Estabilishment And Application Of A Model For Drug Screening Targeting Neprilysin

Despite significant improvements in diagnosis and management, the global health and socioeconomic burden of cardiovascular disease(CVD), in particular that of hypertension and heart failure (HF), remains a major public health concern. A study found in an animal model of heart failure, the content and activity of Neprilysin (NEP) in kidney increased. NEP (EC 3.4.24.11) also known as neutral endopeptidase and enkephalinase, is a type Ⅱ zinc-containing transmembrane metalloproteinase present at the surface of many cell types. It’s main physiological function is cleaving on the N-terminal side of hydrophobic residues. NEP is involved in the physiological degradation of the peptides modulating blood pressure, such as natriuretic peptide, bradykinin and endothelin. It suggests that selective inhibitors of NEP can increase the activity of these vasoactive peptides, therefore, NEP becomes an important target to cure cardiovascular disease.This paper aims to establish the high throughput drug screening model of NEP and apply the model to screen inhibitors in order to develop new drugs.Target protein was obtained using Pichia expression system.The gene of NEP was amplified with PCR and cloned into the expression vector pPICZα-A, and using the X-33 Pichia strain as the host for expression of recombinant proteins.Then we purified the recombinant protein using the Ni2+-NTA column to obtain high purity NEP protein. Then, activity of target protein was examined by fluorescence resonance energy transfer(FRET)assays. Finally, a model for drug screening was established and optimized before screening abundant inhibitors.784 compounds and 2000 natural products segment component were screened with the established drug screening system. We obtained 2 compounds and determined the IC50 value. The IC50 value of MDCR02 was 101.3μmol/L.The IC50 value of MDCR04 was 0.494μmol/L. Furthermore, we found 8 natural products whose inhibition rate higher than 90%, and obtained 2 natural products is MDCNCL01000241, MDCNCL01000242 by comparing inhibitory effect of serial dilution. Notably, the IC50 of 2 natural products were less than 10μmol/mL. The discovery of primer to develop new drug.