c-Fyn (IC50 = 0.035 nM); c-Abl (IC50 = 0.6 μM); v-Src (IC50 = 1.0 μM); CDK2 (IC50 = 18 μM); CAMKII (IC50 = 22 μM)
Protein Kinase D (PKD) isoforms (PKD1, PKD2, PKD3) (IC₅₀≈100 nM for each isoform) [2]
Analog-sensitive Protein Kinase C epsilon (AS-PKCε) (no definite IC₅₀, Ki, or EC₅₀ data provided; does not inhibit wild-type PKCε) [3]
Engineered gatekeeper mutant PKD1(M659G) (12-fold higher sensitivity to 1-Naphthyl PP1 (1-NA-PP1) HCl compared to wild-type PKD1) [2]

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 PKD isoforms: Recombinant human PKD1, PKD2, and PKD3 activities were inhibited by 1-Naphthyl PP1 (1-NA-PP1) HCl in a concentration-dependent manner in an in vitro radiometric kinase assay, with IC₅₀ values of approximately 100 nM for each isoform. The inhibition was ATP-competitive, as demonstrated by Lineweaver-Burke plots of PKD1 activity measured at increasing ATP concentrations and varying 1-Naphthyl PP1 (1-NA-PP1) HCl concentrations [2]
2. Selectivity for PKD: 1-Naphthyl PP1 (1-NA-PP1) HCl did not inhibit closely related kinases including PKCα, PKCδ (tested at 10 nM, 100 nM, 1 μM, 10 μM) or CAMKIIα, whereas the control PKC inhibitor GF109203X potently inhibited PKCα and PKCδ [2]
3. Inhibition of PKD activation in cells: Pretreatment of LNCaP cells with 1-Naphthyl PP1 (1-NA-PP1) HCl (different doses) for 45 min blocked PMA-induced phosphorylation of endogenous PKD1 at S⁹¹⁶ and S⁷⁴⁴/⁷⁴⁸ (measured by Western blot). Densitometric analysis of Western blots yielded an IC₅₀ for inhibiting PKD1 activation [2]
4. Antiproliferative and cell cycle effects: 1-Naphthyl PP1 (1-NA-PP1) HCl (10 μM) potently blocked PC3 prostate cancer cell proliferation when measured by daily cell counting over 5 days (fresh medium and inhibitor added every 2 days). It also induced G2/M phase arrest in PC3 cells (10 μM, 48 h treatment) as determined by flow cytometry after propidium iodide (PI) labeling [2]
5. Inhibition of cell migration and invasion: 1-Naphthyl PP1 (1-NA-PP1) HCl (30 μM) reduced PC3 cell migration (measured by wound healing assay: 22 h treatment, percentage of wound closure calculated) and DU145 cell invasion (measured by Matrigel invasion assay: 20 h treatment, cell counts in 6 fields relative to control) [2]
6. Target validation via 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) HCl-induced growth arrest (10/30 μM, 72 h, MTT assay) and inhibition of invasion (30 μM), confirming PKD as the target [2]
7. Sensitization of mutant PKD1: Engineering a gatekeeper mutation (M659G) in PKD1 increased sensitivity to 1-Naphthyl PP1 (1-NA-PP1) HCl by 12-fold, as shown by inhibition of PMA-induced phosphorylation of Flag-PKD1(M659G) in HEK293 cells (serum-starved for 24 h, 45 min pretreatment with increasing inhibitor concentrations) [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) HCl (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) HCl (25 mg/kg) to AS-PKCε mice significantly decreased phosphorylation of the GABA_A γ2 receptor subunit at S327 (measured by Western blot, n=5 per group, P=0.0175) compared to vehicle treatment [3]
3. Reduction of ethanol consumption: 1-Naphthyl PP1 (1-NA-PP1) HCl (30 mg/kg, intraperitoneal) reversibly reduced ethanol consumption in AS-PKCε knock-in mice (n=18 per group) after habituation to vehicle injections. The inhibitory effect was no longer present 48 h post-administration. The inhibitor did not alter ethanol preference over water, water intake, or ethanol clearance, but slightly reduced saccharin intake (30 mg/kg) without affecting quinine intake [3]
4. Modulation of ethanol-induced behaviors: 1-Naphthyl PP1 (1-NA-PP1) HCl (30 mg/kg, intraperitoneal) prolonged recovery from ethanol-induced ataxia (1.5 g/kg ethanol, n=11 for vehicle, n=13 for inhibitor, P=0.0014) and increased the duration of ethanol-induced loss of righting reflex (LORR, 3.6 g/kg ethanol, n=25 for vehicle, n=26 for inhibitor) in AS-PKCε mice. No such effects were observed in wild-type C57BL/6NTac or C57BL/6J mice (n=7-10 per group) [3]
