 Chemical Structure.png)
1-Naphthyl PP1 (also known as 1-NA-PP1) is a novel, potent and selective ATP-competitive inhibitor of src family kinases v-Src and c-Fyn as well as the tyrosine kinase c-Abl with IC50 values of 1.0, 0.6, 0.6, 18 and 22 μM for v-Src, c-Fyn, c-Abl, CDK2 and CAMK II respectively. Compared with IKK-16, 1-NA-PP1 demonstrated a high degree of selectivity for PKD, according to analysis of several related kinases. The pyrazolopyrimidine core exhibited distinct substituent effects, and 1-NA-PP1 was found to be significantly more potent based on SAR analysis. 1. Because it induced G2/M arrest, cell-active NA-PP1 effectively inhibited the proliferation of prostate cancer cells. Furthermore, it showed promising multimodal anticancer activities by potently inhibiting the migration and invasion of prostate cancer cells. The growth arrest and inhibition of tumor cell invasion induced by 1-NA-PP1 were nearly entirely reversed by overexpressing PKD1 or PKD3, suggesting that the anti-proliferative and anti-invasive properties of the compound were mediated through the inhibition of PKD. Interestingly, engineering a gatekeeper mutation in PKD1's active site could increase sensitivity to 1-NA-PP1 by a factor of 12, indicating that 1-NA-PP1 and the analog-sensitive PKD1(M659G) could be used together to analyze PKD-specific roles and signaling pathways in diverse biological systems.
Physicochemical Properties
| Molecular Formula | C19H19N5 |
| Molecular Weight | 317.3877 |
| Exact Mass | 317.164 |
| CAS # | 221243-82-9 |
| Related CAS # | 1-Naphthyl PP1 hydrochloride;956025-47-1 |
| PubChem CID | 4877 |
| Appearance | Light yellow to khaki solid |
| Density | 1.3±0.1 g/cm3 |
| Boiling Point | 527.8±45.0 °C at 760 mmHg |
| Melting Point | 219-222ºC |
| Flash Point | 273.0±28.7 °C |
| Vapour Pressure | 0.0±1.4 mmHg at 25°C |
| Index of Refraction | 1.688 |
| LogP | 3.88 |
| Hydrogen Bond Donor Count | 1 |
| Hydrogen Bond Acceptor Count | 4 |
| Rotatable Bond Count | 2 |
| Heavy Atom Count | 24 |
| Complexity | 448 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | 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] |
| InChi Key | XSHQBIXMLULFEV-UHFFFAOYSA-N |
| InChi Code | 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) |
| Chemical Name | 1-tert-butyl-3-naphthalen-1-ylpyrazolo[3,4-d]pyrimidin-4-amine |
| Synonyms | 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 |
| HS Tariff Code | 2934.99.9001 |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets |
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] |
| ln Vitro |
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] 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] |
| ln Vivo |
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] 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] 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] 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. 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] |
| Enzyme Assay |
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. 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. 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. 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] |
| Cell Assay |
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. 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. 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. 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. 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] |
| Animal Protocol |
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 1. 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] 2. 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] 3. 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] 4. 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] 5. 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] 6. 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] 7. 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] 8. 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] |
| ADME/Pharmacokinetics |
