Molecular Docking Study and ADMET Pro le: Manipulation of Angiotensin II Pathophysiology in COVID-19 by Potentilla Reptans Root Compounds


 In the novel SARS-CoV-2 (COVID-19) as a global emergency event, the main reason of the cardiac injury from COVID-19 is angiotensin-converting enzyme 2 (ACE2) targeting in SARS-CoV-2 infection. The inhibition of ACE2 induces an increase in the angiotensin II (Ang II) and the angiotensin II receptor type 1 (AT1R) leading to impaired cardiac function or cardiac inflammatory responses. The ethyl acetate fraction of Potentilla reptans L. root can rescue heart dysfunction, oxidative stress, cardiac arrhythmias and apoptosis. Therefore, isolated components of P. reptans evaluated to identify natural anti-SARS-CoV-2 agents via molecular docking. In silico molecular docking study were carried out using the Auto Dock software on the isolated compounds of Potentilla reptans root. The protein targets of selective ACE and others obtained from Protein Data Bank (PDB). The best binding pose between amino acid residues involved in active site of the targets and compounds was discovered via molecular docking. Furthermore, ADMET properties of the compounds were evaluated. The triterpenoids of P. reptans showed more ACE inhibitory potential than catechin in both domains. They were selective on the nACE domain, especially compound 5. Also, the compound 5 & 6 had the highest binding affinity toward active site of nACE, cACE, AT1R, ACE2, and TNF-α receptors. Meanwhile, compound 3 showed more activity to inhibit TXA2. Drug likeness and ADMET analysis showed that the compounds passed the criteria of drug likeness and Lipinski rules. The current study depicted that P. reptans root showed cardioprotective effect in COVID-19 infection and manipulation of angiotensin II-induced side effects.


Introduction
A novel coronavirus (SARS-CoV-2, COVID-19) has caused a great threat to the public healthcare systems and international concern in the world [1][2][3][4]. This virus affected seriously endangers human health by causing susceptibility, disease severity, various mutations and high mortality [5,6]. As of now, there is not exact conventional medications for the SARS-CoV-2 treatment [5]. It seems that there are several mechanisms involved in SARS-CoV-2 infection in different organs including lung, heart, kidney, and brain [2,7]. The main target of SARS-CoV-2 propagates is the angiotensin-converting enzyme 2 (ACE2) receptor and renin-angiotensin system (RAS) of host as a vehicle to entry human cells and viral replication [8]. However, SARS-CoV-2 exhibits cardiac dysfunctions (owing to ACE2 abundant in cardiac tissue) including acute myocardial injury, hypokalemia, arrhythmia, myocarditis, and sudden cardiac death [8][9][10][11][12]. At that point, there is a speci c attention to manage ACE2 expression or COVID-19 cardiac adverse and its associated targets such as cardiac in ammatory, Ang II induction, cardiac arrhythmia signaling and oxidative stress. Viral binding to ACE2 leads to ACE2 shedding and an accumulation of Ang II and the angiotensin II receptor type 1 (AT1R), thereby causing an incidence of vasoconstriction, brosis, arrhythmogenesis, hypertrophy, proliferation, oxidative stress, cardiac dysfunction and myocardium sensitization [8,13,14]. On the other hand, there is the relationship between elevated ACE2 and some cardiac comorbidities such as heart failure, secondary hypertension, coronary artery disease, and cardiomyopathies, which may increase COVID-19 susceptibility and severity [8]. Likewise, angiotensin-I converting enzyme (ACE) inhibitors such as captopril, enalapril, and lisinopril are used to management of COVID-19 [15]. Owing to the non-selectivity of applied ACE inhibitors, they cause some side effects via associated dysregulatory of bradykinin in the patients [16].
To the best our knowledge, P. reptans, as a natural cardioprotective agent may be a promising complementary candidate of COVID-19 for warrant therapeutic intention of ACE2 targeting-COVID-19-induced cardiac adverse. Therefore, we aimed to evaluate isolated components of P. reptans to identify natural anti-SARS-CoV-2 agents via molecular docking pointing the selective ACE inhibition and some properties including physicochemical, absorption, distribution, metabolism, excretion, and toxicity (ADMET).
The docking was performed on the target proteins, which were removed water molecules/non-polar hydrogen atoms and added polar hydrogens/kollman charges. Lamarckian genetic algorithm (GA) was used for local search method with a grid box of 60×60×60 and point spacing of 0.375 Å that was set for creating of autogrid module [25,26]. 150 GA runs were accomplished for each docking. Maestro 11.0 Schrodinger suit and Discovery Studio Visualizer software was applied for visualization of 2D and 3D presentation.