5. Lack of effect on wild-type mice: 1-Naphthyl PP1 (1-NA-PP1) HCl (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 [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. PKD isoform kinase activity assay (radiometric): Prepare recombinant human PKD1, PKD2, or PKD3. Set up reaction mixtures containing the kinase, substrate, ATP (with radioactive label), and 10 different concentrations of 1-Naphthyl PP1 (1-NA-PP1) HCl. Incubate the mixtures under optimal conditions for kinase activity. Measure the incorporation of radioactive phosphate into the substrate to determine kinase activity. Calculate IC₅₀ values as the mean ± SEM of at least three independent experiments with triplicate determinations per inhibitor concentration. Plot activity data against inhibitor concentration to generate concentration-response curves [2]
2. ATP-competitiveness assay for PKD1: Prepare reaction mixtures with recombinant PKD1, substrate, and increasing concentrations of ATP (to assess ATP dependence). Include varying concentrations of 1-Naphthyl PP1 (1-NA-PP1) HCl in each ATP concentration group. 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 inhibition) [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 PKD activation in LNCaP cells: Seed LNCaP cells and allow attachment. Pretreat cells with different doses of 1-Naphthyl PP1 (1-NA-PP1) HCl for 45 min, followed by stimulation with 10 nM PMA for 20 min. Lyse cells and extract proteins. Perform Western blot analysis using antibodies against phosphorylated PKD1 (p-S⁹¹⁶, p-S⁷⁴⁴/⁷⁴⁸) and tubulin (loading control). Quantify blots via densitometry to calculate IC₅₀ for inhibiting PKD activation [2]
2. PC3 cell proliferation assay: Seed PC3 cells in triplicate in 24-well plates and allow attachment overnight. Count cells on day 1 (baseline). Add vehicle (DMSO) or 10 μM 1-Naphthyl PP1 (1-NA-PP1) HCl to the wells. Replace medium and inhibitor every 2 days. Count cells daily for 5 days and plot cell number over time [2]
3. MTT cell viability assay: Seed PC3/DU145 cells (3000 cells/well) in 96-well plates. Incubate cells with 1-Naphthyl PP1 (1-NA-PP1) HCl (0.3-100 μM) for 72 h. Add MTT solution and incubate for 4 h. Measure optical density at 570 nm to determine cell viability. Calculate IC₅₀ as the mean of two independent experiments [2]
4. Cell cycle analysis via flow cytometry: Treat PC3 cells with vehicle (DMSO) or 10 μM 1-Naphthyl PP1 (1-NA-PP1) HCl for 48 h. Fix cells and label DNA with propidium iodide (PI). Analyze cell cycle distribution using flow cytometry. Determine statistical significance via unpaired t-test [2]
5. Wound healing migration assay: Grow PC3 cells to confluence in 6-well plates. Create a uniform wound in the cell monolayer and image immediately (0 h). Incubate cells in growth medium containing vehicle or 30 μM 1-Naphthyl PP1 (1-NA-PP1) HCl for 22 h. Image again and calculate percentage wound healing (healed wound area relative to original wound area) [2]
6. Matrigel invasion assay: Seed DU145 cells in Matrigel-coated inserts with medium containing 30 μM 1-Naphthyl PP1 (1-NA-PP1) HCl. Incubate for 20 h, then remove non-invasive cells from the top of the insert. Fix invasive cells in 100% methanol, stain with 0.4% hematoxylin, and photograph. Count cells in 6 fields and calculate percentage invasion (invasive cells relative to total cells migrated through control inserts) [2]
7. PKD overexpression rescue assay: Seed PC3/DU145 cells (0.5 million cells/60 mm dish). The next day, infect cells with adenoviruses carrying PKD1 (Adv-PKD1), PKD3 (Adv-PKD3), or empty vector (Adv-null) at 50-100 MOI. After 24 h, seed 3000 infected cells/well in 96-well plates (for MTT) or Matrigel inserts (for invasion). Treat with 10/30 μM 1-Naphthyl PP1 (1-NA-PP1) HCl for 72 h (MTT) or 20 h (invasion). Measure viability/invasion and confirm PKD overexpression via Western blot [2]
8. Mutant PKD1 phosphorylation assay in HEK293 cells: Transfect HEK293 cells with Flag-tagged wild-type PKD1 or gatekeeper mutant PKD1(M659G)/PKD1(M659A). Two days post-transfection, serum-starve cells for 24 h. Pretreat with increasing concentrations of 1-Naphthyl PP1 (1-NA-PP1) HCl in serum-free medium for 45 min, then stimulate with 10 nM PMA for 20 min. Lyse cells and perform Western blot with antibodies against p-S⁹¹⁶-PKD1, PKD1, and tubulin [2]

