GSK484 hydrochloride

Fluorescence Polarization (FP) binding affinity studies ^[1]:

Preparation method

PAD4 was serially diluted in the presence of 10 nM GSK484 in Assay Buffer (100 mM HEPES, pH 8, 50 mM NaCl, 5% glycerol, 1 mM CHAPS, 1 mM DTT) at varying concentrations of calcium (0 mM, 0.2 mM, 2 mM and 10 mM). Following incubation for 50 min, apparent K_d values for each calcium concentration were determined using a single site saturation curve.

For IC₅₀ determination, test compounds were serially diluted in DMSO (1% final assay concentration) and tested at the same range of calcium concentrations in the presence of PAD4 (at the calculated K_d for each calcium condition) and 10 nM GSK484 in the same assay buffer and volume. Reactions were incubated for 50 min, after which IC₅₀ values were calculated.

Applications

GSK484 demonstrated high affinity binding to the low-calcium form of PAD4 with IC₅₀ 50 nM(no Ca⁺).

Cell experiment ^[2]:

Cell lines

MDA-MB-231 cells (Triple-negative breast cancer (TNBC))

Preparation method

GSK484 was dissolved in DMSO. The DMSO concentration of the solution used was consistently lower than 0.1%. Cells were grown to 70% -80% confluence on plates and cultivated with a low dose of GSK484 for 1 h before irradiation.

Reaction Conditions

25 nM; 1 h before irradiation

Applications

GSK484 facilitates cell apoptosis and promotes the radiosensitivity of MDA-MB-231 cells.

Animal experiment ^[2]:

Animal models

Female BALB/c mice (6-week-old)

Preparation method

MDA-MB-231 cells were injected subcutaneously into the 4th mammary fat pads of lactiferous ducts the in mice. Mice were randomly divided into four groups (Vehicle, 15 Gy alone, GSK484 4 mg/kg, and 15 Gy + GSK484 4 mg/kg). GSK484 was administered at a dose of 4 mg/kg ip in 2-hydroxypropyl-β-cyclodextrin at 1 hour before irradiation. The tumor areas were irradiated with 6 MV X-rays (15 Gy in one fraction) in 15 Gy alone and 15 Gy + GSK484 4 mg/kg groups.

Dosage form

4 mg/kg; i.p.; 1 hour before irradiation

Applications

Treatment with GSK484 and ischemia reperfusion (IR), the tumor volume and body weight of mice decreased significantly.

References:

[1]. Lewis HD, Liddle J, et,al. Inhibition of PAD4 activity is sufficient to disrupt mouse and human NET formation. Nat Chem Biol. 2015 Mar;11(3):189-91. doi: 10.1038/nchembio.1735. Epub 2015 Jan 26. PMID: 25622091; PMCID: PMC4397581.
[2]. Wei L, Wang X, et,al. The PAD4 inhibitor GSK484 enhances the radiosensitivity of triple-negative breast cancer. Hum Exp Toxicol. 2021 Jul;40(7):1074-1083. doi: 10.1177/0960327120979028. Epub 2020 Dec 23. Erratum in: Hum Exp Toxicol. 2021 Nov;40(11):2022. PMID: 33355008.