Inhibition of CDC25B phosphatase through disruption of protein-protein interaction

Abstract

CDC25 phosphatases are key cell cycle regulators and represent very attractive but challenging targets for anticancer drug discovery. Here, we explored whether fragment-based screening represents a valid approach to identify inhibitors of CDC25B. This resulted in identification of 2-fluoro-4-hydroxybenzonitrile, which directly binds to the catalytic domain of CDC25B. Interestingly, NMR data and the crystal structure demonstrate that this compound binds to the pocket distant from the active site and adjacent to the protein-protein interaction interface with CDK2/Cyclin A substrate. Furthermore, we developed a more potent analogue that disrupts CDC25B interaction with CDK2/Cyclin A and inhibits dephosphorylation of CDK2. Based on these studies, we provide a proof of concept that targeting CDC25 phosphatases by inhibiting their protein-protein interactions with CDK2/Cyclin A substrate represents a novel, viable opportunity to target this important class of enzymes.

Figure 1

Identification and characterization of compound 1 as a novel CDC25B ligand. (A) A portion of the ¹H–¹⁵N HSQC spectrum for the CDC25B catalytic domain in the presence (red) and absence (black) of 2 mM 1. (B) Crystal structure of 1 bound to CDC25B. Dark gray surface denotes the enzymatic active site. Two arginine residues involved in interaction with CDK2/Cyclin A substrate are labeled and shown in red. The distance between the catalytic cysteine and 1 is shown. (C) Molecular details of the interaction of 1 with CDC25B binding pocket. 1 binds in two equally populated orientations with symmetry along CN, OH axis. Distance between position 6 of 1 and the sulfate ion is given (PDB ID: 4WH7). The hydrogen bond network between the hydroxyl of 1 and four waters in the binding pocket is also shown. (D) AlphaLISA signal due to the protein–protein interaction between CDC25B and the CDK2/Cyclin A complex. CDC25B WT is shown in black, and the hotspot mutation R492L is shown in red.

Figure 2

Structure activity relationship (SAR) for 2-fluoro-4-hydroxybenzonitrile analogs. (A) Structures of compounds tested for binding to CDC25B. Sum Δσ was calculated as a sum of the chemical shift perturbations for eight of the most significantly perturbed amide resonances (in Hz) at 2 mM compound concentration. (B) Schematics of the synthesis of compounds 7 and 8. (C) Crystal structure showing the details of the interaction of 7 with CDC25B (PDB ID: 4WH9). Distances between the sulfate oxygens and the side-chain nitrogens of Arg488 and Arg492 are given.

Figure 3

Small molecule ligand binding to the protein–protein interaction site inhibits CDC25B activity. (A) Activity of compound 1 and 7 in an AlphaLISA-based protein–protein interaction assay. (B) In vitro phosphatase assay utilizing phosphorylated CDK2/Cyclin A as a substrate for CDC25B in the presence or absence of 1 or 7. Remaining phosphorylated CDK2/Cyclin A is shown as detected by Western blot.