Volume 13, Issue 4, Pages 399-407 (April 2006) Selective Kinase Inhibition by Exploiting Differential Pathway Sensitivity  Charles Kung, Denise M. Kenski, Kristin Krukenberg, Hiten D. Madhani, Kevan M. Shokat  Chemistry & Biology  Volume 13, Issue 4, Pages 399-407 (April 2006) DOI: 10.1016/j.chembiol.2006.02.004 Copyright © 2006 Elsevier Ltd Terms and Conditions

Figure 1 Functional Assays for Cdk1 and Pho85 Inhibition (A) Oxindole CDK inhibitors were screened in functional assays for inhibition of Cdk1 and Pho85. (Left) Halo assays of GW400426 (1-TEG) or GW297361 (1) showing differential effects on cell proliferation of YRP1 yeast. A 10 μl aliquot of a 2 mM DMSO stock of each compound was added to the disc. (Right) 1-TEG and 1 both induce nuclear localization of Pho4-GFP when added at 20 μM to YRP1 cells. Shown are fluorescence images overlaid with brightfield edge traces of the cells. (B) 1-TEG induces cell cycle arrest in YRP1 cells with an EC50 = 20 μM, while 1 has no effect at concentrations up to 50 μM. (C) Time course of Pho4-GFP assay as depicted in (A). 1-TEG and 1 are nearly indistinguishable at all time points. Chemistry & Biology 2006 13, 399-407DOI: (10.1016/j.chembiol.2006.02.004) Copyright © 2006 Elsevier Ltd Terms and Conditions

Figure 2 Validation of Target Specificity of 1 and 1-TEG (A) Cluster of 213 genes whose expression is repressed upon inhibition of Cdk1. Quantitation of mean log expression ratios is shown below each column. 1 exhibits partial inhibition of Cdk1-dependent gene expression compared to full inhibition achieved by 1-TEG or in a Cdk1-as1 strain inhibited with 1-NA-PP1. (B) Western blot quantitating Orc6 phosphoisoforms present in Cdk1-as1 or YRP1 cells following treatment for 15 min with DMSO, 1-NA-PP1, or 1-TEG or 1. The effect of 1 is consistent with partial inhibition of Cdk1 within cells. (C) Cluster of 27 genes whose expression is induced upon inhibition of Pho85. Both 1 and 1-TEG upregulate Pho4-dependent genes. Comparison to a Pho85-as1 strain inhibited with 1-NA-PP1 suggests partial inhibition of Pho85 by 1 and 1-TEG within cells. The columns are the same as in (A). (D) Effects of 1-TEG and 1 on expression of genes involved in the yeast ESR. A 20 μM treatment with 1 results in a milder response than 40 μM 1 or treatment with 1-TEG, providing indirect evidence that 1 has reduced bioavailability in YRP1 yeast. (E) Quantitative PCR measurements of expression changes in three markers of Pho85 inhibition (SPL2, PHO11, PHO89) and Cdk1 inhibition (CLB2, CDC20, HOF1) upon treatment of YRP1 yeast for 10 min with 1-TEG or 1. Values represent the change in expression compared to DMSO treatment. For the washout experiment on the right, cells were treated with 1-TEG for 10 min, pelleted, and resuspended in fresh media containing 1% DMSO for 10 min. Chemistry & Biology 2006 13, 399-407DOI: (10.1016/j.chembiol.2006.02.004) Copyright © 2006 Elsevier Ltd Terms and Conditions

Figure 3 Probing the Molecular Basis for the Difference in Target Specificity between 1 and 1-TEG (A) (i) TsCl, TEA, −10°C; (ia) LiBr, NaHCO3 then H-1-NM, K2CO3, DMF; (ii) NaN3, TBAHS, DMF; (iii) H2, Pd/C, MeOH; (iiia) FAM-SE, DMF. Further synthesis details in Experimental Procedures. (B) Halo assays showing effects on cellular proliferation of 1-NM-PP1-, 2-NM-PP1-, and 1-NM-TEG-treated Cdk1-as1 cells. In this series of molecules, in vitro potency closely tracks the cellular efficacy. (C) Neither TEG-Me nor TEG-F is able to penetrate the cell wall of YRP1 yeast. FDA treatments demonstrate compartmentalization of fluorescein molecules following hydrolysis of acetyl groups. Chemistry & Biology 2006 13, 399-407DOI: (10.1016/j.chembiol.2006.02.004) Copyright © 2006 Elsevier Ltd Terms and Conditions

Figure 4 Inhibitor Selectivity via Differential Pathway Sensitivity Model to explain cellular effects of 1-TEG and 1 within the cell. The x and y axes show the percent inhibition of Cdk1 and Pho85, respectively. All values are for schematic purposes only. The level of inhibition of Pho85 necessary to induce Pho85 localization (∼30%, shaded in red) is relatively low compared to that necessary to block cellular proliferation through inhibition of Cdk1 (∼75%, shaded in blue). 1-TEG is able to block both kinases within yeast cells and falls within the purple area. If an inhibitor is dosed at a concentration to achieve >30% inhibition of Pho85 and <75% inhibition of Cdk1, than it would be able to selectively inhibit Pho85, despite having no apparent selectivity by in vitro biochemical assays (as illustrated by 1). Chemistry & Biology 2006 13, 399-407DOI: (10.1016/j.chembiol.2006.02.004) Copyright © 2006 Elsevier Ltd Terms and Conditions