FG-2216 (YM-311) is a potent HIF-prolyl hydroxylase inhibitor for the PDH2 enzyme (IC50: 3.9 uM); orally bioavailable and induced reversible and significant Epo induction in vivo.

PHD2:3.9 μM

FG-2216 shows the ability to stabilize HIF-α to stimulate EPO secretion. [1]

In male rhesus macaques, FG-2216 (60 mg/kg, p.o.), induces significant and reversible Epo induction, and induces a small elevation of HbF expression. [2]

Metabolic Labeling and Gel Electrophoresis: COLO 320DM cells (200,000) are injected into each well of 12-well plastic plates 2 days before incubation in the presence of KNK437 for 1 h before heat shock. The cells are then heat-shocked at 42°C for 90 min or kept at 37°C for the same length of time and incubated at 37°C for 2 h. For metabolic labeling, cells are washed with PBS without Ca2+ or Mg2+ and incubated for 1 h with 1.22 MBq of [35S]methionine in 250 μL of methionine-free DMEM supplemented with 10% dialyzed fetal bovine serum. After metabolic labeling, cells are washed twice with PBS and lysed in a buffer containing 1% NP40, 0.15 M NaCl, 50 mM Tris-HCl (pH 8.0), 5 mM EDTA, and protease inhibitors [0.2 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, 2 mM N-ethylmaleimide, 1 μg/mL pepstatin, and 1 μg/mL leupeptin]. After centrifugation at 12,000×g for 20 min, cell extracts containing equal amounts of trichloroacetic acid-insoluble radioactivity are analyzed by two-dimensional gel electrophoresis (the one-dimensional gel electrophoresis is a nonequilibrium pH gradient gel electrophoresis, and the two-dimensional gel electrophoresis is 10% SDS-PAGE).