Cytochrome B5 Reductase 3 Can Be Approached As A Contemporary Therapeutic Target to Restrain Hypertension

Abstract

Hypertension or high blood pressure is attributed as a critical health problem. Currently available therapeutics are indeed effective in combating hypertension, but are not always bereft of complications. Hence, to pursue alternate anti‐hypertensive therapeutics, the present study investigated human Cytochrome B5 Reducatse3 (hCYB5R3) enzyme as a “contemporary” therapeutic target, since this enzyme regulates the bioavailability of nitric oxide, a vasodilator involved in governing vascular tone; and hence hypertension. In the present investigation, small molecule databases were virtually screened against the crystal structure of the enzyme to identify potential inhibitors as well as to obtain binding energies and inhibitory constants. The enzyme was purified to homogeneity from recombinant sources and the inhibitors so obtained were tested in vitro using potassium ferricyanide assay in order to calculate IC50 values as a measure of inhibitor efficacy of these compounds against hCYB5R3. Further, the effect of these inhibitors was tested using various biophysical techniques including fluorescence and CD spectroscopy so as to obtain binding affinity, number of binding sites and effect on secondary structure, respectively. Fluorescence analysis has revealed promising binding constants (in micro molar ranges) and stoichiometric ratios (≈1:1) for some of the inhibitors. CD analysis revealed that these inhibitors do not alter the secondary structure of the FAD containing enzyme. Further, selected inhibitors were able to curb hypertension in vivo in spontaneously hypertensive rats. These encouraging findings have poised us to investigate the inhibitors further since overall, the identified and screened leads showed promising potential to be developed as novel anti‐hypertensive drugs against hCYB5R3.Support or Funding InformationThe funding support to conduct this study was provided by Department of Biotechnology (DBT) and Council of Scientific and Industrial Research (CSIR), Government of India. For experiments in rats, ethical clearance was obtained from Rajiv Gandhi Centre for Biotechnology (RGCB), India (IAEC/2015/23/CCKartha/RGCB/20.05.2015 (Protocol No. IAEC/570/CCK/2016)