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Discovery of synthetic bioactive novel hydroxyanisole pharmacophore series as a promising myeloperoxidase inhibitor (MPOI) targeting atherosclerotic CVD

Premkumar Jayaraj1, Sampath Parthasarathy2, Sanjay Rajagopalan 3, Chandrakala Aluganti 2, *, Rajagopal Desikan 1,2, *

1[anonimizat], [anonimizat] 632014, India

2[anonimizat], [anonimizat], FL 32832, USA

3[anonimizat], Cleveland, Ohio, USA.

(*) Corresponding authors: [anonimizat] & [anonimizat]

Abstract: Cardiovascular complications are established to have happened due to oxidative stress and inflammatory mechanism and myeloperoxidase (MPO) is known to involve in these biochemical pathways. MPO mediated oxidation of lipoproteins leads to dysfunctional entities altering the landscape of lipoproteins functionality. Specificity of anisole derivatives toward preventing MPO mediated oxidation to limit its harmful effects is scantly available in the literature. Diligent computation docking is done to accomplish a portfolio of compounds keeping hydroxyanisole as a building block. [anonimizat]. [anonimizat] (LDL and HDL) is shown to connect with our approach of developing powerful MPO inhibitors. The mechanism of MPO inhibition is demonstrated to be reversible mode. [anonimizat] a [anonimizat].

Keywords: Myeloperoxidase; Hypochlorous acid; Heme protein; Low-density lipoprotein (LDL); Chlorotyrosine; Reversible inhibitor

1. Introduction

Myeloperoxidase (MPO) is a [anonimizat] [1]. [anonimizat] (HOCl) and sometime minor amount of hypobromos (HOBr) is also produced [2]. Specific chemical reaction between hydrogen peroxide (H2O2) and halides (Bromide-Br, Chloride-Cl, Thiocyanate-SCN) to generate corresponding hypohalous acid. These are extremely powerful oxidants responsible for oxidation of biomolecules. [anonimizat] a chemically stable intermediate for a short duration of time. [anonimizat] 3-chlorotyrosine (3-ClTyr) and sometime forms dityrosine (Di-Tyr) [3,4,5]. [anonimizat]. [anonimizat]. [anonimizat], other reactive oxygen species (ROS) react aggressively with biomolecules and structurally modify them altogether. ROS perform a crucial role in the inflammatory network. [anonimizat], migration of white blood cells, and increases the extracellular matrix production [6]. Other roles of ROS including growth and differentiation, apoptotic signaling pathway, gene expression etc., [7]. Oxidative stress is the main reason for organ dysfunction in human subjects and it may lead to complications such as atherosclerosis, Parkinson’s, heart failure, stroke, etc.,

It also acts as a mediator of lipid accumulation and can react with unsaturated fatty acids (UFA) of lipid membranes and induce lipid peroxidation. Lipoprotein, LDL (Low-density lipoprotein) and HDL (High-density lipoprotein), a combination of lipids and proteins, moves through the blood vessel [8,9,10]. Non-atherogenic LDL can deposit artery wall until it stays in native form. Consuming excessive food, smoking, poorly controlled diabetes and stress are some of the risk factors to increase the level of oxidized-LDL (ox-LDL) [11,12]. This will lead to excessive deposition of ox-LDL within the artery wall. This triggers biochemical reactions that will have a cascading effect in the formation of atherosclerotic plaque [13] and subsequently initiate several cardiovascular complications. Oxidative enzymes in presence of H2O2 can promote unfavorable reactions within the premises of LDL leading to the formation of ox-LDL [14].

High-density lipoprotein (HDL), another member of lipid family, has a contrasting behavior compare to LDL. The main structural component of HDL is apolipoprotein A-1 (apoA-1) which clears bad cholesterols from macrophages foam cell via kidney. Mainly, HDL plays a significance part in RTC (Reverse cholesterol transport) mechanism and modulation of inflammation. HDL is established to protect biomolecules thereby preventing atherosclerosis manifestation. In recent literature reports suggest that HDL loses its cardioprotective properties when it is oxidatively damaged [15]. Literature reports also suggest the effect of MPO-mediated oxidation of HDL to transform it as ox-HDL in the human artery wall. The existence of 3-ClTyr and 3-NO2Tyr (3-nitro tyrosine) [14] within the atherosclerotic plaque, characteristic biomarkers of MPO-mediated products was established by high resolution mass spectrometry. Modified HDL may involve in pro-inflammatory process causing extensive damage to biological systems [16].

Atherosclerosis causes plaque formation in large or medium artery blood vessel causing vaso-constriction followed by restricted or decreased blood flow. The constituents of atherosclerotic plaque are ox-lipid, cell remains, SMC (smooth muscle cells), endogenous collagen and calcium deposit on the surface of blood vessels [17]. These depositions may lead to the formation of atherosclerotic lesion. MPO is known to participate in the atherosclerotic event. It includes MPO induced oxidation of lipids, tissue damages, oxidative stress markers, and endothelial dysfunctions, etc. [18,19].

