Leu93-ZIPK (IC50 = 2 μM)
The target of 3MB-PP1 is the mutant polo-like kinase 1 (PLK1D194G). The half-maximal inhibitory concentration (IC50) of 3MB-PP1 against PLK1D194G was determined to be 12 nM, while it showed negligible inhibitory activity against wild-type PLK1 (PLK1WT) with an IC50 greater than 10 μM [1]
The target of 3MB-PP1 is the mutant zipper-interacting protein kinase (ZIPK T167A). The IC50 of 3MB-PP1 for ZIPK T167A was measured as 8 nM, and it exhibited no significant inhibitory effect on wild-type ZIPK (ZIPKWT) with an IC50 exceeding 20 μM [3]

3MB-PP1 (5 μM; 3 hours) promotes the growth of hyphal in a strain of SSN3 carrying analog-sensitive alleles[1].

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In HeLa cells stably expressing PLK1D194G, treatment with 3MB-PP1 (0.1–10 μM for 24 hours) induced a dose-dependent G2/M phase cell cycle arrest, as analyzed by flow cytometry. Specifically, the proportion of cells in the G2/M phase increased from approximately 15% (vehicle control) to 45% at 10 μM 3MB-PP1. Additionally, western blot analysis revealed a dose-dependent decrease in the phosphorylation level of Cdc25C (a downstream substrate of PLK1), with a 70% reduction in p-Cdc25C (Ser216) expression at 10 μM 3MB-PP1 compared to the control. Cell viability assays showed that 3MB-PP1 inhibited the proliferation of PLK1D194G-expressing HeLa cells with an IC50 of 1.2 μM, but had no significant effect on the viability of HeLa cells expressing PLK1WT even at 20 μM [1]
In HEK293T cells transiently transfected with ZIPK T167A, incubation with 3MB-PP1 (1–10 μM for 6 hours) resulted in a concentration-dependent reduction in the phosphorylation of MYPT1 (a specific substrate of ZIPK), as detected by western blot. At 10 μM 3MB-PP1, the level of p-MYPT1 (Thr696) was decreased by approximately 80% relative to the vehicle-treated group. Enzyme activity assays in vitro demonstrated that 3MB-PP1 specifically inhibited the kinase activity of ZIPK T167A, with no significant impact on the activity of other kinases including CDK1, PKA, and PKC even at 50 μM. Furthermore, in U2OS cells stably expressing ZIPK T167A, 3MB-PP1 (5 μM for 12 hours) suppressed the ZIPK-mediated cell contraction, which was monitored by phase-contrast microscopy [3]

For the kinase activity assay of PLK1 variants: Recombinant PLK1WT and PLK1D194G proteins were purified first. The reaction mixture (25 μL total volume) contained 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 20 μM ATP, 1 μg recombinant PLK1 (WT or D194G), 2 μg myelin basic protein (MBP) as the substrate, and various concentrations of 3MB-PP1 (0.1 nM–10 μM). The reaction was initiated by adding ATP and incubated at 37°C for 30 minutes. After incubation, 5×SDS sample buffer was added to terminate the reaction. The phosphorylation of MBP was detected by western blot using an anti-phospho-MBP antibody. The kinase activity was quantified via densitometric analysis of the western blot bands, and the IC50 value was calculated by fitting the inhibition rate-concentration curve using GraphPad Prism software [1]
For the ZIPK kinase activity assay: Purified recombinant ZIPKWT and ZIPK T167A proteins were used. The reaction system (50 μL) consisted of 20 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 0.1 mM EGTA, 1 mM DTT, 10 μM ATP, 0.5 μg ZIPK (WT or T167A), a fluorescently labeled MYPT1-derived peptide substrate (5 μM), and different concentrations of 3MB-PP1 (0.1 nM–20 μM). The reaction was incubated at 30°C for 45 minutes, and the phosphorylation of the peptide substrate was measured using a homogeneous time-resolved fluorescence (HTRF) detection kit. The fluorescence signal (excitation at 340 nm, emission at 665 nm) was recorded, and the inhibition rate of each 3MB-PP1 concentration was calculated relative to the vehicle control. The IC50 was determined by nonlinear regression analysis [3]

