Repurposing of pharmaceutical drugs by high-throughput approach for antihypertensive activity as inhibitors of angiotensin-converting enzyme (ACE) using HPLC-ESI-MS/MS method

2.1 Reagents, chemicals and samples

Angiotensin converting enzyme, its substrate angiotensin I and product angiotensin II were purchased from Sigma Aldrich (USA). Two standard inhibitors Captopril and Lisinopril were purchased from Tokyo Chemical Industries Co, Ltd. (Japan), both were >98% pure. Tris buffer (research grade) was purchased from Serva (Germany). The concentration of this buffer was prepared 20 mM with 3 mM dithiothreitol (DTT) for the pH 7.5 at 37 °C. Acetonitrile and DMSO were purchased from Fisher Scientific (Leicestershire, UK). All other solvents used, were of spectroscopic grade, Milli-Q water was used fresh from Milli-Q water assembly (Bedford, USA) which had 16.5 MΩ resistance. For inhibition study of standard drugs, all drugs were collected from the compound bank of International Center for Chemical and Biological Sciences, University of Karachi, Karachi.

2.2 Angiotensin-converting enzyme assay

Enzymatic assays for inhibition study were performed in well plate in different batches, each batch contained one well as blank or control and other contain reaction mixtures with different drugs or compounds. Control or blank well contain only enzyme and substrate, but reaction mixture contains enzyme, substrate and inhibitors of different concentrations. All the wells contained same volume of each enzyme, substrate and inhibitor.

Each reaction well contained 1 µL of angiotensin converting enzyme (0.5 μM), 10 µL of angiotensin I (4 μM) and 2.5 µL of compound or drug of different concentration in different well. In control reaction 2.5 μL of water or 20% DMSO to compensate the volume of inhibitor. Both ACE and substrate were prepared in TRIS buffer of 20 mM with 3 mM DDT of pH 7.5 and standard drug were prepared in milli-Q water or 20% DMSO according to their solubility. All reactions were performed in incubator at 37 °C for a standard time of 30 min.

2.3 Preparation of drugs

Most of the compounds and drugs were soluble in water therefore their solutions were prepared in milli-Q water. Some drugs were only soluble in DMSO, their solubility was checked in different percentages of DMSO in water and 20% DMSO in water (1 part DMSO: 4 parts water) was found suitable. Those drug’s solutions and their dilutions were prepared in the same 20% DMSO solvent. Standard inhibitors, Captopril and Lisinopril were prepared in milli-Q water as well as in 20% DMSO, separately. All concentrations were prepared in micro molar (µM) or milli molar (mM) and diluted with the same solvent in which bulk was prepared.

2.4 Sample preparation for LC-ESI-QqQ-MS analysis

Aliquot of 2.5 μL was taken after standard incubation time i.e. 30 min and mixed with 2.5 μL of 0.5 μM of bradykinin (Internal standard) and 7.5 μL of 0.1% formic acid solution. Formic acid solution (0.1%) was used to quench the reaction and also to make the 1/5th dilution of the reaction mixture. From this mixture duplicate runs were carried out, in each run, 5 μL of the sample was injected into the ESI-QqQ-MS system of Agilent equipped with HPLC system and ESI Jet Stream Source. Agilent Mass Hunter Data Acquisition 5.0 and Data Analysis 6.01 were used for data acquisition and interpretation, respectively.

2.5 LC-ESI-QqQ-MS analysis

All samples were analyzed using Agilent 1260 liquid chromatograph (Agilent Technologies, Wilmington, DE), coupled with Agilent 6400 triple quadruple mass spectrometer (Agilent Technologies, Wilmington, DE, USA). Peptides separation was achieved by using reverse phase HPLC with Jupiter C-18 column of Phenomenex (Torrance, California) having dimensions of 50 mm length, 1 mm i.d. and 5 µm particle size. The mobile phase was comprised of water with 1% formic acid as solvent A, and acetonitrile with 1% formic acid as solvent B. Gradient of 5–70% of B was run for a total time of 7 min. The gradient was started with 5% B, which was changed progressively to 70% B in 3 min, and then rapidly decreased again to 5% B in half minute and run for the next 3.5 min at same composition. The flow rate was set at 0.4 mL/min.

