Abstract Fri010: Expanding High Throughput CaMKII Inhibitor Screen to Commercial “Hinge Binder” Drug Library

Ca2+/calmodulin-dependent protein kinase II (CaMKII) hyperactivity is proven to contribute to the pathogenesis of cardiovascular diseases, including atrial fibrillation. There is currently no curative treatment for atrial fibrillation, and medical therapies often have serious side effects. Numerous studies have shown the therapeutic benefit of CaMKII inhibition; however, there is no FDA-approved CaMKII inhibitor drug. Previously, our lab designed a fluorescent CaMKII activity reporter (CaMKAR) to conduct a drug repurposing screen of FDA-approved drugs. From this initial screen, ruxolitinib emerged as a previously unrecognized CaMKII inhibitor due to its targeting of the ATP-binding site. This study aimed to expand on the previous high throughput screen to a larger, commercial library of 24,000 “Hinge Binders,” which target the ATP-binding domain essential for kinase function. K562 cells were infected with lentivirus containing constitutively active CaMKII (T287D mutant) and optimized CaMKAR with an improved dynamic range. A pre-assembled hinge-binder drug library was used, with DMSO and ruxolitinib as controls. CaMKAR fluorescence was measured at 488 nm and 405 nm for ratiometric quantification of CaMKII activity. Fluorescence ratios were standardized using the DMSO control, with significant candidates selected based on a Bonferroni-adjusted p-value threshold. Outliers were removed according to the 405 nm signal to account for the expected stability. The average 488/405 ratio for DMSO was 2.77 compared to ruxolitinib of 1.85. After standardization of CaMKII inhibition, 181 drugs were selected as initial candidates. Once outliers were removed, 18 drugs remained as candidates for secondary testing. The average 488/405 ratio for the 18 candidates was 2.13. This expanded high-throughput screen successfully identified 18 novel candidate drugs with potential CaMKII inhibitory activity, highlighting the utility of coupling commercial drug libraries with CaMKAR for in-cellulo drug discovery. Moving forward, specificity testing with other kinases, cell-free assays, and validation in cardiomyocytes and a mouse model of atrial fibrillation will be essential to assess the therapeutic potential of these novel compounds.