In vitro activity: Enalaprilat has high affinity for human endothelial ACE with IC50 of 1.94 nM in vitro binding assay by displacing a saturating concentration of [125I]351A, a radiolabeled lisinopril analogue from ACE binding sites, and shows bradykinin/angiotensin I selectivity ratio of 1.00 calculated from double displacement experiments. Enalaprilat has the strong inhibitory effect on Aβ42-to-Aβ40-converting activity found in the N-domain of ACE, exhibiting a 10-fold lower IC50 (0.003~0.01 μM) than captopril (0.03~0.1 μM). Enalaprilat (100 nM) blocks protein kinase C epsilon by directly activating bradykinin B1 receptor at the canonical Zn2+ binding site, leading to prolonged nitric oxide (NO) production in cytokine-treated human lung microvascular endothelial cells. Enalaprilat attenuates the IGF-I induced neonatal rat cardiac fibroblast growth (30% reduction) in a concentration-dependent fashion, with IC50 of 90 mM.

Kinase 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.

Cell Assay: 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.

Eur J Pharmacol. 2007 Dec 22;577(1-3):1-6; Br J Anaesth. 2006 Apr;96(4):437-43.