Ligand Preparation
The 3D structure of each phytochemical ( Figure 1) was retrieved from PubChem database in SDF format and then converted into PDB format using Open Babel software. Chem3D software was utilized for energy minimization of ligands. Captopril, an ACE inhibitor not selective inhibitor, was used as a control.

Drug Likeness And Admet Prediction
E cacy and safety pro le of the mentioned natural compounds including absorption, distribution, metabolism, excretion and toxicity (ADMET) and their pharmacokinetics were predicted using admetSAR database [27] and swissADME [28]. Furthermore, we investigated topological polar surface area (TPSA) as an important descriptor predicting oral bioavailability and absorption of the compounds. Also, the compound effects on permeability of blood-brain barrier (BBB), inhibition of cytochrome P450 (CYP3A4, CYP2C9 and CYP2D6), AMES toxicity and carcinogenicity were evaluated.

Molecular docking
According to Table 1, the triterpenoids of P. reptans showed more ACE inhibitory potential than catechin in both domains. Also, they were selective on the nACE domain based on their lower binding energies on 6F9V and 6EN5 targets, especially compound 5 had the lowest binding energy. Between assessed-PDB IDs of cACE domain, the triterpenoids revealed their lower binding energy in the binding pocket of 6F9U, mainly compound 6 (-10.2 kcal/mol), and also, it acted as more selective for cACE with lower binding energy for 6F9T and 2OC2 (as cACE domain complexes).
In addition, the results indicated that P. reptans root compounds are ACE inhibitor regards to the binding energy of ATR-inhibitor complexes 4YAY and 4ZUD, but compounds 5 and 6 showed suitable energy for ATR inhibition and acted as angiotensin receptor blocker (ARB). In ACE2 inhibitory investigation, triterpenoids were effective in the binding pocket of 1R4L protein especially compounds 5 and 2 ( Table 1). In general, compounds 5 and 6 were more active in the inhibition of ACE, ACE2 and AT1R with their hydroxyl groups and electron donor -OMe substitution, thereby a preparation condition of interaction with the target Zn 2+ ion.
In the AT1R inhibitory analysis, compound 5 and 6 showed the lowest bonding energy in 4ZUD and 4YAY, respectively ( Fig. 3A-B, Supplementary material Figure S5, S6). In addition, the result of this study indicated the ACE2 inhibitory energy for compound 5 is -9.9 kcal/mol in the interaction with key amino acid residues of 1R4L and binding to Zn 2+ ion at a distance of 2.9 Å (Fig. 3C, Table 1, Supplementary material Figure S7).

Drug Likeness And Admet Prediction
Pharmacokinetic and toxicity properties of compounds were determined. The results of predicted ADMET properties of compounds and toxicity pro les presented in Table 2. To get an insight about the compliance of compounds with Lipinski's Rule of Five, the compounds screened for more analysis. Notably, most compounds passed Lipinski rule and did not show any violation of standardized Lipinski rule of ve. Values of calculated solubility of compounds ranging from -3.08 to -3.89 indicating moderate solubility of compounds, and also calculated Log P indicated between 1.29 and 3.26. Moreover, molecular weight was more than 500 Dalton, number of hydrogen bond donors were more than 5, while number of H-acceptors of compound 5 and 6 exhibited violation of Lipinski rule and others obey Lipinski rule.
Molar refractivity of all compounds were more than 130. According to data presented in Table 2 and 3 pharmacokinetic parameters and ADMET pro les (absorption, distribution, metabolism, excretion, and toxicity) evaluated to predict the toxicity parameters of the compounds. However, all the compounds are following the criteria of drug likeness rules. All the compounds indicated good absorption and permeability demonstrating moderate absorption. Evaluation of two key ADMET descriptor like Human Intestinal Absorption (HIA) and blood brain barrier (BBB) exhibited appropriate pro les. Furthermore, the obtained results revealed that all of the compounds were non-inhibitors of CYP450 enzymes (3A4, 2D6, and 2C9) acting as an effective factor in drug metabolism. Additionally, most compounds were non-carcinogenic, non-hepatotoxicity and non-AMES toxicity.

