To identify novel inhibitors of dopamine beta hydroxylase (DBH) and evaluate their antihypertensive properties in L-NAME induced hypertensive rat model.Design and method:
An experimentally validated computational model for hDBH, built in our lab, was used for structure-based, rational drug-design. The three-dimensional model was used for virtual-screening against small molecule databases from NCI, USA and elsewhere. Identified top hits were then tested in vitro against DBH with known inhibitors nepicastat and disulfiram as controls. Binding of the inhibitors to DBH 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 anti-hypertensive efficacies were evaluated in L-NAME induced hypertensive rat model.Results:
Virtual screening of NCI libraries revealed 69 hits which were then assessed in vitro using a repertoire of biochemical and biophysical methods. UDSC171, UDSC180 and UDSC142 were discovered to be potent inhibitors of DBH with IC50s of 1 μM, 5.5 μM and 18 μM, respectively. The inhibitors displayed KD values against DBH in the range of 100 nm to 1 μM. In silico pharmacokinetic analysis indicated that the molecules are unable to cross the BBB. High doses (up to 50 μM) 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.Conclusions:
Present study successfully developed rapid, systematic and non-expensive biophysical, biochemical and computational tools for the identification, characterization and validation of DBH inhibitors. Moreover, UDSC171, UDSC180 and UDSC142 have been discovered and validated as novel anti-hypertensives against L-NAME induced hypertensive rats.