Abstract 267: Identification And Validation Of New Inhibitors Based On Rational Design Against Dopamine-β-hydroxylase To Combat Hypertension

Abstract

Human dopamine β-hydroxylase (hDBH), expressed in noradrenergic nerve terminals of nervous system and in chromaffin cells of adrenal medulla, is a key constituent of catecholamine biosynthetic pathway. DBH inhibition has been shown to help the treatment of hypertension and cardiac heart failure, which are major causes of mortality and morbidity worldwide. Existing hDBH inhibitors are too few, often result in side effects and are frequently non-responsive to specific population. Since no three-dimensional structure existed for full-length hDBH, structure based rational drug design has been elusive till date, an issue to which we provided solution lately by building an experimentally validated in silico model for hDBH. The model was used in Autodock, Glide SP and XP software for structure based virtual screening against small molecule databases from NCI, USA. The docked structures were scored using Autodock, X-Score and Prime MMGBSA. Thus, 69 compounds were identified as prospective inhibitors of DBH, which were then tested in vitro against human serum DBH and its nearly identical homologue, bovine DBH (586 of 617 amino acids homologous), with known inhibitors nepicastat and disulfiram as positive controls. Three lead molecules (NSC637578, NSC99657 and NSC379555) were discovered in the process as potent inhibitors of DBH with IC50s of 1μM, 5.5μM and 18μM, respectively. The binding of the inhibitors to the enzyme were validated using fluorescence and CD spectroscopy as well as ITC, revealing KD values in the range of 100nm to 1μM. In silico pharmacokinetic analysis indicated the molecules to be latest generation of DBH inhibitors having very high cell permeability and inability to cross the blood brain barrier. High doses (up to 50μM) of the lead compounds showed acceptable cellular tolerance against HEK 293 cell line and insignificant hemo-toxicities against human RBCs. Hence, in vivo evaluation of the lead molecules has been initiated in model systems like C. elegans and D. melanogaster. These studies reconfirmed their nontoxic properties up to 15μM doses. CombiGlide and Ligand Based Core Hopping methods are being employed to optimize the lead compounds computationally to improve their pharmacokinetic properties.