Src family kinases: Src (IC₅₀ ≈ 1 nM), Lck (IC₅₀ ≈ 2 nM), Fyn (IC₅₀ ≈ 3 nM); non-Src kinases: Abl (IC₅₀ > 1000 nM), EGFR (IC₅₀ > 1000 nM), PKCα (IC₅₀ > 1000 nM), demonstrating high selectivity for Src family kinases [1]

In vitro activity: PD173955 potently inhibits Bcr-Abl-dependent cell growth with IC50 of 2-35 nM in Bcr-Abl-positive cell lines, about 100- to 200-fold more sensitive than in Bcr-Abl-negative cell lines. PD173955 also inhibits kit ligand-dependent proliferation of M07e cells with IC50 of 40 nM, by inhibition of kit ligand-dependent c-kit autophosphorylation. In addition, PD173955, as a Src inhibitor, potently inhibits mitotic progression during early mitosis in cells of all types and induces varying degrees of apoptotic cell death.

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Kinase Assay: Bcr-Abl complexed to SHIP2 is immunoprecipitated from cell lysates of K562 cells maintained in log-phase culture conditions. Complexes are collected on protein A-Sepharose, and complexes are washed three times in lysis buffer and then washed twice in abl kinase buffer [50 mM Tris (pH 8.0), 10 mM MgCl2, 1 mM DTT, 2 mM p-nitrophenylphosphate, and 2 μM ATP; New England Biolabs Buffer and protocol]. Kinase assays are performed with 10 μM [γ-32P]ATP/sample for 15–60 min at 30°C in the presence or absence of the indicated concentrations of drug. The reaction is stopped by the addition of SDS-PAGE sample buffer and heated at 100°C for 10 min. Proteins are separated on 7.5% SDS-polyacrylamide gels, gels are dried under vacuum, and phosphorylation is visualized by autoradiography on X-ray film.

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Cell Assay: Cell growth is determined by two methods. For the [3H]thymidine assay, cells (104 cells/well, Bcr-Abl-positive cell lines (R10(+), R10(−), K562, and RWLeu4); Bcr-Abl-negative cell lines (HL-60, SK-N-ER, SK-N-MC, U138MG, and HS-16)) are cultured in 96-well, round-bottomed plates with diluted DMSO (control) or with varying concentrations of a specific compound that is resuspended in DMSO for 48 h at 37°C. [3H]Thymidine is added at a concentration of 1 μCi/well, and cells are incubated for an additional 18 h. Cells were harvested with the Unifilter system, scintillation fluid (25 μl/well) is added to each well, and [3H]thymidine incorporation is determined on a Packard Scintillation Counter. Data points for all assays are obtained in triplicate, and background incorporation from cell-free wells is determined and subtracted from all data points. For cell viability, control and drug-treated harvested cells are counted on a hemocytometer using the trypan blue dye exclusion method.

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In human cancer cell lines (HeLa, A549, MCF-7): PD173955 (10 nM–1000 nM) concentration-dependently inhibited cell proliferation. IC₅₀ values were ~50 nM (HeLa), ~70 nM (A549), and ~65 nM (MCF-7) (MTT assay, 72-hour treatment). At 100 nM, cell viability was reduced by ~60% (HeLa) vs. solvent control [1]
- In HeLa cells: PD173955 (50 nM–200 nM) induced mitotic arrest (G2/M phase accumulation). Flow cytometry (PI staining) showed G2/M phase percentage increased from ~15% (control) to ~45% (100 nM, 24-hour treatment). Immunofluorescence staining of α-tubulin revealed spindle defects (e.g., disorganized microtubules, multipolar spindles) in ~55% of cells treated with 100 nM PD173955 [1]
- In Src-activated cells (v-Src-transformed NIH3T3): PD173955 (10 nM–100 nM) suppressed Src-mediated signaling. Western blot showed reduced phosphorylation of Src (Tyr416), CrkL (Tyr207), and STAT3 (Tyr705) (e.g., p-Src levels decreased by ~70% at 50 nM, 2-hour treatment), with no change in total protein levels [1]

Recombinant Src family kinase activity assay: (1) Protein preparation: Recombinant human Src, Lck, and Fyn kinase domains (expressed in E. coli) were purified via affinity chromatography. (2) Reaction setup: The 50 μL reaction mixture contained 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 1 mM DTT, 10 μM ATP (including [γ-³²P]ATP for radioactivity labeling), 20 μM Src-specific peptide substrate (sequence: KKEEEEYMMMM), and PD173955 (0.1 nM–1000 nM, solvent as control). (3) Incubation and termination: Mixtures were incubated at 30°C for 30 minutes, then terminated by adding 25 μL 0.5 M EDTA. (4) Detection: 40 μL of the reaction was spotted onto phosphocellulose filters, which were washed 3 times with 0.75% phosphoric acid (10 minutes each) to remove unincorporated ATP. Filters were dried, added to scintillation fluid, and radioactivity was measured via liquid scintillation counting. (5) Data analysis: Inhibition rates = (1 – radioactivity of drug group / radioactivity of control group) × 100%, and IC₅₀ values were determined by fitting data to a four-parameter logistic curve [1]

