SD-208 was a cell-active, ATP-competitive PKD inhibitor and did not inhibit PKC and CAMK.

<p><b>A. Determination of PKD kinase activity <i>in vitro</i></b>. Inhibition of recombinant human PKD1, 2 and 3 was assayed in the presence of 10 different concentrations of SD-208 by an <i>in vitro</i> radiometric PKD kinase assay. The IC₅₀ values were calculated as the mean ± SEM of at least three independent experiments with triplicate determinations at each compound concentration in each experiment. The data were plotted as a function of inhibitor concentration and a representative graph is shown. <b>B</b>. <b>SD-208 inhibited PMA-induced PKD1 activation in prostate cancer cells</b>. LNCaP cells were pretreated with different doses of inhibitors for 45 min, followed by PMA stimulation at 10 nM for 20 min. Cell lysates were subjected to immunoblotting for p-S⁹¹⁰-PKD1 and p-S^738/742-PKD1. Tubulin was blotted as loading control. The experiment was repeated three times and the representative blots are shown. <b>C. Determination of cellular IC</b>_<b>50</b><b> </b>. Western blots from ‘B” were quantified using densitometry analysis. The data were plotted and IC₅₀ values were derived from the concentration-response curves using GraphPad. One of the three concentration-response curves is shown. <b>D. SD-208 is an ATP-competitive kinase inhibitor</b>. PKD1 kinase activity was measured as a function of increasing concentrations of ATP in the presence of varying concentrations of SD-208. Lineweaver-Burke plots of the data are shown. Data presented were the mean ± S.E. of three independent experiments with triplicate determinations at each data point in each experiment. <b>E-F. Selectivity of SD-208 against related kinases</b>. Inhibition of PKCα (<b>B</b>) or PKCδ (<b>C</b>) was determined at 10 nM, 100 nM, 1μM, and 10 μM. As controls, the PKC inhibitor GF109203X potently inhibited PKCα and PKCδ activity. Data are the mean ± SEM of two independent experiments. <b>G</b>. Inhibition of CAMKIIα was measured by the radiometric CAMK kinase assay. Data are the mean ± S.E. of two independent experiments with triplicate determinations at each data point in each experiment. Statistical significance was determined using the unpaired t-test. ns, not significantly significant; *, p<0.05; **, p<0.01; ***, p<0.001.