binds to the DDK ATP binding pocket but at higher concentrations disrupts Cdc7- Dbf4 binding. Each of the other 11 compounds has positive DTms. Examination of the compound titrations reveals that three inhibitors had comparable profiles to PHA-767491 in that they induced a DTm of ,2 or more beginning at a 1 mM concentration: a Rho kinase inhibitor , a protein kinase R inhibitor, and a Chk1 kinase inhibitor . Four additional compounds, the JAK3 inhibitor VI, PI3-Ka inhibitor VIII, UCN- 01, and K-252a gave a 3-fold or higher DTm at 5, 10 and 20 mM concentrations. The structures of the top compounds in the TSA screen are shown in Figure 7, revealing a broad range of structural classes. K- 252a is naturally occurring alkaloid related to staurosporine that inhibits a broad variety of protein kinases including serine/ threonine kinases and tyrosine kinases of the Trk family . So, inclusion of K-252a in this list is MEDChem Express 1252003-15-8 perhaps not surprising. Since it is very likely that the inhibitors we recovered in the TSA screen stabilize DDK by their ability to bind in the ATP binding pocket and inhibit DDK, we performed kinase assays using the top six compounds. Kinase assays revealed that they are 1445379-92-9 indeed DDK inhibitors . The Chk1 and the PKR inhibitors were the best compounds in vitro and inhibited DDK with IC50s of 19.3 nM and 67.5 nM, respectively . Interestingly, the DTm profiles of the Chk1 and PKR inhibitors look strikingly like PHA-767491, raising the possibility that these compounds inhibit DDK in cells. Although SB218078 is derived from staurosporine, it is a potent inhibitor of Chk1 . The structures of the other top hits, the PKR inhibitor and Rockout, are not derived from staurosporine and also differ from known DDK inhibitors . We tested whether the PKR and Chk1 inhibitors would alter cell growth and inhibit Mcm2 phosphorylation in the HCC1954 breast cancer cell line, which would be strong evidence that they inhibit DDK in cells. Increasing amounts of the PKR inhibitor were incubated with HCC1954 cells over 72 hours, which resulted in a large decrease in the number of viable cells relative to vehicle control . The large decrease in cell viability was likely the result o
