Fig. 2. CaMKAR-based screen identifies CaMKII inhibitors among drugs in clinical use.

(A) Schematic depiction of CaMKAR-based high-throughput screen. K562 cells were coinfected with CaMKAR- and CA CaMKIIδ^T287D–encoding lentiviruses. CaMKII expression could be induced with doxycycline. K562^{CaMKII-CaMKAR} cells were screened against the Johns Hopkins Drug Library v3.0. Primary hits were further screened using in vitro assays to eliminate false positives and narrow the list to five CaMKII inhibitors. (B) Drugs ranked according to in cellulo CaMKII inhibition in primary screen. CaMKII inhibition percentage defined by minimum-maximum normalization using means of control groups [CTRL (untreated) and AS100397; same data as fig. S4B]. A total of 118 selected hits are shown in blue. Positive control staurosporine is shown in magenta. The dashed line represents hit selection threshold (see Materials and Methods). Data shown are subsets (those scoring between 0 and 140% for visualization of hits) from the complete dataset in data file S1. (C) Hits from (B) ranked according to in vitro CaMKII inhibition as detected by CaMKAR secondary screen. CaMKII inhibition normalized against untreated control (CTRL). AS100397 was used as a positive control. Thirteen identified hits are in blue. Staurosporine is shown in magenta. Data shown are subsets from the complete dataset (see Supplementary Materials). (D) Five CaMKII inhibitory drugs and their intended targets within the human kinase homology dendrogram. Kinase families: AGC, containing PKA, PKG, and PKC families; CK1, casein kinase 1; CMGC, containing CDK, MAPK, GSK3, and CLK; STE, homologs of yeast sterile 7, sterile 11, and sterile 20; TK, tyrosine kinase; TKL, tyrosine kinase–like. (E) IC₅₀ (left) and cell viability (right) curves from 293T cells expressing CaMKAR and CaMKII^T287D and exposed to five candidate drugs and CaMKII inhibitor (AS100397, 10 mM). Measurements in (E) are done in biological triplicate; complete dataset is shown in fig. S5.