Structural Basis of Wee Kinases Functionality and Inactivation by Diverse Small Molecule Inhibitors

Abstract

Members of the Wee family of kinases negatively regulate the cell cycle via phosphorylation of CDK1 and are considered potential drug targets. Herein, we investigated the structure–function relationship of human Wee1, Wee2, and Myt1 (PKMYT1). Purified recombinant full-length proteins and kinase domain constructs differed substantially in phosphorylation states and catalytic competency, suggesting complex mechanisms of activation. A series of crystal structures reveal unique features that distinguish Wee1 and Wee2 from Myt1 and establish the structural basis of differential inhibition by the widely used Wee1 inhibitor MK-1775. Kinome profiling and cellular studies demonstrate that, in addition to Wee1 and Wee2, MK-1775 is an equally potent inhibitor of the polo-like kinase PLK1. Several previously unrecognized inhibitors of Wee kinases were discovered and characterized. Combined, the data provide a comprehensive view on the catalytic and structural properties of Wee kinases and a framework for the rational design of novel inhibitors thereof.

Development of Potent Pyrazolopyrimidinone‐Based WEE1 Inhibitors with Limited Single‐Agent Cytotoxicity for Cancer Therapy

“…At the time of writing this manuscript, the 1.9Å co-crystal structure of the human WEE1 kinase domain in complex with AZD1775 was published (PDB: 5V5Y). [13] The proposed binding orientation of AZD1775 in the ATP-binding site of WEE1 in our computational model was in good agreement with the co-crystal structure (Supporting Information, Figure S4), with only minor differences observed in the orientation of the side chains. Importantly, both our computational model and the WEE1-AZD1775 co-crystal structure equally support the proposed structural modifications to AZD1775 described above.…”

“…The WEE1-AZD1775 co-crystal structure indicates that the allyl group may have a hydrophobic contact with Lys328. [11c] Further compounds may need to be developed to more thoroughly investigate the scope of replacing the allyl group to include substituents that maintain the electronic distribution of negative electrostatic and hydrophobic fields and that can be accommodated in the small hydrophobic pocket in the ATP-binding site of WEE1.…”

“…A kinome interaction network study identified ABL1, LCK, LRRK2, TNK2, and SYK as targets of AZD1775 with Ki values below 1 μM. [11a] Recently, it has been reported that AZD1775 has potent nanomolar activity against PLK1, and that AZD1775 should now be considered a dual WEE1 and PLK1 inhibitor. [11b, 11c] A full kinome profile for AZD1775 across a panel of 468 human kinases at a concentration of 500 n m identified PLK1, FLT3 (D835V), JAK3, GCN2 (S808G), ABL1 (H396P), and JAK2 as additional kinase targets.…”

“…[11a] Recently, it has been reported that AZD1775 has potent nanomolar activity against PLK1, and that AZD1775 should now be considered a dual WEE1 and PLK1 inhibitor. [11b, 11c] A full kinome profile for AZD1775 across a panel of 468 human kinases at a concentration of 500 n m identified PLK1, FLT3 (D835V), JAK3, GCN2 (S808G), ABL1 (H396P), and JAK2 as additional kinase targets. [11c] Furthermore, the authors suggested that WEE1 and PLK1 were the most relevant targets for mediating the anticancer effects of AZD1775; however, our studies support that WEE1 inhibition can be achieved with little impact on cytotoxicity.…”

“…In preclinical studies, AZD1775 demonstrated potent cytotoxicity in a broad panel of cancer cell lines and led to xenograft tumor growth inhibition; [10] however, these anticancer activities cannot be attributed to WEE1 inhibition alone since AZD1775 is known to inhibit other kinases and may have off-target effects outside of the kinome. [6, 11] The combination of WEE1 inhibitors with DNA-damaging agents has been proposed to be more effective in p53-mutant cancer cells that rely on DNA repair at the G2-M checkpoint. However, several preclinical studies have demonstrated that AZD1775 has anticancer activity independent of p53 function, and this has also been attributed to the alternative functions of WEE1 and/or the off-target effects of AZD1775.…”