Targeting Protein-Protein Interactions in Kinase Domains with DNA-Encoded Library Approaches for Therapeutics and Diagnostics

Protein kinases are essential in cell signaling pathways and are well-validated targets for cancer therapeutics and detection of activity levels. Yet, there remains a critical need for kinase inhibitors with high specificity and potency. The development of DNA-encoded library (DEL) technology dramatically facilitates the discovery of ligands to therapeutically relevant proteins. The preparation of combinatorial libraries followed by stringent selections can be exploited to rapidly generate hit molecules that bind to a large variety of targets.

A combinatorial library of peptidomimetics is prepared and subjected to a selection for enriching molecules that can serve as substrates for tyrosine kinase Src. Non-natural substrate molecules are recognized by the anti-phosphotyrosine antibody during the selection. Using biophysical characterization assays including ADP-Glo and NMR, the resulting hits are investigated as novel peptide-substrate competitive inhibitors, as well as specific chemical probes that would benefit kinase activity detection. An ester derivative of the lead compound SrcDEL10 demonstrates cellular activity with inhibition of Src-dependent signaling in cell culture. Subsequently, our effort extends to parallel selections with a highly diverse-scaffold DEL on three cancer-related tyrosine kinases. Several hit molecules are validated with differential phosphotransfer activities among Src, Lyn, and Syk. Studies on the structure activity relationship of hit molecules produce selective kinase substrates with the lowest molecular weights reported to date. Potential bisubstrate inhibitors, showing above 8-fold Src selectivity over Lyn, are designed based on structures of selective substrates.

Meanwhile, high sensitivity of DNA sequence analysis enables the development of specific and multiplexed activity assays. Using the substrate selection strategy, we develop a DNA-based kinome activity profiling assay using DNA conjugates of tyrosine kinase peptide substrates. Selective enrichment of phosphorylated probes enables activity detection by either quantitative PCR (qPCR) or parallel DNA sequencing. Results with detecting recombinant kinases demonstrated a low (~50 pM kinase) limit of detection. A library of 96 DNA-substrate conjugates enabled multiplexed tyrosine kinase assays in cell lysates in a manner analogous to peptide microarrays. This DNA-based assay potentially empowers the detection of tumor biomarkers with high specificity, lower detection limit, multiplexing capability, and high cost-effectiveness.

Together, this research uses DNA-based technologies to assist developing new therapeutics and diagnostics, drug target validation, unveiling drug mechanisms of action, and understanding the role of protein phosphorylation in disease progression.