Enalaprilat Dihydrate (MK-422 Dihydrate) （IC50=1.94 nM） is a potent angiotensin-converting enzyme (ACE) inhibitor.

ACE:1.94 nM

Enalaprilat在体外结合实验中对人类内皮ACE具有高亲和力，通过置换饱和浓度的[125I]351A（一种放射性标记的利尿酶类似物）从ACE结合位点上，显示出1.94 nM的IC50，并且其在双重置换实验中计算出的缓激肽/血管紧张素I选择性比率为1.00。[1] Enalaprilat对ACE N-末端的Aβ42向Aβ40转换活性表现出强烈的抑制效果，其IC50远低于卡托普利（0.003~0.01 μM相比0.03~0.1 μM），显示出约10倍的差异。[2] Enalaprilat（100 nM）通过在典型的Zn2+结合位点直接激活缓激肽B1受体，阻断了蛋白激酶C epsilon，导致在细胞因子处理的人类肺微血管内皮细胞中产生持续的一氧化氮（NO）生成。[3] Enalaprilat以浓度依赖性的方式减少了IGF-I诱导的新生大鼠心脏成纤维细胞生长30%，IC50为90 mM。[4]

Enalaprilat由于其不利的电离特性，不适合通过口服来达到足够的药效，因此只能通过静脉注射给药。通过与乙醇进行酯化反应，可以生产出Enalapril，从而克服了Enalaprilat的这一局限性。研究表明，在大鼠出血性休克期间，相比安慰剂组，Enalaprilat的注射能显著降低70分钟时的平均动脉压(MAP)，并导致心输出量(CO)减少50%，一般趋势显示EB（Evans Blue）渗漏在肾脏和肺部尤为显著，以及在小肠中EB渗漏显著增加(53%)。Enalaprilat对非肥大心脏无效，但能显著减缓肥大心脏中左心室舒张末压的增加，相比之下无化合物时的增加幅度更大。

Single displacement binding assay: The binding assay is based on the competitive displacement of [125I]351A by Enalaprilat performed on whole endothelial cells. Subconfluent HUVECs in 6-well plates are rinsed with 2 mL binding buffer (140 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl2, 1.03 mM MgCl2, 0.42 mM NaH2PO4, 10 mM HEPES, 2 mM sodium pyruvate and 5 mM glucose, pH 7.4), and the culture medium is replaced with 2.5 mL fresh binding buffer containing 5% fetal bovine serum (FBS). The Enalaprilat (2.5-12.5 μL, 0.1-50 nM) or equivalent volumes of diluent are added to the binding buffer. A saturating amount of [125I]351A (10 μL, typically 106 cpm) is then added to each sample and the plates are incubated at 37 °C for 2 hours in a thermostatic bath. The cells are then rinsed twice with 1.5 mL binding buffer. Finally, the cells are extracted with 0.5 mL NaOH 1 N, incubated for 5 minutes, and the radioactivity is counted with a gamma counter. The ratio of specific [125I]351A bound to total bound activity (B/B0) is calculated, and the inhibitory potency of Enalaprilat expressed as the concentration of ACE inhibitors able to displace 50% of the bound radioligand, i.e. the IC50.

After 24 hours incubation in serum-free medium (DMEM), cells are stimulated with IGF-I (1-100 nM) and coincubated with Enalaprilat (1 nM-10 μM) for 24 hours. Cellular proliferation is assessed by 5-bromo-2'-deoxyuridine (BrdU) incorporation during the last 4 hours of the 24 hours incubation period using a colorimetric immunoassay. The extinctions are measured at 450 nm in an ELISA plate reader. All values consist of an n=9.(Only for Reference)