Table 1.
Primary hits identified in a PKD1 inhibitor screen of a targeted library.
Figure 1.
Inhibition of PKD isoforms by 1-NA-PP1 and IKK-16.
Inhibition of recombinant human PKD1, 2 and 3 was assayed in the presence of 10 different concentrations of 1-NA-PP1 (A) and IKK-16 (B) by an in vitro 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.
Figure 2.
IKK-16 and 1-NA-PP1 were ATP-competitive inhibitors of PKD.
PKD1 kinase activity was measured as a function of increasing concentrations of ATP in the presence of varying concentrations of 1-NA-PP1 (A) and IKK-16 (B). Lineweaver-Burke plots of the data are shown. Data presented were representative of three independent experiments.
Figure 3.
1-NA-PP1 did not inhibit PKC and CAMK.
Inhibition of PKCα (A) or PKCδ (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. C. 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; *, p<0.05; **, p<0.01; ***, p<0.001.
Figure 4.
Synthesis and SAR analysis of 1-NA-PP1 analogs.
A. 4-Zone model for 1-NA-PP1 analog synthesis. B. Synthesis of 1H-pyrazolo[3,4-d]pyrimidines 1.
Table 2.
Structures of 1-NA-PP1 analogs.
Figure 5.
Inhibition of PMA-induced activation of endogenous PKD1 by 1-NA-PP1 in cells.
A. LNCaP cells were pretreated with different doses of inhibitors for 45 min, followed by PMA stimulation at 10 nM for 20 min. Cell lysates were subjected to immunoblotting for p-S916-PKD1 and p-S744/748-PKD1. Tubulin was blotted as loading control. The experiment was repeated three times and the representative blots are shown. B. Determination of the IC50. Western blots were quantified using densitometry analysis. The data were plotted and IC50 values were derived the concentration-response curves using GraphPad. One of the three concentration-response curves was shown.
Figure 6.
1-NA-PP1 inhibited PC3 cell proliferation, survival, and arrested cells in G2/M.
A.1-NA-PP1 blocked PC3 cell proliferation. PC3 cells were plated in triplicates in 24-well plates. Cells were allowed to attach overnight. A cell count at day 1 was made, and then either a vehicle (DMSO) or 1-NA-PP1 at 10 µM was added. Cells were counted daily for a total of 5 days. Fresh media and inhibitor were added every 2 days. The means of triplicate determinations were plotted over time. The experiment was repeated twice and results from one representative experiment are shown. B. 1-NA-PP1 induced cell death 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 IC50 was determined as the mean of two independent experiments for each compound. C. 1-NA-PP1 caused G2/M phase cell cycle arrest. PC3 cells were treated with either vehicle (DMSO), or 10 µM 1-NA-PP1 for 48 h. Cell cycle distribution was determined by flow cytometry after propidium iodide labeling of fixed cells. Statistical significance was determined by unpaired t-test and is indicated. **, p<0.01; ***, p<0.001.
Figure 7.
1-NA-PP1-induced growth arrest was mediated through targeted inhibition of PKD.
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) (A) and (Adv-PKD3) PKD3 (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
Figure 8.
1-NA-PP1 blocked prostate cancer cell migration and invasion.
A.1-NA- PP1 blocked prostate cancer cell migration. PC3 cells were grown to confluence in 6-well plates. Monolayer was wounded and imaged immediately (0 h). Cells were then treated in growth media containing a vehicle (DMSO) or 30 µM of 1-NA-PP1 for 22 h and wound closure was measured. Percentage wound healing was calculated as the percent of healed wound area as compared to the original wound. B. 1-NA-PP1 inhibited prostate cancer cell invasion. DU145 cells were incubated with 30 µM 1-NA-PP1 in Matrigel inserts. After 20 h, noninvasive cells were removed and invasive cells were fixed in 100% methanol, stained in 0.4% hematoxylin solution, and photographed. The number of cells that invaded the Matrigel matrix was determined by cell counts in 6 fields relative to the number of cells that migrated through the control insert. Percentage invasion was calculated as the percent of the cells invaded through Matrigel inserts vs. the total cells migrated through the control inserts. C. Overexpressed PKD1 and PKD3 reversed the inhibitory effects of 1-NA-PP1 on tumor cell invasion. DU145 cells were infected with null, PKD1, and PKD3 adenoviruses (Adv-null, Adv-PKD1, and Adv-PKD3) at 100 MOI. After 24 h, cells were replated in control and Matrigel inserts, and a Matrigel invasion assay was conducted as described above. The overexpression of PKD1 and PKD3 was confirmed by Western blotting analysis (images below the graphs). All the above experiments were repeated at least three times and data from a representative experiment are shown.
Figure 9.
Mutating the gatekeeper amino acid sensitized PKD1 to the inhibition of 1-NA-PP1.
A.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. Expression of wild-type and mutant PKDs. HEK293 cells were transfected with wild-type and two gatekeeper mutants of Flag-PKD1 (Flag-PKD1M659G and Flag-PKD1M659A). Two days after transfection, cells were lysed and subjected to Western blotting for PKD1 and tubulin (loading control). C. 1-NA-PP1 concentration-dependently inhibited PMA-induced activation of Flag-PKD1 and Flag-PKD1M659G. HEK293 cells transfected with Flag-PKD1 and Flag-PKD1M659G 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.