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

Enolase:0.576 μM

AP-III-a4在Huh7肝细胞和HEK肾细胞中促进葡萄糖摄取并抑制磷酸烯醇式丙酮糖激酶(PEPCK)的表达。在低氧条件下，AP-III-a4诱导细胞死亡，并通过降低AKT和Bcl-xL的表达，在HCT116细胞中抑制癌细胞的迁移和侵袭。

AP-III-a4（10 μM）在HCT116-异种移植斑马鱼肿瘤模型中抑制癌细胞迁移和侵袭过程。在体内，AP-III-a4（10 μM）还抑制脂肪生成和泡沫细胞形成，并导致PEPCK表达下调和葡萄糖摄取的诱导。

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.