Administration of 1-NA-PP1 [3]
For 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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1. AS-PKCε knock-in mouse generation: Generate mice harboring a point mutation (M486A) in the ATP-binding site of PKCε (AS-PKCε) via gene targeting (excision of Neo cassette using Cre-recombinase). Confirm genotype via PCR of tail DNA and PKCε expression via Western blot (hippocampus) and immunohistochemistry (brain distribution) [3]
2. Pharmacokinetic assay: Administer 1-Naphthyl PP1 (1-NA-PP1) HCl (30 mg/kg) to AS-PKCε mice via intraperitoneal injection. At different time points post-injection, collect plasma and brain samples (n=3 per time point). Measure 1-Naphthyl PP1 (1-NA-PP1) HCl concentrations in plasma and brain to generate pharmacokinetic profiles [3]
3. GABA_A γ2 phosphorylation assay: Inject AS-PKCε mice with 1-Naphthyl PP1 (1-NA-PP1) HCl (25 mg/kg, intraperitoneal) or vehicle. After a specified time, harvest brain tissue, prepare extracts, and perform Western blot with antibodies against phosphorylated GABA_A γ2 (p-S327) and total GABA_A γ2 (n=5 per group) [3]
4. Ethanol consumption assay: Habituate AS-PKCε mice to vehicle injections until stable baseline ethanol consumption is achieved. Administer 1-Naphthyl PP1 (1-NA-PP1) HCl (30 mg/kg, intraperitoneal) and monitor ethanol consumption, ethanol preference, water intake, saccharin intake, and quinine intake. Assess reversibility by measuring consumption 48 h post-injection (n=18 per group for ethanol-related parameters; n=14 per group for saccharin/quinine) [3]
5. Ethanol clearance assay: Inject AS-PKCε mice with 1-Naphthyl PP1 (1-NA-PP1) HCl (30 mg/kg, intraperitoneal) or vehicle. Administer ethanol (1.5 g/kg) and measure blood ethanol concentrations over time to assess clearance (n=7 per group) [3]
6. Ethanol-induced ataxia assay: Inject AS-PKCε or wild-type mice with 1-Naphthyl PP1 (1-NA-PP1) HCl (30 mg/kg, intraperitoneal) or vehicle. Thirty minutes later, administer ethanol (1.5 g/kg) and measure the time to recover from ataxia (ability to right themselves repeatedly) (n=7-13 per group) [3]
7. Ethanol-induced LORR assay: Inject AS-PKCε or wild-type mice with 1-Naphthyl PP1 (1-NA-PP1) HCl (30 mg/kg, intraperitoneal) or vehicle. Thirty minutes later, administer ethanol (3.6 g/kg) and measure the duration of LORR (inability to right themselves) (n=8-26 per group) [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 intraperitoneally injected with 1-naphthylPP1 (1-NA-PP1) HCl (30 mg/kg), and concentrations were detected in both plasma and brain. Plasma concentrations peaked early after injection (e.g., about 1 hour after injection) and decreased over time, and brain tissue concentrations showed a similar trend, confirming that the drug could penetrate the central nervous system.[3]
2. Concentration curves: At specific time points after injection (e.g., 0.25, 0.5, 1, 2, 4, 8, 12 hours), the average plasma concentration of 1-naphthylPP1 (1-NA-PP1) HCl ranged from approximately 100 to 1000 ng/mL, and the average brain tissue concentration ranged from approximately 50 to 500 ng/mL (specific values are from Figure 2 of [3]).[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-naphth-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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In this study, we employed a chemogenetic strategy to determine whether a highly effective and selective PKCε inhibitor could mimic the phenotype we observed in PKCε knockout mice, namely reduced ethanol intake and prolonged duration of ethanol intoxication (Hodge et al., 1999). We constructed a novel AS-PKCε mouse strain with a point mutation at the ATP-binding site, making it highly sensitive to nanomolar concentrations of the PP1 analog 1-NA-PP1. Systemic administration of 1-NA-PP1 crossed the blood-brain barrier and reached concentrations in the brain sufficient to inhibit AS-PKCε. 1-NA-PP1 prolonged ethanol-induced ataxia and hypnotic effects and reduced ethanol intake in 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 PKCε catalytic activity may contribute to reducing ethanol intake. [3] Pharmacokinetic analysis showed that 1-NA-PP1 was rapidly and readily detectable in plasma and brain tissue after parenteral administration. We also detected significant amounts of 1-NA-PP1 in brain tissue after long-term oral administration. 1-NA-PP1 inhibited phosphorylation of the GABAA γ2 subunit S327 site in the mouse striatum, indicating that 1-NA-PP1 can inhibit PKCε-mediated phosphorylation in vivo. We previously found that the immunoreactivity of GABAA γ2-S(P)327 was reduced by 60 ± 6% in the prefrontal cortex of Prkce−/− mice (Qi et al., 2007), and phosphatase treatment did not further reduce this residual immunoreactivity, indicating that the antibody can also detect dephosphorylated proteins. Therefore, a 60% reduction in the immunoreactivity of GABAA γ2-S(P)327 represents a 100% reduction in the phosphorylation level at this site. Based on these results, we conclude that intraperitoneal injection of 25 mg/kg 1-NA-PP1 reduces PKCε-mediated GABAA γ2 phosphorylation levels in AS-PKCε mice by approximately 50% [3]. 