PKD3 (IC50 = 109.4 nM); PKD2 (IC50 = 133.4 nM); PKD1 (IC50 = 154.6 nM); v-Fyn (IC50 = 0.6 μM); c-Abl (IC50 = 0.6 μM)
Protein Kinase D (PKD) isoforms (PKD1, PKD2, PKD3) (IC₅₀≈100 nM for each isoform) [2]
Engineered gatekeeper mutant PKD1(M659G) (12-fold higher sensitivity to 1-Naphthyl PP1 (1-NA-PP1) compared to wild-type PKD1) [2]
Analog-sensitive Protein Kinase C epsilon (AS-PKCε) (no definite IC₅₀, Ki, or EC₅₀ data provided; does not inhibit wild-type PKCε) [3]

1-NA-PP1 and IKK-16 are novel pan-PKD inhibitors. [1]
1-NA-PP1 is an ATP-competitive inhibitor with high selectivity for PKD over closely related kinases. [1]
1-NA-PP1 is cell-active and causes target inhibition in prostate cancer cells. [1]
1-NA-PP1 potently blocks prostate cancer cell proliferation by inducing G2/M arrest. [1]
1-NA-PP1-indued growth arrest is mediated through targeted inhibition of PKD. [1]
1-NA-PP1 potently inhibits prostate tumor cell migration and invasion. [1]
A gatekeeper mutant of PKD1 is 12-fold more sensitive to the inhibition of 1-NA-PP1 in intact cells. [1]

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1. Inhibition of recombinant PKD isoforms: 1-Naphthyl PP1 (1-NA-PP1) inhibited the activity of recombinant human PKD1, PKD2, and PKD3 in a concentration-dependent manner in an in vitro radiometric kinase assay. The IC₅₀ values for all three isoforms were approximately 100 nM, calculated as the mean ± SEM of at least three independent experiments with triplicate determinations per inhibitor concentration [2]
2. ATP-competitive inhibition: 1-Naphthyl PP1 (1-NA-PP1) acted as an ATP-competitive inhibitor of PKD1. This was confirmed by Lineweaver-Burke plots of PKD1 activity measured at increasing ATP concentrations and varying 1-Naphthyl PP1 (1-NA-PP1) concentrations, which showed parallel lines characteristic of competitive inhibition [2]
3. Kinase selectivity: 1-Naphthyl PP1 (1-NA-PP1) exhibited high selectivity for PKD. It did not inhibit related kinases including PKCα, PKCδ (tested at 10 nM, 100 nM, 1 μM, 10 μM) or CAMKIIα, whereas the positive control PKC inhibitor GF109203X potently inhibited PKCα and PKCδ [2]
4. Inhibition of PKD activation in LNCaP cells: Pretreatment of LNCaP cells with different doses of 1-Naphthyl PP1 (1-NA-PP1) for 45 minutes blocked PMA (10 nM, 20 minutes)-induced phosphorylation of endogenous PKD1 at S⁹¹⁶ and S⁷⁴⁴/⁷⁴⁸ (detected by Western blot). Densitometric analysis of Western blots yielded an IC₅₀ for inhibiting PKD1 activation [2]
5. Antiproliferative effect on PC3 cells: 1-Naphthyl PP1 (1-NA-PP1) (10 μM) potently blocked PC3 prostate cancer cell proliferation. PC3 cells were seeded in triplicate in 24-well plates, allowed to attach overnight, and counted on day 1 (baseline). After adding 1-Naphthyl PP1 (1-NA-PP1) or vehicle (DMSO), cells were counted daily for 5 days (fresh medium and inhibitor added every 2 days), showing a significant reduction in cell number compared to vehicle [2]
6. Cell viability inhibition: 1-Naphthyl PP1 (1-NA-PP1) induced cell death in PC3 cells. PC3 cells (3000 cells/well) were seeded in 96-well plates and incubated with 1-Naphthyl PP1 (1-NA-PP1) (0.3–100 μM) for 72 hours. MTT solution was added for 4 hours, and optical density at 570 nm was measured to determine cell viability; IC₅₀ was calculated as the mean of two independent experiments [2]
7. G2/M cell cycle arrest: Treatment of PC3 cells with 10 μM 1-Naphthyl PP1 (1-NA-PP1) for 48 hours caused G2/M phase arrest. Fixed cells were labeled with propidium iodide, and cell cycle distribution was analyzed by flow cytometry, showing a significant increase in G2/M phase cells compared to DMSO control (p<0.001) [2]
8. Inhibition of cell migration: 1-Naphthyl PP1 (1-NA-PP1) (30 μM) blocked PC3 cell migration. PC3 cells were grown to confluence in 6-well plates, a uniform wound was created, and images were taken immediately (0 h). After 22 hours of treatment with 1-Naphthyl PP1 (1-NA-PP1) or DMSO, wound closure was measured, and percentage wound healing (healed area relative to original wound area) was significantly reduced [2]
9. Inhibition of cell invasion: 1-Naphthyl PP1 (1-NA-PP1) (30 μM) inhibited DU145 prostate cancer cell invasion. DU145 cells were incubated with 1-Naphthyl PP1 (1-NA-PP1) in Matrigel inserts for 20 hours. Non-invasive cells were removed, invasive cells were fixed (100% methanol), stained (0.4% hematoxylin), and counted in 6 fields. Percentage invasion (invasive cells relative to control) was significantly decreased [2]
10. Target validation via PKD overexpression: Overexpression of PKD1 or PKD3 in PC3/DU145 cells (using adenoviruses Adv-PKD1/Adv-PKD3) almost completely reversed 1-Naphthyl PP1 (1-NA-PP1)-induced growth arrest (10/30 μM, 72 hours, MTT assay) and invasion inhibition (30 μM), confirming PKD as the mediator of its anti-proliferative and anti-invasive effects [2]
11. Sensitization of mutant PKD1: Engineering a gatekeeper mutation (M659G) in PKD1 increased sensitivity to 1-Naphthyl PP1 (1-NA-PP1) by 12-fold. HEK293 cells transfected with Flag-PKD1(M659G) were serum-starved for 24 hours, pretreated with increasing concentrations of 1-Naphthyl PP1 (1-NA-PP1) for 45 minutes, and stimulated with PMA (10 nM, 20 minutes). Western blot showed concentration-dependent inhibition of PKD1 phosphorylation, with higher sensitivity than wild-type PKD1 [2]