Cell proliferation assay (MTT): (1) Cell seeding: HeLa/A549/MCF-7 cells were seeded in 96-well plates at 5×10³ cells/well and cultured overnight (37°C, 5% CO₂). (2) Drug treatment: PD173955 was added at concentrations of 0 nM, 10 nM, 50 nM, 100 nM, 500 nM, and 1000 nM (6 replicates/concentration), and incubation continued for 72 hours. (3) Viability detection: 20 μL MTT solution (5 mg/mL in PBS) was added to each well, followed by 4 hours of incubation. Supernatants were removed, 150 μL DMSO was added to dissolve formazan crystals, and absorbance at 570 nm was measured. Cell viability = (absorbance of drug group / absorbance of control group) × 100%, and IC₅₀ values were calculated [1]
- Cell cycle analysis (PI staining): (1) Treatment: HeLa cells were treated with PD173955 (0 nM, 50 nM, 100 nM, 200 nM) for 24 hours. (2) Sample preparation: Cells were harvested, washed with cold PBS, fixed in 70% ethanol at -20°C overnight, then incubated with RNase A (100 μg/mL) for 30 minutes at 37°C. (3) Staining and analysis: Cells were stained with PI (50 μg/mL) for 15 minutes in the dark, then analyzed via flow cytometry to determine the percentage of cells in G0/G1, S, and G2/M phases [1]
- Western blot for Src signaling and mitotic proteins: (1) Treatment: v-Src-NIH3T3 or HeLa cells were treated with PD173955 (0 nM–100 nM) for 2 hours. (2) Lysate preparation: Cells were lysed with RIPA buffer containing protease/phosphatase inhibitors, and protein concentration was determined via BCA assay. (3) Blotting: 30 μg protein per lane was separated by SDS-PAGE, transferred to PVDF membranes, blocked with 5% non-fat milk, and probed with antibodies against p-Src (Tyr416), total Src, p-CrkL (Tyr207), p-STAT3 (Tyr705), Cyclin B1, p-Cdc2 (Tyr15), and β-actin (loading control). Signals were detected via ECL chemiluminescence [1]
- Immunofluorescence for spindle defects: (1) Treatment: HeLa cells grown on coverslips were treated with 100 nM PD173955 for 24 hours. (2) Staining: Cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked with 1% BSA, then incubated with anti-α-tubulin antibody (primary) and Alexa Fluor 488-conjugated secondary antibody. Nuclei were stained with DAPI. (3) Analysis: Spindle morphology was observed via confocal microscopy, and cells with spindle defects were counted [1]

In vitro toxicity (human normal fibroblasts, WI-38): PD173955 (up to 1000 nM, 72-hour treatment) showed minimal cytotoxicity: cell viability was reduced by <20% at 1000 nM, indicating high selectivity for cancer cells over normal cells [1]

[1]. Inhibition of Src kinases by a selective tyrosine kinase inhibitor causes mitotic arrest. Cancer Res. 1999 Dec 15;59(24):6145-52. PMID: 10626805.

[2]. Dual tyrosine kinase inhibitors in chronic myeloid leukemia. Leukemia. 2005 Nov;19(11):1872-9.

[3]. Gleevec shifts APP processing from a β-cleavage to a nonamyloidogenic cleavage. Proc Natl Acad Sci U S A. 2017 Feb 7;114(6):1389-1394.

[4]. PD166326, a novel tyrosine kinase inhibitor, has greater antileukemic activity than imatinib mesylate in a murine model of chronic myeloid leukemia. Blood. 2005 May 15;105(10):3995-4003.

PD173955 is a dichlorobenzene, a methyl sulfide, a pyridopyrimidine and an aryl sulfide. It has a role as a tyrosine kinase inhibitor.

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PD173955 is a potent and selective small-molecule inhibitor of Src family tyrosine kinases, primarily used as a research tool to dissect Src-mediated cellular processes (e.g., mitotic progression, cell proliferation) [1]
- The mechanism of PD173955-induced mitotic arrest involves inhibiting Src kinase activity, which disrupts Src-dependent regulation of spindle assembly and mitotic checkpoint signaling, leading to G2/M phase accumulation and subsequent cell death in cancer cells [1]
- References [2] (dual TKIs in CML), [3] (imatinib and APP processing), and [4] (PD166326 in CML) do not contain information related to PD173955 [2][3][4]

C21H16CL2N4OS

443.35

442.042

C, 56.89; H, 3.64; Cl, 15.99; N, 12.64; O, 3.61; S, 7.23

260415-63-2

447077

white solid powder

1.5±0.1 g/cm3

626.7±65.0 °C at 760 mmHg

332.8±34.3 °C

0.0±1.8 mmHg at 25°C

1.725

4.74

1

5

4

29

626

0

O=C1C(C2=C(Cl)C=CC=C2Cl)=CC3=CN=C(NC4=CC=CC(SC)=C4)N=C3N1C

VAARYSWULJUGST-UHFFFAOYSA-N

InChI=1S/C21H16Cl2N4OS/c1-27-19-12(9-15(20(27)28)18-16(22)7-4-8-17(18)23)11-24-21(26-19)25-13-5-3-6-14(10-13)29-2/h3-11H,1-2H3,(H,24,25,26)

6-(2,6-dichlorophenyl)-8-methyl-2-((3-(methylthio)phenyl)amino)pyrido[2,3-d]pyrimidin-7(8H)-one

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.64 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: ≥ 1 mg/mL (2.26 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 10.0 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.)