AZD7545 is a potent PDHK inhibitor.

PDHK2:6.4 nM, PDHK1:36.8 nM

在肥胖的胰岛素抵抗的Zucker大鼠中,AZD7545消除了血糖的餐后升高.AZD7545增加Wistar大鼠中肝脏和骨骼肌中活性PDH的百分比.

AZD7545通过破坏PDHK2与PDC的二氢硫辛酸乙酰转移酶成分的内部硫辛酰基结构域的相互作用抑制PDHK活性。AZD7545增加原代大鼠肝细胞中PDH活性,EC50为105 nM。在重组人PDHK2存在下,AZD7545增加PDH活性,EC50为5.2 nM。

Cellular Kinase Assays: NIH3T3 cells prepared by transfection of human KDR. The cells are cultured in a collagen type I coated 96-well plate in an amount of 1.5 × 104 per well. The medium is then replaced by a DMEM medium containing 0.1% FCS. Ki8751 diluted in DMSO is added to each well and cultured. rhVEGF is added to a final concentration of 100 ng/mL, and the stimulation of cells is carried out at 37 °C. The cells are washed with PBS (pH 7.4), 50 μL of a solubilization buffer (20 mM HEPES (pH 7.4), 150 mM NaCl, 0.2% Triton X-100, 10% glycerol, 5 mM Na3VO4, 5 mM disodium ethylenediamine tetraacetate, and 2 mM Na4P2O7) is then added and a cell extract is prepared. Separately, PBS (50 μL, pH 7.4) containing 5 μg/mL of antiphosphotyrosine antibody (PY20) is added to a microplate for ELISA. After washing of the plate, 300 μL of a blocking solution is added. The cell extract is transferred to the plate. An anti-VEGFR2 antibody and a peroxidase-labeled anti-rabbit Ig antibody are added. Next, a chromophoric substrate for peroxidase is added, and the absorbance at 450 nm is measured with microplate reader. The VEGFR2 phosphorylation activity for each well is determined by presuming the absorbance with the addition of VEGF and without the addition of the test sample to be 100% VEGFR2 phosphorylation activity and VEGF to be 0% VEGFR2 phosphorylation activity. The concentration of the inhibition (%) of VEGFR2 Phosphorylation is determined for each case, and IC50 value is calculated.