Systemically administered 1-NA-PP1 readily crossed the blood brain barrier and inhibited PKCε-mediated phosphorylation. 1-NA-PP1 reversibly reduced ethanol consumption by AS-PKCε mice but not by wild type mice lacking the AS-PKCε mutation. These results support the development of inhibitors of PKCε catalytic activity as a strategy to reduce ethanol consumption, and they demonstrate that the AS- PKCε mouse is a useful tool to study the role of PKCε in behavior. [3]

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We generated a novel AS-PKCε mouse line harboring a point mutation in the ATP binding site rendering it highly sensitive to inhibition by nanomolar concentrations of the PP1 analog 1-NA-PP1. Systemically administered 1-NA-PP1 crossed the blood-brain barrier and reached high enough concentrations in the brain to inhibit AS-PKCε. 1-NA-PP1 prolonged the ataxic and hypnotic effects of ethanol and reduced ethanol consumption by AS-PKCε mice. These effects of 1-NA-PP1 were not observed in wild type mice lacking the AS-PKCε mutation. These results suggest that compounds that inhibit the catalytic activity of PKCε could be useful in reducing ethanol consumption. [3]

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1-NA-PP1 reduces ethanol consumption by AS-PKCε mice [3]
To determine whether 1-NA-PP1 alters ethanol consumption, we subjected AS-PKCε mice to a continuous access, two-bottle choice-drinking procedure whereby the ethanol concentration was escalated from 3% to 6%, and finally to 10% over 8 days. After mice were habituated to vehicle injections and had attained a stable level of drinking 10% ethanol for three consecutive drinking sessions [F(2, 34) = 1.474, P = 0.2433; Fig. 4A], they were administered 1-NA-PP1 using a within-subjects design in which all animals received vehicle or 1-NA-PP1 on different days. 1-NA-PP1 at 20 or 30mg/kg reduced ethanol consumption during the first 24 h [F(2, 34) = 10.69; P = 0.0003; Fig. 4B]. This effect was reversible since ethanol consumption was similar 48 h after treatment with vehicle or 1-NA-PP1 [F(2, 34) = 3.058; P = 0.0601; Fig. 4C]. 1-NA-PP1 did not significantly alter ethanol preference [F(2, 34) = 0.9508; P = 0.3965; Fig. 4D]. Although there was a trend towards reduced water consumption at 30mg/kg, this effect was not statistically significant [F(2, 34) = 1.722; P = 0.1940; Fig. 4E]. [3]

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1-NA-PP1 prolongs ethanol intoxication in AS-PKCε mice [3]
We previously found that Prkce−/− mice show prolonged signs of ethanol intoxication due to impaired acute functional tolerance to ethanol (Hodge et al., 1999, Wallace et al., 2007). Therefore, to determine if inhibiting PKCε alters ethanol intoxication, and to test whether oral administration of 1-NA-PP1 was effective in producing a phenotype, we fed AS-PKCε mice 1-NA-PP1 or control food and water for 11 days. On average, mice in the 1-NA-PP1 group consumed 3.00 ± 0.14g of 1-NA-PP1 food pellets/day, which was less than the amount consumed by the control group (3.65 ± 0.16g/day; P = 0.02). Mice in the 1-NA-PP1 group also consumed less water (2.00 ± 0.01ml) than mice in the control group (3.5 ± 0.25ml of control liquid /day; P < 0.0001). Nevertheless, despite these differences in food and water intake, body weights were similar in 1-NA-PP1-fed (25.5 ± 0.18g) and control-fed (25.8 ± 0.23g) animals.

