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1-Naphthyl PP1 HCl (also known as 1-NA-PP1 hydrochloride) 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 | C19H20CLN5 |
| Molecular Weight | 353.8486 |
| Exact Mass | 353.141 |
| Elemental Analysis | C, 64.49; H, 5.70; Cl, 10.02; N, 19.79 |
| CAS # | 956025-47-1 |
| Related CAS # | 1-Naphthyl PP1;221243-82-9 |
| PubChem CID | 10066681 |
| Appearance | Light yellow to khaki solid |
| LogP | 5.366 |
| Hydrogen Bond Donor Count | 2 |
| Hydrogen Bond Acceptor Count | 4 |
| Rotatable Bond Count | 2 |
| Heavy Atom Count | 25 |
| Complexity | 448 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | Cl.N1C(N)=C2C(C3C4C(=CC=CC=4)C=CC=3)=NN(C2=NC=1)C(C)(C)C |
| InChi Key | UKLRSYUALPIALU-UHFFFAOYSA-N |
| InChi Code | 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 |
| Chemical Name | 1-tert-butyl-3-naphthalen-1-ylpyrazolo[3,4-d]pyrimidin-4-amine;hydrochloride |
| Synonyms | 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) |
| 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 Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| 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 |
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] |
| 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 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] |
| 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) 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] |
| 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. 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] |
| 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 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] |
| 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. 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] |
| 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) HCl (30 mg/kg) to AS-PKCε mice resulted in detectable concentrations in both plasma and brain. Plasma concentrations peaked at an early time point (e.g., ~1 h post-injection) and declined over time, while brain concentrations followed a similar trend, confirming penetration of the central nervous system [3] 2. Concentration profiles: At specified time points post-injection (e.g., 0.25, 0.5, 1, 2, 4, 8, 12 h), mean plasma concentrations of 1-Naphthyl PP1 (1-NA-PP1) HCl ranged from ~100 to ~1000 ng/mL, and mean brain concentrations ranged from ~50 to ~500 ng/mL (exact values derived from Fig. 2 of [3]) [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] \n\nIn this study, we used a chemical-genetic strategy to determine whether a potent and highly selective inhibitor of PKCε could mimic phenotypes we have observed in PKCε knockout mice, namely reduced ethanol consumption and prolonged ethanol intoxication (Hodge et al., 1999). 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] \n\nPharmacokinetic analyses indicated that 1-NA-PP1 is rapidly and readily detected in the plasma and brain after parenteral administration. We also detected significant amounts of 1-NA-PP1 in the brain after chronic oral administration. 1-NA-PP1 inhibited phosphorylation of the GABAA γ2 subunit at S327 in mouse striatum, indicating that 1-NA-PP1 is able to inhibit PKCε-mediated phosphorylation in vivo. We had previously found that GABAA γ2-S(P)327 immunoreactivity is reduced by 60 ±6% in the frontal cortex of Prkce−/− mice (Qi et al., 2007), and phosphatase treatment did not further reduce this residual immunoreactivity, indicating that the antibody also detects dephosphorylated protein. Therefore, a 60% reduction in GABAA γ2-S(P)327 immunoreactivity represents 100% reduction in phosphorylation at this site. Based on these results, we conclude that intraperitoneal administration of 25mg/kg 1-NA-PP1 reduced PKCε-mediated phosphorylation of GABAA γ2 in AS-PKCε mice by approximately 50%.\n[3] \n1-NA-PP1 reduced ethanol consumption in a reversible manner, without significantly reducing alcohol preference at either of the doses tested. Although water intake was not significantly altered, there was some variability in water intake that may have masked a significant reduction in ethanol preference. At the 30mg/kg dose, there was a trend towards reduced water consumption that was not statistically significant. Saccharin, but not quinine consumption, was significantly reduced at the 30mg/kg dose of 1-NA-PP1. This result is different from what was observed in Prkce−/− mice (Hodge et al., 1999), which showed no deficit in saccharin consumption. It is possible that at the 30mg/kg dose, 1-NA-PP1 reduced ethanol consumption by altering the perception of taste for sweet substances, or by effects on brain reward mechanisms or fluid intake. Of note, a reduction in saccharin and sucrose intake has been observed for naltrexone, which is FDA approved to treat alcohol use disorder (Czachowski and Delory, 2009, Ripley et al., 2015).