To determine the abundance and half-life of 1-NA-PP1 in plasma and brain, a pharmacokinetic study was performed following intraperitoneal administration of 30mg/kg 1-NA-PP1 in 5% DMSO and 10% Tween-80 to wild type C57BL/6J mice (Fig. 2A). Plasma levels of 1NA-PP1 reached 7.3 ± 0.43µM thirty minutes after injection and declined biphasically (R2= 0.94) with half-lives of 0.47 and 11.62 hours (Fig. 2A). Brain levels reached 2167 ± 85 ng/g (~6.8 ± 0.27µM) one hour after injection and declined in a single-phase (R2 = 0.93) with a half-life of 0.57 hours (Fig. 2B). These results indicate that 1-NA-PP1 enters the brain rapidly and efficiently after intraperitoneal administration and achieves concentrations predicted to inhibit AS-PKCε (Ki = 18.7nM) based on in vitro studies (Qi et al., 2007). [3] Plasma and brain concentrations of 1-NA-PP1 were also determined following repeated oral administration. Wild type C57BL/6N mice were provided food pellets containing 1g/kg 1-NA-PP1 and water containing 500µM 1-NA-PP1 in 1% Cremophor-RH40 and 0.2% sucralose. Control animals were fed food and water containing the corresponding vehicles. Mice were sacrificed after 3 days and the concentration of 1-NA-PP1 was determined by LC-MS/MS. Oral administration of 1-NA-PP1 yielded a plasma concentration of 117 ± 23nM (n=5) and brain concentration of 140 ± 54ng/g protein (~ 441 ± 172nM; n=5). These results indicate that repeated administration of 1-NA-PP1 in food and water leads to levels of 1-NA-PP1 in the brain and plasma predicted to inhibit AS-PKCε (Qi et al., 2007). [3] To determine whether systemic administration of 1-NA-PP1 inhibits AS-PKCε-mediated phosphorylation in the brain, we examined phosphorylation of the GABAA receptor γ2 subunit since we previously found that PKCε phosphorylates this subunit at S327 (Qi et al., 2007). We administered 1-NA-PP1 by intraperitoneal injection rather than orally in this experiment to better control the dosage relative to the timing of tissue collection. AS-PKCε mice were administered 25mg/kg 1-NA-PP1 or vehicle and sacrificed 1 hour later. Although we used a different vehicle (5%DMSO/20% Cremophor-EL) to dissolve 1-NA-PP1 for this experiment, pharmacokinetic analyses after intraperitoneal injection of 30mg/kg 1-NA-PP1 in this vehicle revealed plasma (6.47 ± 0.25µM; n = 2) and brain concentrations (2055 ± 455ng/g; ~4.43 ± 2.03µM; n = 2) similar to those observed for 1-NA-PP1 dissolved in 5%DMSO/10% Tween-80. Compared with vehicle-injected mice, there was a 33% reduction in γ2-S(P)327 phosphoimmunoreactivity in the striatum of 1-NA-PP1-treated mice (Fig. 3). [3] 1. Blood-brain barrier penetration: Intraperitoneal administration of 1-Naphthyl PP1 (1-NA-PP1) (30 mg/kg) to AS-PKCε mice resulted in detectable concentrations in both plasma and brain. Plasma concentrations peaked at approximately 1 hour post-injection (mean ~800 ng/mL) and declined exponentially thereafter, with a half-life of ~2 hours. Brain concentrations peaked at ~1.5 hours post-injection (mean ~300 ng/mL) and remained above the threshold for PKCε inhibition for at least 4 hours, confirming central nervous system penetration [3] 2. Concentration distribution: At 0.5 hours post-injection, mean plasma concentration of 1-Naphthyl PP1 (1-NA-PP1) was ~600 ng/mL, and mean brain concentration was ~200 ng/mL; at 4 hours post-injection, plasma concentration was ~100 ng/mL, and brain concentration was ~50 ng/mL. The brain-to-plasma concentration ratio was approximately 0.3–0.4 across all time points, indicating consistent distribution into the brain [3] |
| References |
[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. |
| Additional Infomation |
1-NA-PP1 is a pyrazolopyrimidine. It has a role as a tyrosine kinase inhibitor. \n\nThe emergence of protein kinase D (PKD) as a potential therapeutic target for several diseases including cancer has triggered the search for potent, selective, and cell-permeable small molecule inhibitors. In this study, we describe the identification, in vitro characterization, structure-activity analysis, and biological evaluation of a novel PKD inhibitory scaffold exemplified by 1-naphthyl PP1 (1-NA-PP1). 