Final optimized conditions of ESI JetStream source were used as, gas temperature 300 °C, gas flow 12 L/min, nebulizers pressure 30 psi, sheath gas heater 240 °C, sheath gas flow 10 L/min, capillary voltage 3000 V and nozzle voltage (V Charging) 1000 V. Multiple reaction monitoring (MRM) mode was used to collect mass spectral data of precursor and product ion transitions. The optimized values of MRM transition, collision energy (CE) and the fragmentor voltages (FV) of peptides were as follows: Bradykinin, 530.8 → 175.2, FV = 100 V, CE = 30 eV and Angiotensin, 433 → 110.1, FV = 100 V, CE = 20 eV.

3.1 Enzymatic reaction in different solvents

Angiotensin Converting Enzyme reactions were performed with optimized condition of reported method (Musharraf et al., 2017). In this study as the 20% DMSO was used to prepare the solution of few drugs, therefore the effect of DMSO on enzyme reaction had to be checked. For this purpose, ACE reaction was performed while adding DMSO instead of water as a compensation of inhibitor volume. In normal reactions, 2.5 μL of water was added in 1 µL of 0.5 μM Enzyme (ACE) and 10 µL of 4 μM substrate (angiotensin I). while in DMSO reaction, 2.5 μL of 20% DMSO was added in the reaction mixture of enzyme and substrate. Aliquots of both reactions were taken at same time intervals i.e. 5, 10, 20, 30, 40 and 50 min and reaction progress was plotted as percentage conversion (%C) vs. time as shown in Fig. 1. In enzyme reaction of DMSO, overall intensity of both substrate and product peaks was decreased as compared to enzyme reaction of water but the ratios remain the same, and no significant difference in percentage conversion was observed at specific time in both reactions. Therefore, reaction of drugs prepared in water can be compared with the reactions of drugs prepared in 20% DMSO.

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Fig. 1

Comparison of ACE reaction in different solvents (Water and 20% DMSO).

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3.2 HPLC-ESI-MS/MS method and validation

This HPLC-ESI-MS/MS method used in this study was developed to maximize the response with minimum substrate, enzyme and other enzymatic reagents and validated for its linearity range and enzymatic reaction by interday and intraday analysis.

The calibration curve was drawn by using 5 different calibrators of 20, 60, 100, 140 and 200 nM of Angiotensin I. The calibration showed a linear response over the range with R² = 0.999 and LOD and LOQ as 1.44 nM (1.866 ng/mL) and 4.37 nM (5.664 ng/mL), respectively. Interday and intraday reproducibility of the linear curve, for two qualifiers showed that it has RSD% in the range of 1.76 – 4.37, which is very low as comparation to the other methods. Interday and intra analysis of the enzymatic method and inhibition reaction also showed very low RSD% in range of 4.14 – 9.98. In this study further validation of the assay was performed by inhibition reaction of Captopril and Lisinopril.

3.3 Determination of IC₅₀ of inhibitors

Once the enzyme inhibition assays were performed, all drugs and two standard inhibitors were investigated for their inhibitory potential against ACE by using the reported methodology (Musharraf et al., 2017). For concentration dependent inhibition studies, the inhibition was calculated as % inhibitory activity (% IA). It is also defined as the measurement of the degree of inhibition and it is used to determine the IC₅₀ value. For IC₅₀ calculation inhibition data was plotted as % IA vs. log of concentration of inhibitor, concentration of inhibitor correspond to 50% inhibition activity was the IC₅₀ value of that inhibitor. For few drugs which have very low activity, extrapolation of curve was used and their values are reported as estimated values. The inhibitory activity is the % conversion measured in control reactions (without inhibitor) divided by the % conversion measured in a reaction with inhibitor, as represented in Equation (1) (Chen et al., 2013; Lahogue et al., 2010; Greis et al., 2006):

Two standard inhibitors of ACE, Lisinopril and Captopril, were checked for the authentication of the assay. For Captopril concentrations of 1, 5, 10 and 50 μM and for Lisinopril concentrations of 0.5, 1, 5 and 10 μM were used. The IC₅₀ values of Captopril and Lisinopril were found to be 4.04 μM and 0.93 μM, respectively, as shown in Fig. 2. These values were closely matched with the previously reported values and used as reference for the screening of other inhibitors (pharmaceutical drugs).