Discussion
The previous studies established cardioprotective effects of P. reptans compounds in docking and animal experimental studies due to glycogen synthase kinase 3β (GSK-3β) and glucocorticoid regulated kinase-1 (SGK1) protein kinase inhibition [20,23]. Likewise, they demonstrated an ischemic preconditioning property via antioxidant activity, nitric oxide release, activating Nrf2 pathway, and decreasing apoptotic markers in an isolated rat heart ischemia/reperfusion model [22]. Consistent with these data, P. reptans compounds may be applied for cardioprotection in COVID-19 by bene cial cardioprotective effects.
In this study, the isolated triterpenoids of P. reptans root showed selective ACE inhibitory effect as well as inhibition of ACE2 and AT1R, especially compound 5 and 6, compared with catechin (Table 1). However, it may be explained by hydroxyl groups and electron donor substances in chemical structure of triterpenoids. In addition, the compound 5 and 6 had the highest binding a nity toward active site of nACE, cACE, AT1R and ACE2 receptors, through -OMe element or hydroxyl groups. Also, their non-covalent (hydrogen bond and hydrophobic) interactions have a signi cant role in occupation of active site of the targets. Therefore, compound 5 and 6 can be considered as a potential selective ACE inhibitor by binding to the key amino acid residues in the active site of the targets.
To the best of our knowledge, the selective inhibition of ACE plays a pivotal role in controlling the RAS and kallikrein-kinin system (KKS), thereby normal hydrolyzing of the anti-in ammatory peptide N-acetyl-SDKP and bradykinin by nACE and cACE, respectively. Therefore, selective cACE inhibitors diminish the production of angiotensin-II (AngII), ACE2 and plausible angioedema. On the other hand, selective nACE inhibitors attenuate in ammation and brosis of heart, renal and vascular through increasing N-acetyl-SDKP levels [16,29]. However, selective ACE inhibitors prevent the activity of cACE or nACE domains.
It seems that AT1R and ACE2 inhibitory effects of P. reptans triterpenoids (as shown Table 1) can explain their suppressing effects in in ammatory and cardiac brosis induced by COVID-19 infection and dysfunction in RAS. It has been reported that AngII induced angiotensin II type-2 receptor (AT2R) and NO production when AT1R blocked and leads to cardioprotection and anti-brosis via inhibiting of norepinephrine, MAPK and ERK1/2 along with inducing vasodilation by bradykinin/NO/cGMP cascades [30,31]. In addition, the previous studies indicated that isolated ingredients of P. reptans inhibited cardiac apoptosis via NO release, inhibiting GSK-3β and activating RISK/SAFE pathways in reperfusion injury [23]. On the other hand, AT1R inhibition may upregulate ACE2 levels as important target of SARS-CoV-2 [15], thereby the P. reptans compounds can exert a protective role against an increase in ACE2 by their a nity to occupation of ACE2 complex.
In the next step, the current study demonstrated that compounds of P. reptans inhibited TXA2 and TNF-α (Table 1) as adverse effects of infection with COVID-19, which can be in line with mentioned properties of compounds and explain by their antioxidant and antiin ammatory or anti-apoptotic effects [22,23]. Furthermore, the recent study reported that AngII acts similar to in ammatory cytokine and induces hypertrophy, brosis, arterial brillation and arrhythmia in the heart [31]. Likewise, it can activate numerous kinases such as SGK1 that plays a pivotal dual role in the pathogenesis of cardiac arrhythmia, in ammation and remodeling in response to progressive elevated AngII [31,32]. Agreeing with this evidence, ethyl acetate fraction of P. reptans exerted inhibitory effects on SGK1 to reduce arrhythmia and cardiac apoptosis in ischemia/reperfusion injury [20,23]. However, AngII and AT1R could cause arterial/ventricular brillation, thus, by blocking ACE or AT1R it would be plausible that inhibition of SGK1 to reduce the prolongation of the action potential duration (APD), hypokalemia, and the proarrhythmic effects of AngII [33]. Therefore, Potentilla reptans compounds may be promising candidate as anti-arrhythmic agent for management of ACE2 targeting-COVID-19-induced arrhythmia due to their ACE or AT1R inhibitory properties.
Taken together, isolated substance of ethyl acetate fraction of P. reptans root showed cardioprotective effect in COVID-19 infection and manipulation of angiotensin II-induced side effects by molecular docking which can be postulated as new selective ACE inhibitors.
Although ethyl acetate fraction of P. reptans root showed bene cial pharmacological properties in the management of COVID-19 and its adverse effects, larger preclinical and clinical studies are needed to determine whether the compounds reveal safety and selective ACE that indicated by molecular docking.

Conclusions
In conclusion, the molecular docking results of present study gave insights into the potential e cacy of triterpenoid compounds from P. reptans root against COVID-19 through selective ACE inhibitory effect, AT1R and ACE2 inhibition. Ursane type triterpenoids and catechins of P. reptans, especially compound 5 and 6, seems to contribute to anti-COVID-19 and cardioprotective effects of this plant in the in silico. Our ndings indicate that P. reptans compounds follow Lipinski rule of ve and had good pharmacokinetic properties and ADMET pro les. However, further studies in animal and clinical areas in an enhanced setting, are needed to indicate other promising targets and mechanisms of anti-COVID-19 from Potentilla reptans root ingredients. Finally, it can be a hopeful natural-based approach for demoliting the pathogenesis of COVID-19 and boosted AngII.
Declarations Figure 1 Structures of isolated compounds from P. reptans root.