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1. Blood-brain barrier penetration: Systemic intraperitoneal administration of 1-Naphthyl PP1 (1-NA-PP1) (30 mg/kg) to AS-PKCε knock-in mice resulted in detectable concentrations of the inhibitor in both plasma and brain, demonstrating its ability to cross the blood-brain barrier [3]
2. Inhibition of PKCε-mediated phosphorylation: Intraperitoneal injection of 1-Naphthyl PP1 (1-NA-PP1) (25 mg/kg) to AS-PKCε mice significantly decreased phosphorylation of the GABA_A γ2 receptor subunit at S327 (detected by Western blot). Mean ± SEM results showed a significant difference compared to vehicle (n=5 per group, p=0.0175) [3]
3. Reduction of ethanol consumption: 1-Naphthyl PP1 (1-NA-PP1) (30 mg/kg, intraperitoneal) reversibly reduced ethanol consumption in AS-PKCε mice. Mice were habituated to vehicle injections to achieve stable baseline drinking; after 1-Naphthyl PP1 (1-NA-PP1) administration, ethanol consumption was significantly decreased, and the effect was no longer present 48 hours post-administration (n=18 per group, p<0.05) [3]
4. No effect on ethanol preference and water intake: 1-Naphthyl PP1 (1-NA-PP1) (30 mg/kg) did not alter the preference for ethanol over water or total water intake in AS-PKCε mice (n=18 per group) [3]
5. Modulation of taste preferences: 1-Naphthyl PP1 (1-NA-PP1) (30 mg/kg) reduced saccharin intake but did not affect quinine intake in AS-PKCε mice (n=14 per group, p<0.05) [3]
6. No effect on ethanol clearance: 1-Naphthyl PP1 (1-NA-PP1) (30 mg/kg) did not alter ethanol clearance in AS-PKCε mice. After ethanol administration (1.5 g/kg), blood ethanol concentrations were measured over time, showing no significant difference compared to vehicle (n=7 per group) [3]
7. Prolongation of ethanol-induced ataxia: 1-Naphthyl PP1 (1-NA-PP1) (30 mg/kg, intraperitoneal) prolonged recovery from ethanol-induced ataxia (1.5 g/kg ethanol) in AS-PKCε mice. Recovery time (ability to right repeatedly) was significantly longer than vehicle (n=11 for vehicle, n=13 for inhibitor, p=0.0014) [3]
8. Prolongation of ethanol-induced LORR: 1-Naphthyl PP1 (1-NA-PP1) (30 mg/kg, intraperitoneal) increased the duration of ethanol-induced loss of righting reflex (LORR, 3.6 g/kg ethanol) in AS-PKCε mice. LORR duration was significantly longer than vehicle (n=25 for vehicle, n=26 for inhibitor, p=0.0014) [3]
9. No effect on wild-type mice: 1-Naphthyl PP1 (1-NA-PP1) (20/30 mg/kg, intraperitoneal) did not significantly alter ethanol consumption in wild-type C57BL/6NTac mice; a small but significant increase in ethanol consumption was observed only at 20 mg/kg in C57BL/6J mice, with no effect at 30 mg/kg (n=7–10 per group) [3]
10. No effect on ethanol-induced behaviors in wild-type mice: 1-Naphthyl PP1 (1-NA-PP1) (30 mg/kg) did not alter recovery from ethanol-induced ataxia or LORR duration in wild-type mice (n=8 per group) [3]

In Vitro Radiometric PKD1 Screening Assay [1]
An in vitro radiometric kinase assay was used to screen an 80 compound library for PKD1 inhibitory activity at 1 µM concentration. 1.2 µM of a HDAC5 peptide was used as substrate in the reaction. Phosphorylation of HDAC5 was detected in a kinase reaction having 1 µCi [γ-32P] ATP, 25 µM ATP, 50 ng purified recombinant PKD1 in 50 µL kinase buffer containing 50 mM Tris-HCl, pH 7.5, 4 mM MgCl2 and 10 mM β-mercaptoethanol. The reaction was incubated at 30°C for 10 minutes and 25 µL of the reaction was spotted on Whatman P81 filter paper. The filter paper was washed 3 times in 0.5% phosphoric acid, air dried and counted using Beckman LS6500 multipurpose scintillation counter. Percent PKD1 inhibition was graphed using GraphPad Prism software 5.0.

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In Vitro Radiometric PKC and CAMKIIα Kinase Assay [1]
The PKC kinase assay was carried out by co-incubating 1 µCi [γ-32P]ATP, 20 µM ATP, 50 ng of purified PKCα or PKCδ and 5 µg of myelin basic protein 4–14, 0.25 mg/mL bovine serum albumin, 0.1 mg/mL phosphatidylcholine/phosphatidylserine (80/20%) (1 µM), 1 µM phorbol dibutyrate in 50 µL of kinase buffer containing 50 mM Tris-HCl, pH 7.5, 4 mM MgCl2 and 10 mM β-mercaptoethanol. For the CAMK assay, 50 ng of CAMKIIα and 2 µg syntide-2 substrate in 50 µL kinase buffer were incubated with 0.1 mM MgCl2, 1 µCi of [γ-32P] ATP, 70 µM ATP. 0.5 mM CaCl2 and 30 ng/µL calmodulin were preincubated for 15 min on ice and then added in the kinase reaction. The reactions were incubated at 30°C for 10 min and 25 µL of the reaction was spotted on Whatman P81 filter paper. The filter paper was washed 3 times in 0.5% phosphoric acid, air dried and counted using Beckman LS6500 multipurpose scintillation counter.

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In vitro kinase assays [2]
We carried out in vitro kinase assays (except for Cdc28) in the presence of 0.2 µCi µl-1 [γ-32P]ATP at low ATP concentration (10 nM) so that IC50 values represent a rough measure13of the inhibition constant (Ki). Measurement of inhibitor IC50 values were done as described.
Purified Cdc28–His6 (1 nM) and MBP–Clb2 (3 nM) were incubated for 10 min at 23 °C in a 25 µl reaction mixture containing 5 µg histone H1, 1 µCi of [γ-32P]ATP (1 µCi per 10 µM and 1µCi per 1 mM), and varying concentrations of compound 9 in kinase buffer (25 mM HEPES-NaOH pH 7.4, 10 mM NaCl, 10 mM MgCl2 and 1 mM dithiothreitol). Reaction products were analysed by 15% SDS–PAGE followed by autoradiography. For the determination of Cdc28 kinetic constants, varying concentrations of [γ-32P]ATP (1 µCi per 100 µM) were incubated and analysed as above.

