New Inhibitors of Angiotensin Converting Enzyme from Plant and Synthetic Origin by Mass Spectrometric Screening Assay

Abstract

Enzymes play a very crucial role in the regulation of almost all process of life. Enzymes are also an important class of drug target; screening of inhibitors for enzymes is the first step in drug discovery process. Angiotensin converting enzyme (ACE) is a pharmacological important enzyme, it converts the Al into All. It plays a key role in Renin angiotensin aldosterone system (RAAS), which regulate the blood pressure and hypertension. Over expression of RAAS can cause many cardiovascular and renal diseases, therefore, inhibition of ACE is a promising way of controlling over expression of RAAS. Many methods have been used for the inhibition study of ACE including spectrophotometric, fluorimetric, HPLC, mass spectrometric, etc. When LC-MS combines with the MS/MS then it becomes very unambiguous and sensitive analysis techniques for detection and identification of peptides in complex samples. In this study, a new sensitive and robust HPLC-ESI-MS/MS method, with very low limit of quantitation was developed for ACE screening assay. In this method, substrate was used instead of product to determine ACE activity. For this purpose, a calibration curve of Angiotensin I was developed in a range of 20-200 nM, linear equation y=0.0888x-0.1932 and R2=0.999. Two commercially available antihypertensive drugs, Captopril and Lisinopril, were checked to validate the method and their IC50 values were found to be 3.969 μM and 0.852 μM, respectively. This newly developed method required very low amount of substrate, enzyme and inhibitor per sample. MALDI was also explored for the potential of enzyme inhibition study, MALDI-TOF-MS is a well-known technique for the analysis of protein and peptides. Relative quantification was performed by using the ratio of substrate and product. In this study, a high-throughput method of ACE inhibition was developed on MADLI-TOF-MS. In this method, a linear curve of AI was developed for 0-100% conversion, linear equation y=9.3175x+3.4177 and R2=0.994. Both standard inhibitors, Captopril and Lisinopril, were checked for their inhibition and their IC50 values were found to be 8.256 μM and 1.962 μM, respectively, which validated the method. This method provides a fast and alternative for the screening of drugs to find their potential against the target enzyme. As a final step, 77 commercial drugs, 40 synthetic compounds, 4 natural products and 3 extracts of plants were investigated for their inhibitory potential. In drugs, 36 showed moderate to good inhibition of as low as IC50=272 µM. In synthetic compounds, 32 compounds showed significant inhibition ranged from IC50 = 320-5123 µM. In the last category, all 4 compounds were moderately active with IC50 value 496-9705 µM; while only one plant extracts showed some inhibition with IC50 =10779 µg/mL (ppm). Current study has proved that direct nature of the MS measurement ensures the minimization of the false results and large number of compounds can be screened without the need for labels or additional internal standards, both MALDl-TOF-MS and LC-ESIMS/MS are valid readout for enzyme inhibitor screening assays.