P023 Development of methods to investigate effects of narrow spectrum kinase inhibitors on cellular phosphorylation of kinase substrates
S. Sirohi*, M. Foster, M. Fyfe, Y. Solanke, S. Webber, C. Walshe
Topivert Pharma, London, United Kingdom
Protein kinases catalyse the transfer of a phosphate group from adenosine triphosphate (ATP) to a serine, threonine or tyrosine residue in their substrates and play a critical role in intracellular signalling. Selective kinase inhibitors have been used as therapies particularly in oncology and chronic inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease (IBD). Most recently, to improve efficacy, targeted multikinase inhibitors have been developed, as exemplified by narrow spectrum kinase inhibitor TOP1288, which targets p38α MAPK, Syk, and Src family kinases. TOP1288 has been proposed as a potential therapy for IBD, and to investigate target engagement, flow cytometric methods have been developed to demonstrate inhibition of target kinases after clinical dosing.
Activation of key kinases by hydrogen peroxide was evaluated using phospho-site-specific antibodies for intracellular flow cytometric analysis. TOP1288-mediated inhibition of P38α & Lck auto-phosphorylation events was assessed in vitro, in both a T-cell line and primary cells derived from human blood or colonic tissue.
Initial studies were performed in Jurkat T-cells, and demonstrated transient phosphorylation of P38α and Lck after stimulation with H2O2 (5–60 min). TOP1288 inhibited activation of both phospho-targets with sub-100nM IC50, and completely inhibited target activity at sub-microMolar concentrations. TOP1288 exhibited equivalent potency and efficacy when assessed in human peripheral blood mononuclear cells (PBMCs) isolated from healthy donors. Investigation of lamina propria mononuclear cells (LPMCs) isolated from colonic biopsies revealed a reduced induction of phospho-P38α in response to H2O2 stimulation. However, there was good induction of Phospho-Lck activation, allowing the investigation of phospho-target down regulation. In this system, TOP1288 achieved equivalent potency of sub-100nM IC50 against phospho-Lck, comparable with both Jurkat T-cells and PBMCs.
In conclusion, the method developed allows semiquantitative measurement of target phosphorylation in LPMCs. TOP1288, after in-vitro addition, has been demonstrated to inhibit auto-phosphorylation in cell subsets of primary origin. Therefore, this methodology has the potential to be used in clinical studies for biomarker measurements in pathologically relevant cells of IBD.