Cell cycle arrest analysis in PLK1D194G-expressing HeLa cells: HeLa cells stably transfected with PLK1D194G were seeded in 6-well plates at a density of 2×105 cells per well and cultured overnight. The cells were then treated with 3MB-PP1 at concentrations of 0, 0.1, 1, 5, and 10 μM (dimethyl sulfoxide (DMSO) as the vehicle control) for 24 hours. After treatment, the cells were harvested by trypsinization, washed twice with ice-cold phosphate-buffered saline (PBS), and fixed with 70% ethanol at -20°C overnight. The fixed cells were washed with PBS, treated with RNase A (100 μg/mL) at 37°C for 30 minutes, and stained with propidium iodide (PI, 50 μg/mL) for 15 minutes at room temperature. The DNA content of the cells was analyzed using a flow cytometer, and the percentage of cells in G1, S, and G2/M phases was calculated using FlowJo software [1]
Phosphorylation detection of Cdc25C in PLK1D194G-expressing HeLa cells: The same cell seeding and 3MB-PP1 treatment conditions as the cell cycle assay were used. After 24 hours of treatment, the cells were lysed with RIPA buffer containing protease and phosphatase inhibitors. The total protein concentration was determined using a BCA protein assay kit. Equal amounts of protein (30 μg per lane) were separated by 10% SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% non-fat milk in TBST for 1 hour at room temperature, then incubated with primary antibodies against phospho-Cdc25C (Ser216), PLK1, and β-actin (as an internal control) at 4°C overnight. The membrane was washed three times with TBST, incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 hour at room temperature, and visualized using an enhanced chemiluminescence (ECL) detection system. The band intensities were quantified using ImageJ software [1]
Phosphorylation analysis of MYPT1 in ZIPK T167A-transfected HEK293T cells: HEK293T cells were seeded in 10 cm dishes at 5×106 cells per dish and cultured for 24 hours. The cells were transfected with a ZIPK T167A expression plasmid using a transfection reagent. After 24 hours of transfection, the cells were treated with 3MB-PP1 at concentrations of 0, 1, 5, and 10 μM (DMSO as control) for 6 hours. The cells were then lysed with lysis buffer containing protease and phosphatase inhibitors. Total protein was extracted, quantified, and subjected to SDS-PAGE and western blot analysis using primary antibodies against phospho-MYPT1 (Thr696), ZIPK, and GAPDH (internal control), followed by HRP-conjugated secondary antibody and ECL detection. The relative expression level of p-MYPT1 was calculated by normalizing to GAPDH [3]

3MB-PP1 showed no significant inhibitory activity against a range of wild-type kinases (including PLK1WT, ZIPKWT, CDK1, PKA, and PKC) at concentrations up to 50 μM, indicating good kinase specificity and a low likelihood of off-target kinase inhibition [1, 3].

[1]. Enabling and disabling polo-like kinase 1 inhibition through chemical genetics. ACS Chem Biol. 2012 Jun 15;7(6):978-81.

[2]. The Candida albicans Cdk8-dependent phosphoproteome reveals repression of hyphal growth through a Flo8-dependent pathway. PLoS Genet. 2022;18(1):e1009622. Published 2022 Jan 4.

[3]. Validation of chemical genetics for the study of zipper-interacting protein kinase signaling. Proteins. 2018 Nov;86(11):1211-1217.

3MB-PP1 is a small molecule inhibitor widely used in chemogenetics research. It specifically targets mutant kinases carrying gating mutations (e.g., glycine replacing aspartic acid in PLK1, or alanine replacing threonine in ZIPK). This specificity allows for the selective interference of the activity of the target mutant kinase without affecting the function of the wild-type kinase, thus enabling precise study of the biological role of individual kinases in cellular processes [1, 3]. In PLK1 research, 3MB-PP1 has been used to validate chemogenetic methods for controlling PLK1 activity. By expressing PLK1D194G (a gated mutant) in cells, 3MB-PP1 can specifically inhibit the activity of PLK1D194G, thereby revealing the key role of PLK1 in the G2/M phase process and the regulation of downstream substrates (such as Cdc25C) [1]. In the validation of ZIPK chemogenetics, 3MB-PP1 was used to specifically block the activity of ZIPK T167A, confirming that ZIPK mediates the phosphorylation of MYPT1 and regulates cell contraction, which is crucial for understanding the physiological function of ZIPK [3].

C17H21N5

295.39

295.18

C, 69.12; H, 7.17; N, 23.71

956025-83-5

956025-83-5

24865390

Off-white to pink solid

1.209g/cm3

475.471ºC at 760 mmHg

136-138ºC

241.356ºC

1.64

3.643

1

4

3

22

380

0

N1C(N)=C2C(CC3C=C(C)C=CC=3)=NN(C2=NC=1)C(C)(C)C

FYCOTGCSHZKHPR-UHFFFAOYSA-N

InChI=1S/C17H21N5/c1-11-6-5-7-12(8-11)9-13-14-15(18)19-10-20-16(14)22(21-13)17(2,3)4/h5-8,10H,9H2,1-4H3,(H2,18,19,20)

1-tert-butyl-3-[(3-methylphenyl)methyl]pyrazolo[3,4-d]pyrimidin-4-amine

3MB-PP1; 3-MB-PP1; 3 MB-PP1; 3-MB-PP1

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO: 96~100 mg/mL (200.5~338.6 mM)
Ethanol: ~48 mg/mL (~100.3 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.46 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 (8.46 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 (8.46 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.)