A novel approach to the discovery of small-molecule ligands of CDK2

Abstract

In an attempt to identify novel small-molecule ligands of cyclin-dependent kinase 2 (CDK2) with potential as allosteric inhibitors, we have devised a robust and cost-effective fluorescence-based high-throughput screening assay. The assay is based on the specific interaction of CDK2 with the extrinsic fluorophore 8-anilino-1-naphthalene sulfonate (ANS), which binds to a large allosteric pocket adjacent to the ATP site. Hit compounds that displace ANS directly or indirectly from CDK2 are readily classified as ATP site binders or allosteric ligands through the use of staurosporine, which blocks the ATP site without displacing ANS. Pilot screening of 1453 compounds led to the discovery of 12 compounds with displacement activities (EC(50) values) ranging from 6 to 44 μM, all of which were classified as ATP-site-directed ligands. Four new type I inhibitor scaffolds were confirmed by X-ray crystallography. Although this small compound library contained only ATP-site-directed ligands, the application of this assay to large compound libraries has the potential to reveal previously unrecognized chemical scaffolds suitable for structure-based design of CDK2 inhibitors with new mechanisms of action.

Figure 1. Exploitation of the CDK2-ANS interaction for the identification of small molecule ligands

A) Type I kinase inhibitors bind to the ATP site, Type II inhibitors extend from the ATP site into the neighboring allosteric pocket, and Type III inhibitors bind in a purely allosteric manner. The activation loop is shown in magenta, the DFG motif in cyan, and inhibitors in green. B) The allosteric site in CDK2 accommodates two ANS molecules, which are displaced upon binding of ligands by direct competition (Type III) or indirectly through conformational changes caused by binding of certain Type I or Type II ligands to the ATP site. C) The approach involves primary screening to measure ANS displacement potential, a follow-up assay using an ATP site blocker such as staurosporine to classify the hit compounds, a secondary assay to evaluate inhibitory activity, and the determination of the molecular mode of action by protein crystallography.

Figure 2. Screening of the NCI library using the CDK2-ANS assay

Example of a 96-well plate CDK2-ANS assay measured at excitation and emission wavelengths of 405 and 460 nm, respectively. Compounds displaying ANS displacement greater than 60% (hits) are shown in black, along with the controls in columns 1 and 12. A) “BLANK” shows the fluorescence signal produced by ANS and compound in the absence of CDK2. Compounds A5, B8, B11, C3, C10, F5, F8, and G6 are self-fluorescent (~2.5 times greater than control shown in grey). These compounds are not to be discarded, but their displacement capability may be difficult to assess using the experimental conditions. B) “RAW” shows the fluorescence signal upon addition of CDK2, identifying A3, B7, D2, and H9 as potential hits. C) “DELTA” is the result of subtracting “BLANK” from “RAW” (Eqn. 1). D) “CORR” is “DELTA” after correction for the inner-filter effect using Eqn. 2, revealing H9 as an unspecific fluorescence quencher (shown in grey). A5 is also highlighted in grey for reason as discussed above. E) “%CORR” is the displacement potential relative to the positive control (SU9516). From this plate, A3 and B7 were identified as hit compounds 6 and 2 respectively, while D2 was discarded following dose-response assays (EC₅₀ > 50 μM).

Figure 3. Assay performance and follow-up assay for classification

A) Z-factors and signal-to-background ratios corresponding to 96-well low volume format CDK2-ANS assay plates. Average Z-factor value: 0.8 +/− 0.1 (▼), average signal-to-background ratio: 12 +/− 2.3 (x). B) The use of staurosporine as a non-displacing Type I inhibitor for the classification of Type I/II and Type III in the CDK2-ANS secondary assay. Titration with staurosporine has little effect on the fluorescence yield of the CDK2-ANS complex (▲). Titration of positive control SU9516 in the presence of 10 μM staurosporine showed a 15-fold increase in EC₅₀ from 0.8 ± 0.05 μM (◆) to 12 ± 1.1 μM (●).

Figure 4. Ternary structure of CDK2 with staurosporine and ANS

A) The co-crystal structure of CDK2 liganded with ANS and staurosporine was determined at 2.5 A resolution (Table 2). Staurosporine (green) binds to the ATP site, along with two ANS molecules (yellow) in the allosteric pocket. The hinge region is shown in orange, the DFG motif in cyan, and the C-helix in magenta. Inset: The 2Fo-Fc electron density map, contoured at 1σ, is shown as blue mesh with hydrogen bonding interactions indicated as black dotted lines. A 1Fo-Fc density map omitting the ligands during refinement is shown in Supplemental Fig. 4. B) Schematic presentation of the binding interactions of staurosporine and ANS in CDK2, with potential van der Waals interactions shown in green.

Figure 5. Molecular mode of action of hit compounds

Co-crystal structures with CDK2 were determined for hit compounds A) 1 (sunitinib), B) 2 (purpurogallin), C) 3, and D) 4 with the hinge region shown in orange, gatekeeper residue Phe80 in red, the DFG motif in cyan, the C-helix in magenta, and the compounds in yellow; the 2Fo-Fc electron density map, contoured at 1σ, is shown as blue mesh. The black dotted lines indicate potential hydrogen bonding interactions, and the red dotted lines indicate potential clashes with gatekeeper residue Phe80 and Asp145 of the DFG motif. 1Fo-Fc density maps omitting the ligands during refinement are shown in Supplemental Fig. 5.