Abstract Body: Small molecule drugs are highly attractive for clinical therapies due to their bioavailability, cell permeability, and clinically compatible half-lives, dominating pharmaceutical treatments for decades. One of the primary barriers to discovering new clinical therapeutics is the difficulty in developing inhibitors that have high specificity and an acceptable safety profile. Our lab recently developed a screening platform to identify drug-like compounds that are inhibitors of CaMKII, an essential mediator of cardiac function and among the most validated pathways driving cardiovascular diseases. Our method utilizes CaMKAR, a highly specific and sensitive biosensor that can measure CaMKII activity in living cells. This cpGFP-based kinase biosensor is modular and dependent on 3 elements: 1) the substrate peptide, 2) the phospho-amino acid binding domain (PAABD) and, 3) the length of the flexible linker between the substrate peptide and cpGFP which allows the PAABD to bind the phosphorylated substrate and re-circularize the GFP barrel.
Our goal is to develop a library of kinase reporters that can be deployed as tools to screen drugs in live cells and characterize their effects on different kinase signaling pathways. As a proof of concept for the future expansion of our kinase activity reporter library, we asked if we could modify CaMKAR, which is a serine-threonine kinase reporter, to create a reporter for other kinase subfamilies such as JAK, a tyrosine kinase. Our main objective in designing JAK activity reporter (JKAR) candidates was to find compatible combinations of the JAK substrate peptides and a PAABD that recognizes phospho-tyrosine. Utilizing the NanoBiT® luciferase system, we tested 24 combinations utilizing 2 PAABDs and 12 amino acid sequences representing potential substrates for JAK phosphorylation. Of these candidates, four have increased luciferase activity compared to a control peptide, and reduced activity following treatment with a JAK inhibitor. The best candidates were used to build a cpGFP-based sensor where the linker length can be optimized. In conclusion, we have developed the first JAK1/2 Activity Reporter, JKAR, for use in live cell assays and use in drug discovery applications.