title: 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 GPVI-mediated 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 IC50 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 IC50 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.
authors.affiliation: Hoffmann-La Roche, Inc., Inflammation Discovery and Therapeutic Area, Nutley, NJ, USA.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Hsu
authors.forename: Jun
authors.initials: J
authors.lastname: Zhang
authors.forename: Chris
authors.initials: C
authors.lastname: Kitson
authors.forename: Seng-Lai
authors.initials: SL
authors.lastname: Tan
authors.forename: Satwant
authors.initials: S
authors.lastname: Narula
authors.forename: Julie A
authors.initials: JA
authors.lastname: DeMartino
authors.forename: Cheng
authors.initials: C
authors.lastname: Liao
mesh_terms: D000077329:Agammaglobulinaemia Tyrosine Kinase; D015703:Antigens, CD / Q000032:analysis; D000945:Antigens, Differentiation, T-Lymphocyte / Q000032:analysis; D001172:Arthritis, Rheumatoid / Q000097:blood / Q000378:metabolism; D001792:Blood Platelets / Q000166:cytology / Q000378:metabolism; D057075:Enzyme Assays / Q000379:methods*; D019337:Hematologic Neoplasms / Q000378:metabolism; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007249:Inflammation / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D037181:Lectins, C-Type / Q000032:analysis; D051966:Phospholipase C gamma / Q000378:metabolism*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015398:Signal Transduction; D000072377:Syk Kinase; D000099192:CD69 Antigens
chemical_list: D000077329:Agammaglobulinaemia Tyrosine Kinase; D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D047908:Intracellular Signaling Peptides and Proteins; D037181:Lectins, C-Type; D051966:Phospholipase C gamma; D011505:Protein-Tyrosine Kinases; D000072377:Syk Kinase; D000099192:CD69 Antigens; C000625949:BTK protein, human; C000606896:SYK protein, human
keywords: BTK; PLCγ2; SYK; pharmacodynamic assay; platelets