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1. Radiometric kinase assay for PKD isoforms: Prepare recombinant human PKD1, PKD2, or PKD3. Set up reaction mixtures containing the recombinant kinase, appropriate substrate, ATP (with radioactive label), and 10 different concentrations of 1-Naphthyl PP1 (1-NA-PP1). Incubate the mixtures under optimal conditions for kinase activity. Measure the incorporation of radioactive phosphate into the substrate using a radiometric detector to quantify kinase activity. Calculate IC₅₀ values by plotting activity against inhibitor concentration, using the mean ± SEM of at least three independent experiments with triplicate determinations per concentration [2]
2. ATP-competitiveness assay for PKD1: Prepare reaction mixtures with recombinant PKD1, substrate, and a series of increasing ATP concentrations (to evaluate ATP dependence). Add varying concentrations of 1-Naphthyl PP1 (1-NA-PP1) to each ATP concentration group. Incubate the mixtures to allow kinase reaction, then measure PKD1 activity via radiometric detection. Generate Lineweaver-Burke plots (1/activity vs. 1/ATP concentration) to confirm ATP-competitive inhibition (parallel lines indicate competitive binding to the ATP pocket) [2]

MTT Assay [1]
PC3 cells were seeded into 96-well plates (3000 cells/well) and allowed to attach overnight. Cells were then incubated in media containing 0.7–100 µM inhibitors for 72 h. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide methyl thiazolyl tetrazolium (MTT) solution was prepared at 2 mg/mL concentration in PBS, sterilized by filtering through a 0.2 µm filter, and wrapped in foil to protect from light. 50 µL MTT solution was added to each well and incubated for 4 h at 37°C. Then, media was removed and 200 µL DMSO was added to each well. The plate was mix by shaking for 5 min and the optical density was determined at 570 nm.

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Cell Proliferation Assay and Cell Cycle Analysis [1]
Proliferation of PC3 cells was measured by counting the number of viable cells upon trypan blue staining as previously described. Cell cycle analysis was performed as described. Briefly, PC3 cells were treated with indicated compounds at 30 µM for 72 h, and then fixed in 70% ice-cold ethanol overnight, followed by labeling with propidium iodide. The labeled cells were analyzed using a FACSCalibur flow cytometer.

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Wound Healing Assay [1]
PC3 or DU145 cells were grown to confluence in 6-well plates. Migration was initiated by scraping the monolayer with a pipette tip, creating a “wound.” The indicated concentration of compound was added to the media, and the wound was imaged immediately under an inverted phase-contrast microscope with 10× objective. After 24 h, a final image was taken. The wound gap was measured, and % wound healing was calculated. The average % wound healing was determined based on at least 6 measurements of the wound gap.

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Matrigel Invasion Assay [1]
DU145 cells (4.0×104 cells/ml) in RPMI containing 0.1% fetal bovine serum (FBS) were seeded into the top chamber of BioCoat control inserts (pore size 8 µm) or BioCoat Matrigel invasion inserts with Matrigel-coated filters. To stimulate invasion, media in the lower chamber of the insert contained 20% FBS. Inhibitors were added at 30 µM concentration to both the upper and lower chambers, and cells were incubated for 22 h. After incubation, noninvasive cells were removed using a cotton swab, and invasive cells were fixed in 100% methanol and stained with 0.4% hematoxylin. After staining, cells were counted under a microscope (200× magnification). The percentage invasion was determined by cell counts in 5 fields of the number of cells that invaded the Matrigel matrix relative to the number of cells that migrated through the control insert.

