ZT-1a is a potent, non-ATP-competitive and selective SPAK inhibitor. ZT-1a inhibits SPAK activity with IC50s of 44.3, 35.0, 46.7 μM at ATP concentrations of 0.01, 0.1 and 1 mM, respectively.IC50 & Target: SPAKIn Vitro: ZT-1a inhibits Na-K-2Cl cotransporter (NKCC1) and stimulates K-Cl cotransporters (KCCs) by decreasing their SPS1-related proline/alanine-rich kinase (SPAK)-dependent phosphorylation.
ZT-1a inhibits phosphorylation of NKCC1 p-Thr203/207/212 by 72±5.2% at 1?μM ZT-1a and phosphorylation of KCC sites 1/2 by 65-77% at 3?μM in HEK-293 cells.
SPAK phosphorylation at Ser373 is inhibited by 70±3.8% inhibition at 3-10?μM ZT-1a.
ZT-1a (10?μM) inhibits NKCC1 but stimulates KCC3 activity.In Vivo: ZT-1a (10-100?mg/kg) inhibits SPAK-dependent cation-Cl? cotransporters (CCC) phosphorylation in vivo.