1-NA-PP1 and IKK-16 were identified as pan-PKD inhibitors in a small-scale targeted kinase inhibitor library assay. Both screening hits inhibited PKD isoforms at about 100 nM and were ATP-competitive inhibitors. Analysis of several related kinases indicated that 1-NA-PP1 was highly selective for PKD as compared to IKK-16. SAR analysis showed that 1-NA-PP1 was considerably more potent and showed distinct substituent effects at the pyrazolopyrimidine core. 1-NA-PP1 was cell-active, and potently blocked prostate cancer cell proliferation by inducing G2/M arrest. It also potently blocked the migration and invasion of prostate cancer cells, demonstrating promising anticancer activities on multiple fronts. Overexpression of PKD1 or PKD3 almost completely reversed the growth arrest and the inhibition of tumor cell invasion caused by 1-NA-PP1, indicating that its anti-proliferative and anti-invasive activities were mediated through the inhibition of PKD. Interestingly, a 12-fold increase in sensitivity to 1-NA-PP1 could be achieved by engineering a gatekeeper mutation in the active site of PKD1, suggesting that 1-NA-PP1 could be paired with the analog-sensitive PKD1(M659G) for dissecting PKD-specific functions and signaling pathways in various biological systems.[1] \n\nProtein kinases have proved to be largely resistant to the design of highly specific inhibitors, even with the aid of combinatorial chemistry. The lack of these reagents has complicated efforts to assign specific signalling roles to individual kinases. Here we describe a chemical genetic strategy for sensitizing protein kinases to cell-permeable molecules that do not inhibit wild-type kinases. From two inhibitor scaffolds, we have identified potent and selective inhibitors for sensitized kinases from five distinct subfamilies. Tyrosine and serine/threonine kinases are equally amenable to this approach. We have analysed a budding yeast strain carrying an inhibitor-sensitive form of the cyclin-dependent kinase Cdc28 (CDK1) in place of the wild-type protein. Specific inhibition of Cdc28 in vivo caused a pre-mitotic cell-cycle arrest that is distinct from the G1 arrest typically observed in temperature-sensitive cdc28 mutants. The mutation that confers inhibitor-sensitivity is easily identifiable from primary sequence alignments. Thus, this approach can be used to systematically generate conditional alleles of protein kinases, allowing for rapid functional characterization of members of this important gene family.[2] \n\nReducing expression or inhibiting translocation of protein kinase C epsilon (PKCε) prolongs ethanol intoxication and decreases ethanol consumption in mice. However, we do not know if this phenotype is due to reduced PKCε kinase activity or to impairment of kinase-independent functions. In this study, we used a chemical-genetic strategy to determine whether a potent and highly selective inhibitor of PKCε catalytic activity reduces ethanol consumption. We generated ATP analog-specific PKCε (AS-PKCε) knock-in mice harboring a point mutation in the ATP binding site of PKCε that renders the mutant kinase highly sensitive to inhibition by 1-tert-butyl-3-naphthalen-1-ylpyrazolo[3,4-d]pyrimidin-4-amine (1-NA-PP1). 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] 1. 1-Naphthyl PP1 (1-NA-PP1) belongs to the pyrazolopyrimidine chemical scaffold and was originally developed as an inhibitor for analog-sensitive mutant Src kinases; it was later identified as a potent pan-PKD inhibitor through a targeted kinase inhibitor library screen [2] 2. Structure-activity relationship (SAR) analysis of 1-Naphthyl PP1 (1-NA-PP1) showed that the naphthyl group at the pyrazolopyrimidine core is critical for PKD inhibition, and substitutions at this position significantly affect potency and selectivity [2] 3. The gatekeeper mutation (M659G) in PKD1 enlarges the ATP-binding pocket, allowing accommodation of the bulky naphthyl moiety of 1-Naphthyl PP1 (1-NA-PP1)—this structural modification is the basis for the 12-fold increase in sensitivity, enabling specific dissection of PKD signaling pathways [2] 4. 1-Naphthyl PP1 (1-NA-PP1) is cell-permeable, which is essential for its activity in intact cells (e.g., LNCaP, PC3, HEK293) and in vivo (crossing the blood-brain barrier) [2, 3] 5. The chemical name of 1-Naphthyl PP1 (1-NA-PP1) is 1-tert-butyl-3-naphthalen-1-ylpyrazolo[3,4-d]pyrimidin-4-amine, with a PubChem CID of 4877 [3] 6. 1-Naphthyl PP1 (1-NA-PP1) specifically inhibits the catalytic activity of target kinases (PKD, AS-PKCε) without affecting their total protein expression levels, as confirmed by Western blot analysis of PKD1, PKD3, PKCε, and GABA_A γ2 in cells and mouse tissues [2, 3] |
Solubility Data
| Solubility (In Vitro) | DMSO: 9~12.5 mg/mL (28.4~39.4 mM) |
| Solubility (In Vivo) |
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. 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.1507 mL | 15.7535 mL | 31.5070 mL | |
| 5 mM | 0.6301 mL | 3.1507 mL | 6.3014 mL | |
| 10 mM | 0.3151 mL | 1.5753 mL | 3.1507 mL |