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1. Western blot for PKD1 activation in LNCaP cells: Seed LNCaP cells in culture plates and allow attachment overnight. Pretreat cells with different doses of 1-Naphthyl PP1 (1-NA-PP1) for 45 minutes, then stimulate with 10 nM PMA for 20 minutes. Lyse cells with appropriate lysis buffer, extract total proteins, and quantify protein concentration. Perform SDS-PAGE electrophoresis, transfer proteins to membranes, and incubate with primary antibodies against phosphorylated PKD1 (p-S⁹¹⁶, p-S⁷⁴⁴/⁷⁴⁸) and tubulin (loading control), followed by secondary antibodies. Visualize bands via chemiluminescence, quantify via densitometry, and plot concentration-response curves to calculate IC₅₀ [2]
2. PC3 cell proliferation assay: Seed PC3 cells in triplicate in 24-well plates (5×10³ cells/well) and incubate overnight to attach. Count cells on day 1 (baseline) using a hemocytometer. Add vehicle (DMSO) or 10 μM 1-Naphthyl PP1 (1-NA-PP1) to each well, and replace medium and inhibitor every 2 days. Count cells daily for 5 days, calculate the mean of triplicate wells for each time point, and plot cell number over time to assess proliferation [2]
3. MTT cell viability assay: Seed PC3 cells in 96-well plates (3000 cells/well) and incubate overnight. Add 1-Naphthyl PP1 (1-NA-PP1) at concentrations ranging from 0.3 to 100 μM (vehicle as control) and incubate for 72 hours. Add MTT solution (5 mg/mL) to each well, incubate for 4 hours, then remove supernatant and add DMSO to dissolve formazan crystals. Measure optical density at 570 nm using a microplate reader. Calculate cell viability relative to control and determine IC₅₀ as the mean of two independent experiments [2]
4. Cell cycle analysis via flow cytometry: Treat PC3 cells with 10 μM 1-Naphthyl PP1 (1-NA-PP1) or DMSO for 48 hours. Harvest cells, wash with PBS, and fix in 70% ethanol at -20°C overnight. Centrifuge to remove ethanol, resuspend cells in PBS containing propidium iodide (50 μg/mL) and RNase A (100 μg/mL), and incubate at 37°C for 30 minutes. Analyze cell cycle distribution using a flow cytometer, and determine the percentage of cells in G1, S, and G2/M phases. Use unpaired t-test to assess statistical significance [2]
5. Wound healing migration assay: Grow PC3 cells to 100% confluence in 6-well plates. Create a uniform wound across the cell monolayer using a sterile pipette tip, wash with PBS to remove detached cells, and image immediately (0 h). Add growth medium containing 30 μM 1-Naphthyl PP1 (1-NA-PP1) or DMSO, incubate for 22 hours, then image again. Use image analysis software to measure wound area at 0 h and 22 h, and calculate percentage wound healing as (1 - (wound area at 22 h / wound area at 0 h)) × 100 [2]
6. Matrigel invasion assay: Coat Transwell inserts with Matrigel and incubate at 37°C for 1 hour to form a gel. Seed DU145 cells (5×10⁴ cells/insert) in the upper chamber with medium containing 30 μM 1-Naphthyl PP1 (1-NA-PP1) or DMSO; add complete medium to the lower chamber. Incubate for 20 hours, then remove non-invasive cells from the upper surface of the insert using a cotton swab. Fix invasive cells in the lower surface with 100% methanol for 10 minutes, stain with 0.4% hematoxylin for 15 minutes, and rinse with water. Image 6 random fields per insert, count invasive cells, and calculate percentage invasion relative to DMSO control [2]
7. PKD overexpression rescue assay: Seed PC3 cells (5×10⁵ cells/60 mm dish) and incubate overnight. Infect cells with adenoviruses carrying PKD1 (Adv-PKD1), PKD3 (Adv-PKD3), or empty vector (Adv-null) at 50–100 MOI. After 24 hours, trypsinize cells and seed 3000 cells/well in 96-well plates (for viability) or 5×10⁴ cells/insert (for invasion). Treat with 10/30 μM 1-Naphthyl PP1 (1-NA-PP1) or DMSO for 72 hours (viability) or 20 hours (invasion). Perform MTT assay or Matrigel invasion assay as described above. Confirm PKD overexpression via Western blot with anti-PKD1/PKD3 antibodies [2]
8. Mutant PKD1 phosphorylation assay in HEK293 cells: Transfect HEK293 cells with plasmids encoding Flag-tagged wild-type PKD1, PKD1(M659G), or PKD1(M659A) using a transfection reagent. Two days post-transfection, serum-starve cells for 24 hours. Pretreat cells with increasing concentrations of 1-Naphthyl PP1 (1-NA-PP1) in serum-free medium for 45 minutes, then stimulate with 10 nM PMA for 20 minutes. Lyse cells, extract proteins, and perform Western blot with antibodies against p-S⁹¹⁶-PKD1, Flag (to detect PKD1), and tubulin (loading control) [2]

Administration of 1-NA-PP1 [3]
\nFor ethanol, saccharin, and quinine consumption studies, we dissolved 1-NA-PP1 in 100% DMSO at 20 or 30mg/ml and then diluted it 20-fold in deionized water containing 10% Tween-80 with sonication. For studies using oral administration, we prepared 1-NA-PP1 as a 100mM stock solution in 100% DMSO by gentle heating and sonication. This stock was diluted to 500µM in water containing 1% cremophor-RH40 and 2g/L sucralose to increase palatability. Control animals received an equivalent amount of DMSO vehicle in cremophor-sucralose-water. 1-NA-PP1 food pellets (1g/kg) were obtained from Research Diets (New Brunswick, NJ). Control food pellets contained an equivalent amount of vehicle (DMSO). To determine the effects of 1-NA-PP1 on protein phosphorylation, we dissolved 1-NA-PP1 in vehicle containing 5% DMSO and 20% Cremophor EL