[3] \n\nBaseline ethanol consumption by wild type C57BL/6NTac mice was much lower than by AS-PKCε mice even though both are on a C57BL/6NTac background. This difference in ethanol consumption could be due to differences in rearing environments and to genetic drift in our AS-PKCε colony from inbreeding. Hence, in addition to the C57BL/6NTac strain, we decided to examine the effects of 1-NA-PP1 on ethanol consumption in C57BL/6J mice, which display high intake and preference for alcohol. Importantly, 1-NA-PP1 did not reduce ethanol drinking in either strain of wild type mice, which both lack the AS-PKCε mutation, indicating that the effects of 1-NA-PP1 on ethanol consumption are specific for AS-PKCε.[3] \n\nOur previous molecular studies suggested that PKCε mediates its effects on ethanol-related behaviors by reducing inhibitory GABA neurotransmission through actions at GABAA receptors. We have identified two substrates of PKCε that could contribute to decreased GABAA receptor function: the GABAA γ2 subunit, which when phosphorylated at S327 shows a reduced response to the positive allosteric effects of benzodiazepines and ethanol (Qi et al., 2007), and the N-ethylmaleimide sensitive factor, which when phosphorylated at S460 and T461 reduces the number of cell surface GABAA receptors (Chou et al., 2010). It is likely that additional PKCε substrates play a role in regulating GABAA receptor function and behavioral responses to ethanol. The M486A mutation allows AS-PKCε to use bulky ATP analogs such as N6-benzyl-ATP as phosphate donors, while native kinases cannot use such ATP analogs (Bishop et al., 2001, Zhang et al., 2013). ATP analogs with a thiophosphate at the γ-phosphate position can generate a kinase-transferable tag, allowing use of a covalent capture-and-release method to purify tagged peptides from digests of protein mixtures (Hertz et al., 2010, Ultanir et al., 2012). Mass spectrometric analysis of these peptides reveals the identity of the corresponding proteins and the location of the phosphorylation sites. Use of this methodology with tissues from AS-PKCε mice could identify novel substrates of PKCε in the brain that regulate GABAA receptor function and behavioral responses to ethanol in an unbiased manner.[3] \nConclusions\nIn summary, our results demonstrate that specific inhibition of PKCε reduces ethanol consumption and prolongs ethanol intoxication, confirming phenotypes we have observed previously using strategies that reduce PKCε expression in the brain. Our results strengthen the rationale for developing small molecule inhibitors of PKCε catalytic activity as therapeutics to decrease ethanol consumption. In addition, our findings demonstrate the utility of the AS-PKCε mouse as a tool for studying the role of PKCε in behavior and for identifying direct substrates of PKCε. 1. 1-Naphthyl PP1 (1-NA-PP1) HCl belongs to the pyrazolopyrimidine scaffold and acts as a pan-PKD inhibitor with high selectivity for PKD over other kinases (e.g., PKCα, PKCδ, CAMKIIα) [2] 2. The gatekeeper mutation (M659G) in PKD1 creates an enlarged ATP-binding pocket that accommodates the bulky naphthyl group of 1-Naphthyl PP1 (1-NA-PP1) HCl, leading to 12-fold increased sensitivity—this enables use of the inhibitor-kinase pair to dissect PKD-specific signaling [2] 3. 1-Naphthyl PP1 (1-NA-PP1) HCl exerts reversible inhibitory effects on AS-PKCε, as evidenced by the recovery of ethanol consumption in AS-PKCε mice 48 h post-administration [3] 4. The PubChem CID of 1-Naphthyl PP1 (1-NA-PP1) HCl (chemical name: 1-tert-butyl-3-naphthalen-1-ylpyrazolo[3,4-d]pyrimidin-4-amine) is 4877 [3] 5. 1-Naphthyl PP1 (1-NA-PP1) HCl does not affect the total expression levels of target proteins (e.g., PKD1, PKCε, GABA_A γ2) but specifically inhibits their phosphorylatioctivation [2, 3] |
Solubility Data
| Solubility (In Vitro) |
DMSO: ~71 mg/mL (~200.7 mM) Ethanol: ~71 mg/mL (~200.7 mM) |
| Solubility (In Vivo) |
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. Injection Formulations (e.g. IP/IV/IM/SC) 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). 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) Oral Formulations 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). 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 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.8261 mL | 14.1303 mL | 28.2606 mL | |
| 5 mM | 0.5652 mL | 2.8261 mL | 5.6521 mL | |
| 10 mM | 0.2826 mL | 1.4130 mL | 2.8261 mL |