Inhibition of p38δ and p38α by the PKD1 inhibitors.

<p>Inhibitory activities of the twenty-eight hits for p38δ at 1 µM (<b>A</b>) and the six most selective inhibitors for p38α at 10 and 100 nM (<b>B</b>) were evaluated using an <i>in vitro</i> p38 kinase assay. The representative graphs show % residual p38 kinase activity calculated based on the total kinase activity measured in the absence of inhibitors (DMSO). The experiment was performed twice with triplicate determinations at 1 µM for each compound and a representative graph is shown.</p

Selectivity of twenty-eight PKD1 inhibitors.

<p>Inhibition of PKCα (<b>A</b>), PKCδ (<b>B</b>), or CAMKIIα (<b>C</b>) by each of the twenty-eight hits was determined at 100 nM, 1 µM and 10 µM concentrations. In the PKC assays, GF109203X, a potent PKC inhibitor was used as control.</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

1-NA-PP1 did not inhibit PKC and CAMK.

<p>Inhibition of PKCα (<b>A</b>) or PKCδ (<b>B</b>) was determined at 10 nM, 100 nM, 1 µM, 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>C.</b> Inhibition of CAMKIIα was measured by the radiometric CAMK kinase assay. The experiment was repeated twice and a representative graph is shown. Statistical significance was determined using the unpaired t-test. ns, not statistically significant; *, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.001.</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

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

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

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

1-NA-PP1-induced growth arrest was mediated through targeted inhibition of PKD.

<p>Overexpression of PKD1 and PKD3 in prostate cancer cells rescued the anti-proliferative effects of 1-NA-PP1. PC3 (0.5 million) cells were seeded in a 60 mm dish and infected the next day with 50 and 100 MOI adenoviruses carrying PKD1 (Adv-PKD1) (<b>A</b>) and (Adv-PKD3) PKD3 (<b>B</b>). Empty adenovirus (Adv-null) was used as control. After 24 h, 3000 cells/well were plated in 96-well plates and treated with and without 10 and 30 µM 1-NA-PP1 for 72 h. MTT solution was added to each well and incubated for 4 h. Optical density was read at 570 nm to determine cell viability. The overexpression of PKD1 and PKD3 was confirmed by Western blotting analysis (images below the graphs). Statistical significance between DMSO and inhibitor treatment for each adenovirus as well as between control and PKD adenoviruses at each inhibitor concentration were determined by unpaired t-test in GraphPad Prism V. ns, not statistically significant; *, p<0.05; **, p<0.01; ***, p<0.001</p