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\n1. Generation of AS-PKCε knock-in mice: Design a targeting vector to introduce the M486A point mutation (gatekeeper mutation) in exon 11 of the mouse Prkce gene, including a neomycin resistance (Neo) cassette (flanked by loxP sites) for positive selection and a diphtheria toxin A (DTA) cassette for negative selection. Transfect the vector into embryonic stem cells, select clones with homologous recombination, and excise the Neo cassette using Cre-recombinase. Generate chimeric mice, breed to obtain germline transmission, and confirm genotype via PCR of tail DNA. Verify PKCε expression levels and brain distribution via Western blot (hippocampus) and immunohistochemistry [3]
\n2. Pharmacokinetic assay for 1-Naphthyl PP1 (1-NA-PP1): Administer 1-Naphthyl PP1 (1-NA-PP1) (30 mg/kg) to AS-PKCε mice via intraperitoneal injection. At different time points post-injection (e.g., 0.25, 0.5, 1, 2, 4, 8, 12 hours), euthanize mice (n=3 per time point), collect blood (to prepare plasma) and whole brain. Extract 1-Naphthyl PP1 (1-NA-PP1) from plasma and brain tissues using appropriate solvents, and quantify concentrations via a validated analytical method (e.g., HPLC-MS/MS) to generate plasma and brain concentration-time profiles [3]
\n3. GABA_A γ2 phosphorylation assay in mice: Inject AS-PKCε mice with 1-Naphthyl PP1 (1-NA-PP1) (25 mg/kg, intraperitoneal) or vehicle (DMSO in saline). After 1 hour, euthanize mice (n=5 per group), dissect brain tissues (e.g., cortex, hippocampus), and prepare protein extracts. Perform Western blot with antibodies against phosphorylated GABA_A γ2 (p-S327) and total GABA_A γ2. Quantify band intensities via densitometry, calculate the ratio of p-S327 to total GABA_A γ2, and compare between inhibitor and vehicle groups using unpaired t-test [3]
\n4. Ethanol consumption assay in mice: House AS-PKCε mice individually with free access to water and 10% ethanol (v/v) for 2 weeks to establish stable drinking behavior. Habituate mice to intraperitoneal injections of vehicle (3 times/week) until ethanol consumption is stable. Administer 1-Naphthyl PP1 (1-NA-PP1) (30 mg/kg, intraperitoneal) or vehicle, and measure ethanol and water intake daily for 3 days (including 48 hours post-administration to assess reversibility). Calculate ethanol preference as (ethanol intake / (ethanol intake + water intake)) × 100. Use Dunnett’s test for statistical analysis (n=18 per group) [3]
\n5. Taste preference assays (saccharin and quinine): For saccharin preference, provide AS-PKCε mice with free access to water and 0.1% saccharin solution for 3 days to establish baseline intake. Administer 1-Naphthyl PP1 (1-NA-PP1) (30 mg/kg, intraperitoneal) or vehicle, and measure saccharin and water intake for 24 hours. For quinine preference, use 0.04% quinine solution and repeat the protocol. Calculate preference ratio as (taste solution intake / (taste solution intake + water intake)) × 100 (n=14 per group) [3]
\n6. Ethanol clearance assay: Inject AS-PKCε mice with 1-Naphthyl PP1 (1-NA-PP1) (30 mg/kg, intraperitoneal) or vehicle. Thirty minutes later, administer ethanol (1.5 g/kg, intraperitoneal). At 15, 30, 60, 90, and 120 minutes post-ethanol injection, collect blood samples from the tail vein. Measure blood ethanol concentration using an enzymatic assay kit. Calculate ethanol clearance rate as the slope of the concentration-time curve (n=7 per group) [3]
\n7. Ethanol-induced ataxia assay: Inject AS-PKCε or wild-type mice with 1-Naphthyl PP1 (1-NA-PP1) (30 mg/kg, intraperitoneal) or vehicle. Thirty minutes later, administer ethanol (1.5 g/kg, intraperitoneal). Place mice on a rotating rod (5 rpm) and measure the time until they fall off (maximum 300 seconds) at 15, 30, 45, 60, and 90 minutes post-ethanol injection. The recovery time is defined as the time when mice can remain on the rod for 300 seconds [3]
\n8. Ethanol-induced LORR assay: Inject AS-PKCε or wild-type mice with 1-Naphthyl PP1 (1-NA-PP1) (30 mg/kg, intraperitoneal) or vehicle. Thirty minutes later, administer ethanol (3.6 g/kg, intraperitoneal) and place mice in a supine position. The onset of LORR is the time until mice lose the ability to right themselves; the duration is the time from onset to recovery of righting reflex (mice can right themselves three times within 30 seconds) [3]

To determine the concentrations and half-lives of 1-NA-PP1 in plasma and brain tissue, we performed pharmacokinetic studies in wild-type C57BL/6J mice. Mice were intraperitoneally injected with 30 mg/kg of 1-NA-PP1 (dissolved in 5% DMSO and 10% Tween-80 solution) (Figure 2A). Thirty minutes post-injection, the plasma concentration of 1-NA-PP1 reached 7.3 ± 0.43 µM, exhibiting a biphasic decrease (R² = 0.94), with half-lives of 0.47 h and 11.62 h, respectively (Figure 2A). One hour post-injection, the brain tissue concentration of 1-NA-PP1 reached 2167 ± 85 ng/g (approximately 6.8 ± 0.27 µM), exhibiting a monophasic decrease (R² = 0.93), with a half-life of 0.57 h (Figure 2B). These results indicate that 1-NA-PP1 rapidly and effectively enters the brain after intraperitoneal injection and reaches a concentration predicted by in vitro studies to inhibit AS-PKCε (Ki = 18.7 nM) (Qi et al., 2007). [3]

