PKD1 108 nM (IC50) Cellular PKD2 94 nM (IC50) PKD3 108 nM (IC50)

3-IN-PP1 exhibits strong pan-PKD inhibitory activity for PKD1, PKD2, and PKD3 with IC50 values of 108, 94, and 108 nM, respectively[1]. Strong anti-proliferative action against PANC-1 cells is demonstrated by 3-IN-PP1 (5 μM, 0-114 h)[1]. 3-IN-PP1 (20 μM, 1 h) effectively inhibits PANC-1 cells' PKD-dependent cortactin phosphorylation[1]. 3-IN-PP1 inhibits the development of several tumor cells, with IC50 values ranging from 1.6 to 39.2μM[1].

PKD screening assay [1]
An in vitro luminescence based assay (ADP-Glo™ from Promega Corporation) was used to determine the PKD phosphorylation activity. Syntide-2 was used as a substrate and assays were performed in the presence of various compounds at a concentration of 1 μM for initial screening, and concentrations ranging from 10 μM to 1 nM for IC50 determinations. Reactions were carried out in white 96-well flat bottom plates (NUNC) with a 10 μL reaction volume containing 50 mM Tris-HCl pH 7.4, 10 mM MgCl2, 8 nM of PDBu stimulated FLAG PKD, 250 μM Syntide-2 and 10 μM ATP. After incubation of 15 min at 30 °C, the reaction was stopped and incubated with ADP-Glo reagent I and II (ratio 1:1:2, i.e. reaction volume: reagent 1: reagent 2, respectively) according to the manufacturer’s protocol. Subsequently, the luminescence was measured using a PerkinElmer Victor X4 Multiple Plate Reader in counts per second (CPS). Percentage of PKD inhibition was determined relative to a DMSO control (0.1% DMSO). Graphs were plotted and IC50 values were determined using the GraphPad Prism 7 software package.

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Cellular screening assay using phospho-cortactin as a readout of PKD inhibition [1]
PANC-1 cells were seeded at a density of 0.3 × 106 cells per condition in a 6-well plate and allowed to attach. Twenty-four hours later, 0.5 μg of pcDNA-FLAG-PRKD1 or pcDNA-FLAG-PRKD2 and 0.5 μg of pcDNA3-Myc-CTTN were transfected using a PEI at a 1:3 (m/m) plasmid/PEI ratio. After 24 h, the cells were treated with the indicated compounds (Fig. 3) at a final concentration of 20 μM or DMSO control for 1 h, followed by either stimulation or no stimulation with 250 nM of phorbol-12,13-dibutyrate (PDBu) for 15 min. Lysis was done in 50 mM Tris, pH 7.4, 150 mM NaCl, 15 mM EDTA, 1% NP-40 supplemented with phosphatase inhibitors, and protease inhibitors. Protein quantification was determined using the BCA protein assay. Next, equal amounts of lysate (10 μg) were resolved in an SDS-PAGE and blotted into a nitrocellulose membrane. Antibody unspecific binding was blocked with either 5% BSA or 5% low-fat milk. Blots were probed with anti-Cortactin pSer-298 antibody (homemade), anti-Myc 9E10 and anti-FLAG M2. Finally, blots were incubated with the appropriate secondary HRP-linked antibody: Goat anti-Rabbit or Horse anti-Mouse. Band intensities were quantified with Image Studio Lite. Ponceau staining was used as a loading control. The sharpness of the ponceau images was improved by using the default settings of the sharpen tool in Image Studio Lite software. Experiments were performed in triplicate (n = 3). The statistical measures used were the mean and SEM. A one-way ANOVA was performed to determine the statistical significance with the control conditions (DMSO). Statistical analysis was conducted using the GraphPad Prism 7 software package.

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CAMKIIα and PKCδ assay [1]
An in vitro radiometric assay was used to determine the inhibition of CAMKIIα and PKCδ in presence of various compounds at the indicated concentrations. Reactions were carried out in 40 μL of a mixture containing 50 mM Tris-HCl pH 7.4, 10 mM MgCl2, 2 mM CaCl2, 30 μg/μL Calmodulin, 50 ng of CAMKIIα, 2 μg Syntide-2, 25 μM ATP and 2 μCi [γ-32P] ATP for the CAMKIIα assay. For the PKCδ assay, reactions were carried out in 40 μL of a mixture containing 50 mM Tris-HCl pH 7.4, 10 mM MgCl2, 4 μL of phosphatidylserine (PS)/PDBu vesicles stock, 50 ng of PKCδ, 5 μg MARCKS peptide (in-house synthesis), 25 μM ATP and 2 μCi [γ-32P] ATP. After 10 min, the kinase reaction was stopped by spotting 30 μL of the reaction on Whatman P81 filter paper. The filter papers were washed three times with an aqueous solution of 0.5% phosphoric acid, followed by one wash with 100% acetone. Subsequently, the papers were air-dried and counted using a Tri-Carb 2810 TR scintillation counter. Percentage of inhibition was determined relative to a condition where no inhibitor was added (0.1% DMSO). Graphs were plotted using the GraphPad Prism 7 software package. PS/PDBu vesicles were prepared as follows: PDBu and PS were mixed to final concentrations of 100 μg/mL PS and 250 nM PDBu and dried using a Savant Speedvac concentrator. Dried lipids were resuspended in 50 mM Tris pH 7.4 and sonicated three times for 10 min until a clear solution was achieved.

