Novel protein kinase D inhibitors cause potent arrest in prostate cancer cell growth and motility

Abstract

Background: Protein kinase D (PKD) has been implicated in a wide range of cellular processes and pathological conditions including cancer. However, targeting PKD therapeutically and dissecting PKD-mediated cellular responses remains difficult due to lack of a potent and selective inhibitor. Previously, we identified a novel pan-PKD inhibitor, CID755673, with potency in the upper nanomolar range and high selectivity for PKD. In an effort to further enhance its selectivity and potency for potential in vivo application, small molecule analogs of CID755673 were generated by modifying both the core structure and side-chains.

Results: After initial activity screening, five analogs with equal or greater potencies as CID755673 were chosen for further analysis: kb-NB142-70, kb-NB165-09, kb-NB165-31, kb-NB165-92, and kb-NB184-02. Our data showed that modifications to the aromatic core structure in particular significantly increased potency while retaining high specificity for PKD. When tested in prostate cancer cells, all compounds inhibited PMA-induced autophosphorylation of PKD1, with kb-NB142-70 being most active. Importantly, these analogs caused a dramatic arrest in cell proliferation accompanying elevated cytotoxicity when applied to prostate cancer cells. Cell migration and invasion were also inhibited by these analogs with varying potencies that correlated to their cellular activity.

Conclusions: Throughout the battery of experiments, the compounds kb-NB142-70 and kb-NB165-09 emerged as the most potent and specific analogs in vitro and in cells. These compounds are undergoing further testing for their effectiveness as pharmacological tools for dissecting PKD function and as potential anti-cancer agents in the treatment of prostate cancer.

Figure 1

Synthesis and chemical structures of CID755673 and CID797718. CID755673, a compound identified and confirmed as a PKD1 inhibitor after interrogation of the PMLSC library, and CID797718, an analog of CID755673 obtained during the synthesis of the latter structure, were synthesized as described in "Methods".

Figure 2

Chemical structures and SAR of CID755673 and its analogs. A, Diagram describing the major structural zones dissected for SAR analysis. B, Chemical structures of the parental compound CID755673, previously identified and confirmed as a pan-PKD inhibitor, and of five analogs of CID755673.

Figure 3

Inhibition of PKD by CID755673 analogs in vitro. A-E, inhibition of recombinant human PKD1 in vitro. PKD kinase activity was assayed by a radiometric kinase assay in the presence of increasing concentrations of the CID755673 analogs. A 10-point concentration curve was generated for each compound for IC₅₀ determination. Each IC₅₀ was determined as the mean ± S.E.M. of three independent experiments with triplicate determinations at each concentration in each experiment. Representative graphs are shown.

Figure 4

Inhibition of PMA-induced endogenous PKD1 activation in LNCaP cells. LNCaP cells were pretreated with indicated concentrations of the five analogs for 45 min, then stimulated with 100 nM PMA for 20 min. Cell lysates were immunoblotted for p-S916-PKD1 and p-S742-PKD1. GAPDH was blotted as a loading control. The experiment was repeated at least three times and representative blots are shown.

Figure 5

Selectivity of the CID755673 analogs. Inhibition of PKCα (A), PKCβI (B), PKCδ (C), or CAMKIIα (D) by each of the 5 analogs was determined at 100 nM, 1 μM, and 10 μM concentrations. In the PKC assays, the potent PKC inhibitor GF109203X was used as a control. Data are the mean ± S.E.M. of three independent experiments. Statistical significance was determined using the unpaired t-test. ns, not statistically significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 6

Cytotoxic effects of the CID755673 analogs in PC3 cells. PC3 cells were seeded into 96-well plates (3000 cells/well) and were then incubated in media containing 0.3-100 μM inhibitors 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 EC₅₀ was determined as the mean ± S.E.M. of three independent experiments for each compound.

Figure 7

Effects of the CID755673 analogs on cell proliferation in PC3 cells. A, The analogs caused potent arrest in cell proliferation. PC3 cells were plated in triplicate in 24-well plates. Cells were allowed to attach overnight. A cell count at day 1 was made, and then either vehicle (DMSO) or the indicated compound at 10 μM concentration was added. Cells were counted daily for a total of 6 days. Media and inhibitor were refreshed every 2 days. The mean cell number ± S.E. was plotted over time. The experiment was repeated twice and a representative graph is shown. Statistical significance versus Day 1 cell count was determined by unpaired t-test and is indicated. *, p < 0.05; **, p < 0.01; ***, p < 0.001. B, The analogs caused G₂/M phase cell cycle arrest. PC3 cells were treated with either vehicle (DMSO), or 10 μM concentration of indicated compound for 48 hours. Cell cycle distribution was determined by flow cytometry after propidium iodide labeling of fixed cells. The experiment was repeated three times and a representative is shown for each compound.

Figure 8

CID755673 and its analogs cause accumulation of cyclin D1 and cyclin D3. A, PC3 cells were treated with increasing concentrations of CID755673 for 48 hrs. Inhibitor and growth media were refreshed after 24 hrs. Western blots for cyclin D1 and cyclin D3 are shown. B, PC3 cells were treated with 25 μM CID755673, 10 μM kb-NB142-70, 10 μM kb-NB165-09, 1 μM kb-NB165-92, or 10 μM kb-NB184-02 for 48 hrs. Note that 1 μM kb-NB165-92 was used in this assay since this compound at 10 μM caused significant cell death. Inhibitors and growth media were refreshed after 24 hrs. Western blots for cyclin D1 and D3 are shown. GAPDH was used as a loading control.

Figure 9

Effects of the CID755673 analogs on prostate cancer cell migration. The analogs inhibited wound healing in prostate cancer cells. DU145 cells (A) or PC3 cells (B) were grown to confluence in 6-well plates. The monolayer was wounded and imaged immediately. Cells were then treated with either vehicle (DMSO) or analogs at indicated concentration for 24 hours. Cells were then fixed and stained with 0.5% crystal violet. Percentage wound closure was calculated as an average of 9 determinations for each concentration/compound as described under "Methods." Data shown are the mean ± S.E.M. for three independent experiments. Statistical significance versus the DMSO control was determined by unpaired t-test in GraphPad Prism V. ns, not statistically significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 10

Analogs of CID755673 inhibit prostate cancer cell invasion. A, The analogs inhibited invasion in DU145 cells. 0.08 M DU145 cells in RPMI 1640 media containing 0.1% FBS and 10 μM of indicated compound were seeded into Matrigel inserts. After 22 hours, noninvasive cells were removed and invasive cells were fixed in 100% methanol, stained in 0.1% crystal violet solution, and photographed. The number of cells that invaded the Matrigel matrix was determined by cell counts in 5 fields relative to the number of cells that migrated through the control insert. The data shown is the mean ± S.E.M. of two independent experiments. Statistical significance versus the control DMSO was determined by unpaired t-test. ***, p < 0.001. B, Representative images comparing invasion of the vehicle (DMSO) and the compounds.