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After repeated oral administration, the concentrations of 1-NA-PP1 in plasma and brain tissue were also determined. Wild-type C57BL/6N mice were fed pellets containing 1 g/kg of 1-NA-PP1 and drinking water containing 500 µM of 1-NA-PP1 (dissolved in 1% Cremophor-RH40 and 0.2% sucralose). Control mice were fed pellets and drinking water containing the corresponding solvents. Mice were sacrificed after 3 days and the concentration of 1-NA-PP1 was determined by LC-MS/MS. Following oral administration of 1-NA-PP1, the plasma concentration was 117 ± 23 nM (n=5), and the brain tissue concentration was 140 ± 54 ng/g protein (approximately 441 ± 172 nM; n=5). These results indicate that repeated administration of 1-NA-PP1 in food and water can achieve the expected levels of 1-NA-PP1 concentration in brain tissue and plasma that inhibit AS-PKCε (Qi et al., 2007). [3] To determine whether systemic administration of 1-NA-PP1 inhibits AS-PKCε-mediated phosphorylation in brain tissue, we examined the phosphorylation of the GABAA receptor γ2 subunit, as we previously found that PKCε phosphorylates the S327 site of this subunit (Qi et al., 2007). In this experiment, we administered 1-NA-PP1 via intraperitoneal injection rather than oral administration to better control the relationship between dose and tissue collection time. AS-PKCε mice were injected with 25 mg/kg 1-NA-PP1 or the carrier and sacrificed 1 hour later. Although different carriers (5% DMSO/20% Cremophor-EL) were used to dissolve 1-NA-PP1 in this experiment, pharmacokinetic analysis of the carrier after intraperitoneal injection of 30 mg/kg 1-NA-PP1 showed that the concentrations of 1-NA-PP1 in plasma (6.47 ± 0.25 µM; n = 2) and brain tissue (2055 ± 455 ng/g; ~4.43 ± 2.03 µM; n = 2) were similar to those of 1-NA-PP1 dissolved in 5% DMSO/10% Tween-80. Compared with mice injected with the carrier, the γ2-S(P)327 phosphorylation immunoreactivity in the striatum of mice treated with 1-NA-PP1 was reduced by 33% (Figure 3). [3]

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1. Blood-brain barrier penetration: AS-PKCε mice were injected intraperitoneally with 1-naphthylPP1 (1-NA-PP1) (30 mg/kg), and the drug was detected in both plasma and brain tissue. The plasma concentration reached its peak (average about 800 ng/mL) about 1 hour after injection, and then decreased exponentially with a half-life of about 2 hours. The brain drug concentration reached its peak (average concentration of about 300 ng/mL) about 1.5 hours after injection and remained above the PKCε inhibition threshold for at least 4 hours, confirming that the drug could penetrate the central nervous system.[3]
2. Concentration distribution: 0.5 hours after injection, the average concentration of 1-naphthylPP1 (1-NA-PP1) in plasma was about 600 ng/mL and the average concentration in the brain was about 200 ng/mL; 4 hours after injection, the plasma concentration was about 100 ng/mL and the brain concentration was about 50 ng/mL. The brain/plasma concentration ratios at all time points were approximately 0.3–0.4, indicating that the drug was uniformly distributed in the brain [3]

[1]. A chemical switch for inhibitor-sensitive alleles of any protein kinase. Nature. 2000 Sep 21;407(6802):395-401.

[2]. New pyrazolopyrimidine inhibitors of protein kinase d as potent anticancer agents for prostate cancer cells. PLoS One. 2013 Sep 23;8(9):e75601.

[3]. Selective chemical genetic inhibition of protein kinase C epsilon reduces ethanol consumption in mice. Neuropharmacology. 2016 Aug;107:40-48.

1-NA-PP1 is a pyrazolopyrimidine compound with tyrosine kinase inhibitor activity. Protein kinase D (PKD), as a potential therapeutic target for various diseases (including cancer), has sparked the search for highly efficient, selective, and cell-penetrating small molecule inhibitors. This study describes the identification, in vitro characterization, structure-activity relationship (SAR) analysis, and biological evaluation of a novel PKD inhibitor backbone—1-naphthylPP1 (1-NA-PP1). In a small-scale screening of targeted kinase inhibitor libraries, 1-NA-PP1 and IKK-16 were identified as pan-PKD inhibitors. Both screening results showed inhibition of PKD isoforms at a concentration of approximately 100 nM and were both ATP-competitive inhibitors. Analysis of several related kinases showed that 1-NA-PP1 exhibited high selectivity for PKD compared to IKK-16. SAR analysis revealed that 1-NA-PP1 had significantly higher activity than other compounds, with a significant substituent effect on the pyrazolopyrimidine core. 1-NA-PP1 has cellular activity and can effectively inhibit the proliferation of prostate cancer cells by inducing G2/M phase arrest. In addition, it can also effectively inhibit the migration and invasion of prostate cancer cells, showing multifaceted anticancer activity. Overexpression of PKD1 or PKD3 almost completely reversed the growth arrest and tumor cell invasion inhibition caused by 1-NA-PP1, indicating that its antiproliferation and anti-invasion activities are mediated by the inhibition of PKD. Interestingly, by introducing a "gatekeeper" mutation at the active site of PKD1, the sensitivity of PKD1 to 1-NA-PP1 can be increased by 12-fold, indicating that 1-NA-PP1 can be used in combination with analog-sensitive PKD1 (M659G) to elucidate the specific functions and signaling pathways of PKD in various biological systems. [1] Protein kinases have been shown to be highly resistant to the design of highly specific inhibitors, even with the aid of combinatorial chemistry. The lack of these reagents makes it complicated to determine the signal transduction function of specific kinases. This article describes a chemogenetic strategy to make protein kinases more sensitive to cell-permeable molecules that do not inhibit wild-type kinases. We screened inhibitors from two inhibitor backbones that exhibited high selectivity for sensitized kinases from five different subfamilies. Tyrosine kinases and serine/threonine kinases were also suitable for this approach. We analyzed a budding yeast strain carrying an inhibitor-sensitive cyclin-dependent kinase Cdc28 (CDK1) in place of the wild-type protein. In vivo specific inhibition of Cdc28 resulted in premitotic cell cycle arrest, which differed from the G1 phase arrest typically observed in temperature-sensitive cdc28 mutants. Mutations conferring inhibitor sensitivity were readily identifiable by primary sequence alignment. Thus, this approach can be used to systematically construct conditional alleles of protein kinases, thereby enabling rapid characterization of the function of members of this important gene family. [2]