1-NA-PP1 reversibly reduces ethanol intake and does not significantly reduce alcohol preference in mice at either of the two doses tested. Although water intake did not change significantly, there were some fluctuations in water intake, which may have masked the significant reduction in ethanol preference. At a dose of 30 mg/kg, water intake showed a decreasing trend, but did not reach statistical significance. At a dose of 30 mg/kg 1-NA-PP1, saccharin intake was significantly reduced, while quinine intake did not change significantly. This result differs from the observations in Prkce−/− mice (Hodge et al., 1999), where saccharin intake was not reduced. 1-NA-PP1 at a dose of 30 mg/kg may reduce ethanol intake by altering the perception of sweet substances or by affecting brain reward mechanisms or fluid intake. Notably, naltrexone (an FDA-approved drug for the treatment of alcohol use disorder) reduces the intake of saccharin and sucrose (Czachowski and Delory, 2009; Ripley et al., 2015) [3]. Baseline ethanol intake in wild-type C57BL/6NTac mice was much lower than in AS-PKCε mice, despite both having a C57BL/6NTac background. This difference in ethanol intake may be due to different housing conditions and genetic drift resulting from inbreeding in our AS-PKCε mouse population. Therefore, in addition to the C57BL/6NTac strain, we decided to investigate the effect of 1-NA-PP1 on ethanol intake in C57BL/6J mice, which exhibited higher alcohol intake and preference. Importantly, 1-NA-PP1 did not reduce ethanol intake in either of the two wild-type mice (both lacking the AS-PKCε mutation), suggesting that the effect of 1-NA-PP1 on ethanol intake is AS-PKCε specific. [3]
Our previous molecular studies have shown that PKCε mediates its effects on ethanol-related behaviors by acting on GABAA receptors to reduce inhibitory GABA neurotransmission. We have identified two substrates of PKCε that may lead to reduced GABAA receptor function: the GABAA γ2 subunit, which, upon phosphorylation at S327, reduces the positive allosteric response to benzodiazepines and ethanol (Qi et al., 2007); and the N-ethylmaleimide sensitizer, which, upon phosphorylation at S460 and T461, reduces the number of GABAA receptors on the cell surface (Chou et al., 2010). Other substrates of PKCε may be involved in regulating GABAA receptor function and behavioral responses to ethanol. The M486A mutation enables AS-PKCε to utilize bulky ATP analogs (e.g., N6-benzyl-ATP) as phosphate donors, which the native kinase cannot (Bishop et al., 2001; Zhang et al., 2013). ATP analogs with thiophosphate groups at the γ-phosphate site can generate kinase-transferable tags, allowing the purification of labeled peptides from digests of protein mixtures using a covalent capture-release method (Hertz et al., 2010; Ultanir et al., 2012). Mass spectrometry analysis of these peptides can reveal the identity of the corresponding proteins and the location of phosphorylation sites. Using this method to analyze tissues of AS-PKCε mice, novel PKCε substrates that regulate GABAA receptor function and ethanol behavioral responses in the brain can be identified unbiasedly. [3] Conclusion: In summary, our results indicate that specific inhibition of PKCε reduces ethanol intake and prolongs the duration of ethanol poisoning, confirming the phenotypes we previously observed using a strategy of reducing PKCε expression in the brain. Our results reinforce the theoretical basis for developing small-molecule inhibitors of PKCε catalytic activity as therapeutic agents to reduce ethanol intake. Furthermore, our findings demonstrate the utility of AS-PKCε mice as a tool for studying the role of PKCε in behavior and identifying direct substrates of PKCε. 1. 1-NaphthylPP1 (1-NA-PP1) HCl belongs to the pyrazolopyrimidine skeleton and is a pan-PKD inhibitor with a much higher selectivity for PKD than other kinases (e.g., PKCα, PKCδ, CAMKIIα) [2] 2. The gate mutation (M659G) in PKD1 forms an enlarged ATP-binding pocket that can accommodate the large naphthyl group in 1-naphthylPP1 (1-NA-PP1) HCl, thereby increasing sensitivity by 12-fold—making this inhibitor-kinase pair usable for elucidating PKD-specific signaling pathways [2] 3. 1-NaphthylPP1 (1-NA-PP1) HCl has a reversible inhibitory effect on AS-PKCε, as evidenced by the recovery of ethanol intake in AS-PKCε mice 48 hours after administration [3] 4. 1-NaphthylPP1 (1-NA-PP1) The PubChem CID of HCl (chemical name: 1-tert-butyl-3-naphth-1-ylpyrazolo[3,4-d]pyrimidine-4-amine) is 4877 [3]
5. 1-NaphthylPP1 (1-NA-PP1) HCl does not affect the total expression level of target proteins (e.g., PKD1, PKCε, GABA_A γ2), but specifically inhibits their phosphorylation activation [2, 3]

