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

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

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

Primary hits identified in a PKD1 inhibitor screen of a targeted library.

<p>A targeted protein kinase inhibitor library of 80 compounds was screened for PKD1 inhibitory activity at 1 µM using an <i>in vitro</i> radiometric PKD1 kinase assay. Sixteen compounds were selected as primary hits based on their ability to inhibit PKD1 at or above 50% at 1 µM. The % PKD1 inhibition referred to the percent inhibition of the total kinase activity measured in the absence of inhibitors (DMSO). Kb-NB142-70, a previously validated PKD inhibitor, was used as a positive control. Experiments were performed with triplicate determinations at 1 µM for each compound.</p

Mutating the gatekeeper amino acid sensitized PKD1 to the inhibition of 1-NA-PP1.

<p><b>A.</b>Alignment of the primary sequences containing the gatekeeper amino acid in PKD. Arrow indicates the consensus gatekeeper amino acid “Methionine” (M) in a shaded rectangle. <b>B.</b> Expression of wild-type and mutant PKDs. HEK293 cells were transfected with wild-type and two gatekeeper mutants of Flag-PKD1 (Flag-PKD1^M659G and Flag-PKD1^M659A). Two days after transfection, cells were lysed and subjected to Western blotting for PKD1 and tubulin (loading control). <b>C.</b> 1-NA-PP1 concentration-dependently inhibited PMA-induced activation of Flag-PKD1 and Flag-PKD1^M659G. HEK293 cells transfected with Flag-PKD1 and Flag-PKD1^M659G were serum-starved for 24 h and pre-treated with 1-NA-PP1 at increasing concentrations in serum-free medium for 45 min, followed by stimulation with PMA at 10 nM for 20 min. The cells were harvested and subjected to immunoblotting for p-S⁹¹⁶-PKD1, PKD1, and tubulin. The experiment was repeated three times and representative images from one experiment are shown.</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

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