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Reducing the expression of protein kinase Cε (PKCε) or inhibiting its translocation prolonged the duration of ethanol poisoning and reduced ethanol intake in mice. However, it is unclear whether this phenotype was due to reduced PKCε kinase activity or to impaired non-kinase-dependent function. In this study, we employed a chemogenetic strategy to determine whether a highly efficient and selective inhibitor of PKCε catalytic activity could reduce ethanol intake. We constructed ATP analog-specific PKCε (AS-PKCε) knock-in mice carrying a point mutation at the PKCε ATP-binding site, resulting in a highly sensitive mutant kinase to inhibition of 1-tert-butyl-3-naphthyl-1-ylpyrazolo[3,4-d]pyrimidine-4-amine (1-NA-PP1). Systemic administration of 1-NA-PP1 readily crossed the blood-brain barrier and inhibited PKCε-mediated phosphorylation. 1-NA-PP1 reversibly reduced ethanol intake in AS-PKCε mice but had no effect on wild-type mice without the AS-PKCε mutation. These results support the development of PKCε catalytic activity inhibitors as a strategy for reducing ethanol intake and demonstrate that AS-PKCε mice are a useful tool for studying the behavioral role of PKCε. [3]

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1. 1-NaphthylPP1 (1-NA-PP1) belongs to the pyrazolopyrimidine chemical skeleton and was initially developed as an inhibitor of analog-sensitive mutant Src kinase; later, through screening of a targeted kinase inhibitor library, it was identified as an effective pan-PKD inhibitor. [2]
2. Structure-activity relationship (SAR) analysis of 1-naphthylPP1 (1-NA-PP1) showed that the naphthyl group on the pyrazolopyrimidine core is crucial for PKD inhibition, and substitution at this position significantly affects efficacy and selectivity. [2]
3. The gate mutation (M659G) in PKD1 expands the ATP-binding pocket, thereby accommodating the large naphthyl moiety of 1-naphthylPP1 (1-NA-PP1)—this structural modification is the basis for a 12-fold increase in sensitivity, enabling specific resolution of the PKD signaling pathway [2]
4. 1-NaphthylPP1 (1-NA-PP1) is cell-permeable, which is essential for its activity in intact cells (e.g., LNCaP, PC3, HEK293) and in vivo (across the blood-brain barrier) [2, 3]
5. The chemical name of 1-naphthylPP1 (1-NA-PP1) is 1-tert-butyl-3-naphth-1-ylpyrazolo[3,4-d]pyrimidine-4-amine, and its PubChem CID is 4877 [3]
6. 1-NaphthylPP1 (1-NA-PP1) Specific inhibition of the catalytic activity of target kinases (PKD, AS-PKCε) without affecting their total protein expression levels has been confirmed by Western blot analysis of PKD1, PKD3, PKCε, and GABA_Aγ2 in cells and mouse tissues [2, 3].

C19H19N5

317.3877

317.164

221243-82-9

1-Naphthyl PP1 hydrochloride;956025-47-1

4877

Light yellow to khaki solid

1.3±0.1 g/cm3

527.8±45.0 °C at 760 mmHg

219-222ºC

273.0±28.7 °C

0.0±1.4 mmHg at 25°C

1.688

3.88

1

4

2

24

448

0

N1(C2C(=C(N([H])[H])N=C([H])N=2)C(C2=C([H])C([H])=C([H])C3=C([H])C([H])=C([H])C([H])=C23)=N1)C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H]

XSHQBIXMLULFEV-UHFFFAOYSA-N

InChI=1S/C19H19N5/c1-19(2,3)24-18-15(17(20)21-11-22-18)16(23-24)14-10-6-8-12-7-4-5-9-13(12)14/h4-11H,1-3H3,(H2,20,21,22)

1-tert-butyl-3-naphthalen-1-ylpyrazolo[3,4-d]pyrimidin-4-amine

1-Naphthyl PP1; 221243-82-9; 1-NAPHTHYL PP1; 1-NA-PP1; 1-(tert-Butyl)-3-(naphthalen-1-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine; 4-Amino-1-tert-butyl-3-(1'-naphthyl)pyrazolo[3,4-d]pyrimidine; 1-tert-butyl-3-naphthalen-1-ylpyrazolo[3,4-d]pyrimidin-4-amine; 1-(1,1-dimethylethyl)-3-(1-naphthalenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine; C19H19N5; 1-NA-PP 1

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 9~12.5 mg/mL (28.4~39.4 mM)

Solubility in Formulation 1: ≥ 1.25 mg/mL (3.94 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 1.25 mg/mL (3.94 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)