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Updated: Feb 7, 2026

Direct Measurement of KDM1A Target Engagement Using Chemoprobe-based Immunoassays
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Homogeneous BTK Occupancy Assay for Pharmacodynamic Assessment of Tirabrutinib (GS-4059/ONO-4059) Target Engagement

Helen Yu1, Hoa Truong1, Scott A Mitchell1

  • 11 Gilead Sciences, Inc., Foster City, CA, USA.

SLAS Discovery : Advancing Life Sciences R & D
|July 17, 2018
PubMed

Insights

A new assay accurately measures Bruton

Area of Science:

  • Biochemistry
  • Pharmacology
  • Oncology

Background:

  • Bruton's tyrosine kinase (BTK) is a validated therapeutic target for B-cell malignancies.
  • Approved BTK inhibitors include ibrutinib and acalabrutinib.
  • Tirabrutinib is a second-generation BTK inhibitor in clinical development for lymphoid malignancies.

Purpose of the Study:

  • To develop and qualify a pharmacodynamic assay for tirabrutinib.
  • To assess tirabrutinib target coverage in clinical studies.
  • To inform dose and schedule selection for tirabrutinib.

Main Methods:

  • Developed a novel duplex homogeneous assay using time-resolved fluorescence resonance energy transfer (TR-FRET).
  • Measured free and total Bruton's tyrosine kinase (BTK) levels in a multiplexed format.
  • Utilized terbium-conjugated anti-BTK antibody as energy donor and distinct fluorescent acceptors.

Main Results:

  • The TR-FRET assay was characterized and qualified using recombinant BTK protein and peripheral blood mononuclear cells.
  • Assay utility was demonstrated in cells from lymph node and bone marrow samples of patients with CLL and DLBCL.
  • The assay accurately quantifies BTK occupancy.

Conclusions:

  • A novel TR-FRET-based BTK occupancy assay was developed and qualified.
  • This assay provides accurate, quantitative assessment of BTK occupancy.
  • The assay is utilized in ongoing clinical studies for tirabrutinib.
Keywords:
BTK inhibitorBruton’s tyrosine kinaseassay developmentlymphoid malignancies

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