PKD1 selective inhibitors with little or no inhibitory activity for PKCα, PKCδ or CAMKIIα.

<p>A list of PKD inhibitors that had ≤50% inhibitory activity for PKCα, PKCδ or CAMKIIα at 10 µM. Compounds 121, 122, 123, 139, 140, 209 (bold) were identified as “inactive” compounds for all three kinases.</p

Selectivity profiling of compounds 122 and 140.

<p>Listed in this table are all the protein kinases that bound compound <b>122</b> at over 50% at 10 µM. Their competition by compound <b>140</b> is listed in parallel. Compound <b>140</b> exhibited greater selectivity as compared to compound <b>122</b>. The data were obtained from profiling of a total of 353 kinases in the kinome. Enzymes competed by compound <b>140</b> at 99–100% are bolded.</p

The selectivity of compounds 122 and 140 presented on a dendrogram of the human kinome.

<p><b>A</b>. Compound 122 at 10 µM. <b>B</b>. Compound 140 at 10 µM. Pink circle represents inhibitory activity: <i>big circle</i>, 99–100% inhibition; <i>intermediate circle</i>, 91–98% inhibition; <i>small circle</i>, 51–90% inhibition.</p

Five models of human PKD1 kinase domain generated by I-TASSER server.

<p>Five models of human PKD1 kinase domain generated by I-TASSER server.</p

Chemical structures of novel PKD1 small molecule inhibitors identified from the screen.

<p>Twenty-eight PKD1 inhibitors were identified as primary hits in a screen using a radiometric PKD1 kinase assay. Hits were selected based on their ability to inhibit PKD1 at or above 50% at 1 µM.</p

Molecular modeling of compound 139 in the active site of a PKD1 homology model.

<p><b>A</b>. The docking result of the bioactive compound <b>139</b> in the ATP binding site of the PKD1 kinase domain. <i>carton ribbon and thick line</i>, PKD1; <i>ball and stick</i>, Compound <b>139</b>; <i>thin line</i>, residues in the binding pocket; <i>magenta line</i>, hydrogen bond. <b>B</b>. The proposed key contacts in the active site. <i>purple line</i>, hydrogen bond; residues in different colors: <i>purple</i>, basic; <i>pink</i>, acidic; <i>green</i>, hydrophobic; <i>gray</i>, hydrophilic.</p

Structures and PKD1 inhibitory activities of selected small molecule inhibitors identified in previous HTS assays.

<p>Structures and PKD1 inhibitory activities of selected small molecule inhibitors identified in previous HTS assays.</p

<i>In vitro</i> IC₅₀, cellular activity, and mode of action of representative compounds.

<p><b>A</b>. Concentration-dependent inhibition of PKD1 <i>in vitro</i> by two representative compounds, <b>139</b> and <b>209</b>. IC₅₀ values were calculated based on the dose-response curve. Data are represented as mean ± SEM of 3 independent experiments. <b>B</b>. Inhibition of endogenous PKD1 activity by the compounds in intact cells. LNCaP prostate cancer cells were pretreated with increasing concentration of compounds <b>139</b> and <b>209</b> for 45 min, followed by PMA stimulation at 10 nM for 20 min. Cell lysates were subjected to immunoblotting for pS⁹¹⁶-PKD1 and pS^744/748-PKD1. Tubulin was blotted as loading control. The experiments were repeated three times and representative blots are shown. <b>C</b>. Both compounds are ATP-competitive inhibitors. Lineweaver-Burk plots of compounds <b>139</b> and <b>209</b>. V_max and K_m values derived from the plots are shown in the table below each plot. Data are representative of three independent experiments.</p

Screen of a kinase inhibitor library for PKD1 activity.

<p>A targeted library of 235 compounds was screened for PKD1 activity at 1 µM using an <i>in vitro</i> radiometric PKD1 kinase assay. The representative graphs show % residual PKD1 kinase activity calculated based on the total kinase activity measured in the absence of inhibitors (DMSO). Kb-NB142-70, a previously known PKD inhibitor, was used as a positive control. Experiments were performed with triplicate determinations at 1 µM for each compound.</p