Dopamine‐β‐monooxygenase inhibitors obtained by structure based methods exhibited anti‐hypertensive effect in L‐NAME induced hypertensive rats

Abstract

IntroductionHypertension is the leading cause of mortality worldwide (Kwan G.F. et. al., Circulation, 2016). While classical anti‐hypertensives like ACE inhibitors, ARBs, calcium channel blockers, beta blockers and diuretics are widely used, they have their own limitations and no new type of drugs emerged in the last few years that use body's own blood pressure lowering mechanism. Dopamine β‐monooxygenase (DBM), expressed in noradrenergic nerve terminals as well as in adrenal medullary chromaffin cells, which convert dopamine into norepinephrine, is a novel target whose inhibition has been shown to help the treatment of hypertension with several advantages over the classical antihypertensives. However, DBM inhibitors are few in number, often result in side‐effects and are frequently non‐responsive to specific population, probably since the known inhibitors so far have been designed on ligand based methods. We hypothesize that rational and three dimensional structure based identification of novel inhibitors may overcome such problems.ObjectiveTo identify novel inhibitors of DBM and evaluate their anti‐hypertensive properties in L‐NAME induced hypertensive rat model.Design and methodAn experimentally validated computational model for human DBM, built in our lab, was used for structure‐based, rational drug design. The three‐dimensional model was used for virtual screening against various small molecule databases. Identified top hits were then tested in vitro against DBM with known inhibitors nepicastat and disulfiram as controls. Binding of the inhibitors to DBM were validated using fluorescence and CD spectroscopy as well as ITC. Pharmacokinetic analysis was performed computationally. Cyto‐ and hemo‐toxicities of the lead compounds were assessed ex vivo. Finally. their antihypertensive efficacies were evaluated in L‐NAME induced hypertensive rat model.ResultsVirtual screening of various compound libraries revealed 69 hits which were then assessed in vitro using a repertoire of biochemical and biophysical methods. UDSC‐171, 180 and 142 were discovered to be potent inhibitors of DBM with IC50s of 1μM, 5.5μM and 18μM, respectively. The inhibitors displayed KD values against DBM in the range of 100nm to 1μM. In silico pharmacokinetic analysis indicated that the molecules are unable to cross the BBB. High doses (up to 50μM) of the lead compounds showed acceptable cellular tolerance against HEK293 cell line and insignificant hemo‐toxicities against RBCs. These three leads were successful in preventing elevated systolic blood pressure in L‐NAME induced hypertensive rat models.ConclusionsPresent study successfully developed rapid, systematic and non‐expensive biophysical, biochemical and computational tools for the identification, characterization and validation of DBM inhibitors. Moreover, UDSC‐171, 180 and 142 have been discovered and validated as novel antihypertensives against L‐NAME induced hypertensive rats. (Indian Patent filed for the present study with app. no. 201711036983)Support or Funding InformationDepartment of Biotechnology, Govt. of India