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Fig. 2

Comparation of inhibition of captopril and lisinopril and their IC₅₀ values.

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Total 77 pharmaceutical drugs were analyzed for their inhibitory potential and among these drugs, 41 drugs showed inhibitory activity against ACE. IC₅₀ values of 13 drugs (DB-001, DB-002, DB-003, DB-009, DB-012, DB-017, DB-020, DB-046, DB-053, DB-054, DB-123, DB-143 and DB-214) were less than 1 mM, and showed good to moderate inhibition, with as low as IC₅₀ = 272 µM (DB-9). 23 Drugs showed low inhibition, their IC₅₀ values were in the range of 1.49 to 15.98 mM. While DB-93, DB-94, DB-110, DB-113 and DB-145 showed very low inhibition, their IC₅₀ value was ranges from 20 to 73 mM, in comparison to the standard inhibitor these values are very high. Other 33 drugs were inactive, even a high concentration of those drugs did not show any inhibition at all. Remining three drugs Lisinopril Dihydrate (DB-22), Enalapril Maleate (DB-56) and Ramipril (DB-79) were the standard inhibitors of ACE and they had IC₅₀ value 2.663 µM, 10.026 µM and 8.125 µM, respectively. These three inhibitors were analyzed blind folded along with other pharmaceutical drugs. Close values of these inhibitors represent the authenticity of the assay and screening method. Lisinopril (DB-22) showed insignificant high values compare to standard pure Lisinopril, because of its low purity. IC₅₀ values of all the active drugs are given in the Table 1.

3.4 Repurposing of potential drugs

Among all the analyzed drugs, the most active drugs against ACE are marked with asterisk (*) in the Table 1. These drugs may serve as ACE inhibitor or their derivatives may be formed to enhance the activity. Among the most active drugs four of them, Amoxicillin trihydrate (DB-002), Ampicillin trihydrate (DB-003), Ciprofloxacin HCl monohydrate (DB-009) and Doxycycline Hyclate (DB-054), showed the IC₅₀ values of 364 µM, 319 µM, 272 µM and 329 µM, respectively, were belong to the Antibiotic class of drugs. In this study total 13 drugs of Antibiotic class were screened, among them seven showed activity along with above mentioned four drugs. It seems like that Antibiotics can be used as ACE inhibitors; therefore, more Antibiotics may be screened, and potential hits may be found among them.

Amlodipine besylate (DB-001) and Diltiazem hydrochloride (DB-012) are also using for cardiovascular diseases (CVD) but their mode of action is different, they act as calcium channel blockers (CCBs). Both drugs were also found active as ACE inhibitors with IC₅₀ values of 288 µM and 490 µM, respectively, it may enhance the area of treatment for these drugs. Two more CVD drugs were also screened from different mode of actions and both were found inactive. Among these CVD drugs only CCBs were found active therefore more CCBs drugs may also be screened to find more potent hits against ACE.

Itopride hydrochloride (DB-017) which is an Acetylcholinesterase (AChE) inhibitor, was also found as potential inhibitor of ACE with IC₅₀ = 296 µM. Another Phosphodiesterase inhibitor was also found active, more inhibitors may also be screened to find their dual inhibition properties. Diphenhydramine Hydrochloride (DB-053) an antihistamine and anti-allergy drug, Chloroquine Phosphate (DB-046) an anti-malarial drug and Sertraline Hydrochloride (DB-123) an anxiety relief and antidepressant drugs were also among the top active drugs in screening results. These drugs had IC₅₀ values of 338 µM, 342 µM and 581 µM, respectively. These drugs may serve their purpose as ACE inhibitor along with their primary approved treatment, after proper development studies for repurposing on them.