Kyunghye Ahn

A permanent human cell line (EA.hy926) preserves the characteristics of endothelin converting enzyme from primary human umbilical vein endothelial cells

Purification of endothelin converting enzyme (ECE) from endothelial cells has been hindered by the difficulty in obtaining primary endothelial cells in large quantity. We therefore tested transformed human umbilical vein endothelial cells (EA.hy926) for ECE activity. Our data clearly demonstrate that this transformed cell line preserves the ECE properties of the primary cell line. These include: (i) one sharp activity optimum at neutral pH; (ii) characteristics typical of a metalloprotease; (iii) IC50 value for phosphoramidon of 1.8 microM (2.7 microM for HUVEC); (iv) no inhibition by captopril and thiorphan, inhibitors of angiotensin converting enzyme and neutral endopeptidase 24.11. The enzyme showed a substrate specificity for big ET-1:big ET-2:big ET-3 in a ratio of 40:2.5:1. This report presents evidence that a permanent human endothelial cell line, EA.hy926, preserves the ECE activity of HUVEC and is useful for the study of ECE and its regulation of ET-1 production.

Synthesis of novel substituted pyridines as inhibitors of endothelin coverting enzyme-1 (ECE-1)

A series of bi-aryl pyridine carboxylic acids has been prepared and evaluated as inhibitors of ECE-1. The analogs were prepared by Pd catalyzed cross couplings of halogenated pyridines with heteroaryl organo-boranes, -tinate or -zincate derivatives.

Chapter 7. Endothelin Inhibitors

Publisher Summary Discovery and development of endothelin inhibitors have been one of the fastest growing research areas in the pharmaceutical industry. With several newly discovered endothelin (ET) inhibitors, investigators soon confirmed the possible involvement of endothelin-1 (ET-1) in number of disease states, including systemic hypertension (HP), pulmonary hypertension (PH), renal and myocardial ischemia (RI and MI), stroke, subarachnoid hemorrhage (SAH), restenosis (RT), congestive heart failure (CHF), and chronic renal failure (CRF). Currently, several ET receptor antagonists have been brought to the early development stages with a few already having advanced into clinical evaluation. Although less advanced, persistent effort in ECE inhibitor area has led to the discovery of several novel inhibitors. This chapter reviews recent progress in the discovery and development of new endothelin converting enzyme (ECE) inhibitors and ET receptor antagonists with a brief introduction of the endothelin system. The production of ET-1 with ECE inhibitors have been the subject of much research. The physiologically relevant ECE is a membrane-bound metalloprotease that is inhibited by phosphoramidon (PA), a nonselective peptidic metalloprotease inhibitor, but is insensitive to the inhibitors of other metalloproteases, such as thiorphan and captopril (angiotensin converting enzyme). Phosphoramidon inhibits both ECE-1 and ECE-2 albeit, with a 250-fold difference in potency. It also potently inhibits other metalloproteases, such as neutral endopeptidase (NEP), with IC 50 values in the low nanomolar range. Another example is thiorphan that is a potent NEP inhibitor but is inactive against ECE-1 and ECE-2. Selective mass screening of zinc protease inhibitors revealed an analog of retrothiorphan to be moderately active to ECE-1. Such ECE inhibitors and ET antagonists of diverse structural types have been identified and optimized with different profiles in terms of potency, selectivity, efficacy, and pharmacokinetics.

Characterization of endothelin converting enzyme from intact cells of a permanent human endothelial cell line EA.hy926

Endothelin converting enzyme (ECE) from intact cells of a permanent human endothelial cell line, EA.hy926, was studied by examining the effects of phosphoramidon, an endothelin converting enzyme inhibitor, on the levels of secreted endothelin‐1 and big endothelin‐1. The specific ECE activity was demonstrated by a phosphoramidon dose‐dependent decrease in ET‐1 level with a concomitant increase in big ET‐1 level. By using a specific neutral endopeptidase 24.11 (NEP 24.11) inhibitor, thiorphan, it was also shown that the phosphoramidon‐sensitive ET‐1 degrading activity in this cell line is due to the NEP 24.11 activity. Other serine, acid, and cysteine protease inhibitors had no effect on the endogenous synthesis of ET‐1 and big ET‐1 supporting the evidence that ECE is insensitive to these protease inhibitors as has been demonstrated with the isolated enzyme.