact on the design of small molecule inhibitors. We measured the dynamic equilibrium between DFG-in-like active and DFG-out-like inactive conformations of the activation loop of unphosphorylated Aurora-A alone, in the presence of the activator TPX2, and in the presence of kinase inhibitors. The unphosphorylated kinase had a shorter residence time of the activation loop in the active conformation and a shift in the position of equilibrium towards the inactive conformation compared with phosphorylated kinase for all conditions measured. Ligand binding was associated with a change in the position of conformational equilibrium which was specific to each ligand and independent of the kinase phosphorylation state. As a consequence of this, the ability of a ligand to discriminate between active and inactive activation loop conformations was also independent of phosphorylation. Importantly, we discovered that the presence of multiple enzyme conformations can lead to a plateau in the overall ligand Kd, despite increasing affinity for the chosen target conformation, and modelled the conformational discrimination necessary for a conformation-promoting ligand.
Original languageEnglish
Pages (from-to)4069-4076
Number of pages8
JournalChemical Science
Volume10
Issue number14
Early online date4 Mar 2019
DOIs
Publication statusPublished - 14 Apr 2019

ASJC Scopus subject areas

  • Physical and Theoretical Chemistry
  • Biophysics
  • Organic Chemistry

Cite this

Ligand discrimination between active and inactive activation loop conformations of Aurora-A kinase is unmodified by phosphorylation. / Gilburt, James ; Girvan, Paul; Blagg, Julian; Ying, Liming; Dodson, Charlotte.

In: Chemical Science, Vol. 10, No. 14, 14.04.2019, p. 4069-4076.

Research output: Contribution to journalArticle

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AB - Structure-based drug design is commonly used to guide the development of potent and specific enzyme inhibitors. Many enzymes – such as protein kinases – adopt multiple conformations, and conformational interconversion is expected to impact on the design of small molecule inhibitors. We measured the dynamic equilibrium between DFG-in-like active and DFG-out-like inactive conformations of the activation loop of unphosphorylated Aurora-A alone, in the presence of the activator TPX2, and in the presence of kinase inhibitors. The unphosphorylated kinase had a shorter residence time of the activation loop in the active conformation and a shift in the position of equilibrium towards the inactive conformation compared with phosphorylated kinase for all conditions measured. Ligand binding was associated with a change in the position of conformational equilibrium which was specific to each ligand and independent of the kinase phosphorylation state. As a consequence of this, the ability of a ligand to discriminate between active and inactive activation loop conformations was also independent of phosphorylation. Importantly, we discovered that the presence of multiple enzyme conformations can lead to a plateau in the overall ligand Kd, despite increasing affinity for the chosen target conformation, and modelled the conformational discrimination necessary for a conformation-promoting ligand.

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