Cell Proliferation Assay[1]
Cell Types: PANC-1 cell
Tested Concentrations: 5 μM
Incubation Duration: 0-114 h
Experimental Results: Dramatically inhibited PANC-1 cell proliferation after incubation for 96 and 144 hrs (hours).

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Western Blot Analysis[1]
Cell Types: PANC-1 cells
Tested Concentrations: 20 μM
Incubation Duration: 1 h
Experimental Results: diminished cortactin phosphorylation in PANC-1 cells.

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Proliferation test [1]
PANC-1 cells were seeded at 2000 cells/well on a 96-well plate and allowed to attach for 4 h. Subsequently, each compound was added to achieve a concentration of 5 μM or DMSO (vehicle) was added to a final concentration of 0.25% as a negative control. Cells were allowed to grow for 0, 48, 96 and 144 h before adding MTT (Thiazolyl Blue Tetrazolium Bromide) at final concentration of 0.5 mg/mL. The cells were incubated for 3 h and thereafter lysed with DMSO. The absorbance at 550 nm was used as a readout of cell proliferation. Proliferation experiments were performed four times (n = 4). The statistical measures used were the mean and SEM. A two-way ANOVA combined with Dunnett’s multiple comparisons test was performed to determine the statistical significance with the control conditions (DMSO). Statistical analysis was conducted using the GraphPad Prism 7 software package.

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Antitumor assays [1]
All cell lines were cultured as recommended by the suppliers. Culture media were purchased from Gibco Life Technologies and supplemented with 10% fetal bovine serum. Adherent cell lines LN-229, HCT-116, NCI-H460, and Capan-1 cells were seeded at a density between 400 and 1250 cells per well, in 384-well, black walled, clear-bottomed tissue culture plates. After overnight incubation, cells were treated with the test compounds at seven different concentrations ranging from 100 to 6.4 × 10−3 μM. Suspension cell lines HL-60, K-562, Z-138, and DND-41 were seeded at densities ranging from 2500 to 5000 cells per well in 384-well, black walled, clear-bottomed tissue culture plates containing the test compounds at the same seven concentration points. The plates were incubated and monitored at 37 °C for 72 h in an IncuCyte for real-time imaging. Images were taken every 3 h, with one field imaged per well under 10 × magnification. All compounds were tested with duplicate data points and averaged.

[1]. Design, synthesis and biological evaluation of pyrazolo[3,4-d]pyrimidine-based protein kinase D inhibitors. Eur J Med Chem.

The multiple roles of protein kinase D (PKD) in various cancer hallmarks have been repeatedly reported. Therefore, the search for novel PKD inhibitors and their evaluation as antitumor agents has gained considerable attention. In this work, novel pyrazolo[3,4-d]pyrimidine based pan-PKD inhibitors with structural variety at position 1 were synthesized and evaluated for biological activity. Starting from 3-IN-PP1, a known PKD inhibitor with IC50 values in the range of 94-108 nM, compound 17m was identified with an improved biochemical inhibitory activity against PKD (IC50 = 17-35 nM). Subsequent cellular assays demonstrated that 3-IN-PP1 and 17m inhibited PKD-dependent cortactin phosphorylation. Furthermore, 3-IN-PP1 displayed potent anti-proliferative activity against PANC-1 cells. Finally, a screening against different cancer cell lines demonstrated that 3-IN-PP1 is a potent and versatile antitumoral agent.[1]

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Based on our previously identified PKD inhibitor 3-IN-PP1, a novel series of pyrazolo[3,4-d]pyrimidines was synthesized. Systematic structural variation at position 1 of this scaffold led to the discovery of 17m, which is the most potent pyrazolo[3,4-d]pyrimidine based pan-PKD inhibitor reported to date with IC50 values of 35, 35 and 17 nM for PKD1, 2 and 3, respectively. Despite the fact that compound 17m was 3- to 5-fold more potent in a biochemical PKD assay, when compared to 3-IN-PP1, compound 17m was less potent in cellular environments. On the other hand, 3-IN-PP1 very potently inhibited PKD activity in cells and blocked the proliferation of PANC-1 pancreatic cancer cells. Finally, a broad antitumoral screening revealed that 3-IN-PP1 is endowed with low-micromolar antiproliferative activity against 7 out of 8 cancer cell lines. Altogether, these data demonstrate that within the pyrazolo[3,4-d]pyrimidine based PKD inhibitors developed to date, 3-IN-PP1 clearly is the most potent antiproliferative agent.[1]

C17H18N6

306.365022182465

306.159

2227110-54-3

162663243

Off-white to light yellow solid powder

2.5

2

4

2

23

435

0

C1=NC(N)=C2C(C3C4=C(NC=3)C=CC=C4)=NN(C(C)(C)C)C2=N1

RBZLXANOQRQGTN-UHFFFAOYSA-N

InChI=1S/C17H18N6/c1-17(2,3)23-16-13(15(18)20-9-21-16)14(22-23)11-8-19-12-7-5-4-6-10(11)12/h4-9,19H,1-3H3,(H2,18,20,21)

1-tert-butyl-3-(1H-indol-3-yl)pyrazolo[3,4-d]pyrimidin-4-amine

3-IN-PP1; 2227110-54-3; CHEMBL4779081; BDBM50565687; PD195878;

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 (e.g. under nitrogen), avoid exposure to moisture and light.

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

DMSO: 100 mg/mL (326.40 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.16 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 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (8.16 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (8.16 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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