AP-III-a4 (ENOblock) (ENOblock) is the first nonsubstrate analogue inhibitor of enolase with IC50 of 0.576 μM.

Enolase:0.576 μM

Enolase activity assay is in the 2.0 mM MgSO4 and 400 mM KCl in the absence or presence of ENOblock or NaF, at 37°C by incubating pure enolase (3–9 U) in a buffer containing 50 mM imidazole-HCl (pH 6.8). The reaction is initiated by adding 1 μmol of 2-phospho-D-glycerate, and the OD is measured after 10 min of reaction time with a spectrophometer at 240 nm.

HCT116 cells(3 x 10E5) are seeded in a 6 well plate. 24 h later, cells are treated with AP-III-a4 (5 μM) for 24hours.

In HCT116-xenotransplanted zebrafish tumor xenograft model, were treated with AP-III-a4 of 10 μM.

ENOblock

DMSO: 257.5 mg/mL (432.98 mM), Sonication is recommended.

H2O: < 1 mg/mL (insoluble or slightly soluble)

10% DMSO+40% PEG300+5% Tween 80+45% Saline: 5 mg/mL (8.41 mM), Sonication is recommended.