C19H20CLN5

353.8486

353.141

C, 64.49; H, 5.70; Cl, 10.02; N, 19.79

956025-47-1

1-Naphthyl PP1;221243-82-9

10066681

Light yellow to khaki solid

5.366

2

4

2

25

448

0

Cl.N1C(N)=C2C(C3C4C(=CC=CC=4)C=CC=3)=NN(C2=NC=1)C(C)(C)C

UKLRSYUALPIALU-UHFFFAOYSA-N

InChI=1S/C19H19N5.ClH/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);1H

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

1-NA-PP 1 hydrochloride; 956025-47-1; 1-Naphthyl PP1 hydrochloride; 1-Naphthyl PP1 (hydrochloride); 1-tert-butyl-3-naphthalen-1-ylpyrazolo[3,4-d]pyrimidin-4-amine;hydrochloride; 1-tert-butyl-3-(naphthalen-1-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine hydrochloride; 1-tert-Butyl-3-(naphthalen-1-yl)-1H-pyrazolo-[3,4-d]pyrimidin-4-amine hydrochloride; 1-tert-Butyl-3-(naphthalen-1-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine HCl; UKLRSYUALPIALU-UHFFFAOYSA-N; 1-Naphthyl PP1 hydrochloride; 1-Naphthyl PP1 (hydrochloride)

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

DMSO: ~71 mg/mL (~200.7 mM)
Ethanol: ~71 mg/mL (~200.7 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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