Abstract C94: A novel binding assay to identify inhibitors that bind to inactive forms of Bruton's tyrosine kinase based on fluorescence resonance energy transfer.
Authors: Asami Tokiko , Kawahata Wataru , Sawa Masaaki 
Published: June 2012

Bruton's tyrosine kinase (Btk) is a member of the Tec family of non-receptor tyrosine kinases. Btk is one of the crucial kinases for the B cell maturation and also involved in mast cell activation through the high-affinity IgE receptor. Therefore Btk is an attractive target for the potential treatment of multiple therapeutic areas that involve B-cell and/or mast cell activation, such as B cell malignances, asthma, and rheumatoid arthritis. In order to develop a selective Btk inhibitor, it is important to identify a highly selective compound as the drug discovery starting point. Generally, activity-based high-throughput screening (HTS) using active kinase has been used to identify hit compounds by measuring the inhibition of substrate phosphorylation. However, the activity-based HTS campaign frequently results in the enrichment of classical ATP competitive inhibitors, which would often require considerable medicinal chemistry efforts to increase kinase selectivity, because the human protein kinase family consists of over 500 enzymes with very similar active sites. Here we describe a novel approach to identify inhibitors of inactive form of Btk.

Method: Several compounds that inhibit Btk kinase activity have been described in the literature. One compound, CGI1746 is reported to bind to the inactive form of Btk, so we synthesized FITC-labeled CGI1746 derivatives with various linker groups as fluorescent probes that preferentially bind to an inactive form of Btk. To develop a screening system targeting the inactive form of Btk, we designed a competitive binding assay based on time-resolved fluorescence resonance energy transfer (TR-FRET) platform that measures the binding of the FITC-labeled CGI1746 probe to biotinylated Btk.

Result: We produced a single endogenous biotinylated Btk protein (BTN-Btk), and the BTN-Btk protein was treated with ATP (autophosphorylation) or with Lambda Protein Phosophatase (dephosphorylation) to obtain the activated and unactivated BTN-Btk respectively. Interestingly, dephosphorylated BTN-Btk still showed weak catalytic activity indicating phosphatase-treatment might not able to remove phosphates of Btk completely. We synthesized various FITC-labeled CGI1746 derivatives with different linkers, and determined their IC50 value against both the activated and unactivated BTN-Btk by measuring substrate phosphorylation. Several compounds were found to maintain the original inhibitory potency even after introducing a FITC fluorophore. To test if the FITC-labeled CGI1746 derivatives can be applied to a competitive binding assay, we determined the Kd value of each probes with the TR-FRET assay platform. Most of the probes synthesized were found to be excellent FRET acceptors with high affinity and low background signals. Detailed data of this novel binding assay targeting the inactive form of Btk will be presented in this poster.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C94.