Development of a Pharmacodynamic Assay Based on PLCγ2 Phosphorylation for Quantifying Spleen Tyrosine Kinase (SYK)–Bruton’s Tyrosine Kinase (BTK) Signaling

Abstract

Spleen tyrosine kinase (SYK) and Bruton's tyrosine kinase (BTK) are key mediators in coupling cell surface receptors, such as the B-cell receptor (BCR), to downstream signaling events affecting diverse biological functions. There is therefore tremendous interest in the development of pharmacological inhibitors targeting the SYK-BTK axis for the treatment of inflammatory disorders and hematological malignancies. A good pharmacodynamic (PD) assay, ideally a blood-based assay that measures proximal events, is warranted for evaluation of such inhibitors. In platelets, collagen-induced activation of membrane glycoprotein GPVI is dependent on the SYK-BTK axis. Here, we report the development of a novel immunoassay that uses the dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) to measure GPVImediated phosphorylation of phospholipase C γ2 (PLCγ2), a direct substrate of SYK and BTK, in platelets. The assay was validated using SYK or BTK inhibitors and generated IC 50 correlated with those from the BCR-induced B-cell activation assay. Furthermore, this assay showed good stability and uniformity over a period of 24 h in different donors. Interestingly, compound IC 50 values using blood from patients with rheumatoid arthritis were slightly higher compared with those produced using samples from healthy donors. This novel platelet PLCγ2 phosphorylation-based immunoassay should serve as a promising PD assay for preclinical and clinical development of inhibitors targeting the SYK-BTK axis.

A Cell-Based Assay for Measuring Endogenous BcrAbl Kinase Activity and Inhibitor Resistance

“…Antibody independent cell-based kinase assays often require genetic manipulation or kinase overexpression systems, affecting the physiological relevance of the assay results [23–25]. Still other methods rely on measuring enzymatic activity from cell lysates, reducing the physiological relevance of the results [23,26]. These limitations have made it difficult to comprehensively measure endogenous tyrosine kinase activity in disease-relevant cellular models, posing limitations in pre-clinical kinase inhibitor development and in translating their use to measuring kinase activity in clinical samples for the purposes of characterizing target-focused response.…”