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

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

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

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

New Pyrazolopyrimidine Inhibitors of Protein Kinase D as Potent Anticancer Agents for Prostate Cancer Cells

<div><p>The emergence of protein kinase D (PKD) as a potential therapeutic target for several diseases including cancer has triggered the search for potent, selective, and cell-permeable small molecule inhibitors. In this study, we describe the identification, <i>in vitro</i> characterization, structure-activity analysis, and biological evaluation of a novel PKD inhibitory scaffold exemplified by 1-naphthyl PP1 (1-NA-PP1). 1-NA-PP1 and IKK-16 were identified as pan-PKD inhibitors in a small-scale targeted kinase inhibitor library assay. Both screening hits inhibited PKD isoforms at about 100 nM and were ATP-competitive inhibitors. Analysis of several related kinases indicated that 1-NA-PP1 was highly selective for PKD as compared to IKK-16. SAR analysis showed that 1-NA-PP1 was considerably more potent and showed distinct substituent effects at the pyrazolopyrimidine core. 1-NA-PP1 was cell-active, and potently blocked prostate cancer cell proliferation by inducing G2/M arrest. It also potently blocked the migration and invasion of prostate cancer cells, demonstrating promising anticancer activities on multiple fronts. Overexpression of PKD1 or PKD3 almost completely reversed the growth arrest and the inhibition of tumor cell invasion caused by 1-NA-PP1, indicating that its anti-proliferative and anti-invasive activities were mediated through the inhibition of PKD. Interestingly, a 12-fold increase in sensitivity to 1-NA-PP1 could be achieved by engineering a gatekeeper mutation in the active site of PKD1, suggesting that 1-NA-PP1 could be paired with the analog-sensitive PKD1^M659G for dissecting PKD-specific functions and signaling pathways in various biological systems.</p></div

Inhibition of PKD isoforms by 1-NA-PP1 and IKK-16.

<p>Inhibition of recombinant human PKD1, 2 and 3 was assayed in the presence of 10 different concentrations of 1-NA-PP1 (<b>A</b>) and IKK-16 (<b>B</b>) 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 concentration of drug in each experiment. The data were plotted as a function of drug concentration and a representative graph is shown.</p

IKK-16 and 1-NA-PP1 were ATP-competitive inhibitors of PKD.

<p>PKD1 kinase activity was measured as a function of increasing concentrations of ATP in the presence of varying concentrations of 1-NA-PP1 (<b>A</b>) and IKK-16 (<b>B</b>). Lineweaver-Burke plots of the data are shown. Data presented were representative of three independent experiments.</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

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

Structures of 1-NA-PP1 analogs.

<p>Structures of 1-NA-PP1 analogs.</p