Last few decades, health care industries have watched tremendous growth in the development of antioxidant therapy. Oxidative stress modulators, vitamins (Vitamin-C, E, etc.,) [20], carotenes (α-Carotene and β-Carotene etc.,) [21,22], and phytonutrients (cinnamyl, curcumin etc.,) are reported in the literature [23,24]. Many pharmaceutical industries devote their drug discovery effort toward developing chemical agents utilizing nature-based supplements without compromising pharmacological effect of the parental structure aiming at specific disease targets. Hydroxyanisoles are important class of organic compounds holding excellent bioactivity properties including modulation of oxidative stress. Hydroxyanisole phytonutrients, such as ferulyl scaffolds are the abundant phytonutrients, mainly available in “Corn” (Zea mays), and “Bamboo shoot” (Phyllostachys edulis), “Cinnamon spice” (Lauraceae), and “Turmeric” (Zingiberaceae). These phytonutrients are shown to have the highest free radical absorbance capacity when compared to other phytonutrient materials.

Recent report from our collaborative labs has demonstrated the usefulness of supplements for atherosclerosis treatment, specific to MPO inhibition [25,26]. Although several scientific reports clearly indicted MPO as one of the main reasons for manifestation of cardiovascular disease (CVD), yet no single drug is available in the market as an MPO inhibitor, though AstraZeneca’s product, AZD4831, is under clinical trial for heart failure with preserved ejection fractions [27]. Considering the importance of developing novel chemical entities as MPOI for CVD treatment, we have used hydroxyanisoles as our basic building block to create novel scaffolds for MPO inhibition. Advantages of this moiety are functionalization with other existing anti-inflammatory compounds, efficacy improvement and non-toxic nature [28,29]. Outcome of this approach seems to show promising results which can be construed for developing active agents as anti-inflammatory and anti-atherosclerotic agent by means of inhibition of MPO.

2. Result

2.1 In silico Validations

The prediction of Lipinski rules for the proposed derivatives are given in Table-1. There is no violation of five Lipinski rules in all designed compounds. It was carried out by Chemdraw Professional-15 software.

Molecular Docking studies

The molecular docking studies were carried out using designed MPOI molecules. These designed molecules were docked at the possible active site of MPO protein (5FIW) and the binding energy of the MPO complexes was found to be between -5.71 to -8.00 kcal/mol. The binding modes of active site of MPO complex structures were illustrated in Figure-S1 (Supplementary data). These illustrations demonstrated that the docked poses get docking scores (Binding energy) of protein and ligand complexes which are depicted in Table-2. Based on the binding energy of docked molecules in the MPO active site pocket, we concluded that all the designed molecules have been shown to possess some potential binding capacity within the active site of protein. These active molecules were successfully synthesized, and lead optimization was performed using SAR study and protein inhibition assay.

Table-1: Evaluation of Drug-Likeness of designed compounds using Lipinski’s Rule

Mm – Molecular mass; DHB – Donor-Hydrogen bonding; HBA – Acceptor – Hydrogen bonding; LogP – Partition Coefficient for n-octanol/water; (#) by Crippen's fragmentation; (##) by Viswanathan's fragmentation; (###) by Broto's method

ADMET Predicted Profiles classifications & Regressions

ADMET properties were evaluated on the parameters of Absorbance of the drug followed by drug distribution property. All designed molecules have no violations in ADMET parameters and data are given in Table-S2 (Provided in supplementary data).

2.2 Chemical Synthesis

Existing commercial drugs, such as Primaquine (Anti-malarial -drug) [30], Niflumic acid (anti-inflammatory agent) [31], Flufenamic acid (Antipyretic and anti-inflammatory agent) [32], Mefenamic acid (anti-inflammatory agent) [33] have the potential chemical functionalities with specific medicinal properties. The concept of drug repurposing is getting prominence and this investigation is focused on chemical combination of existing drug with hydroxylanisole based phytonutrients to realis synergism in the bioactivity.

We thus posited that this may represent a scaffold that can be further modified with an above-mentioned commercially available drugs to potentially improve the affinity of the compounds leading to greater MPOI.

Scheme-I: Synthesis of compounds 1 to 9 by synthetic route-I

The designed compounds 1, 2, 3, 4, 5, 6, 7, 8, 9 contain diverse chemical bonding such as amide, ester, hydrazide and hydroximic acid functional groups. It is well documented that amide and ester bonds can exert pertinent biological response and it also offers avenues for new chemical development [34,35]. Several commercial drugs [36] hold these two types of chemical functional groups. Also, hydrazide and hydroximic acid moieties are shown to possess crucial role in the MPO inhibition [37,38].

Salicylic hydroximic acid and hydrazide molecules showed prominent bio efficacy as MPOIs [39]. In order to achieve targeted drugs with good yield, high purity and scalable methodology, ester and amide functionality can be considered as reasonable choice. Two synthetic routes are invoked namely Route-I (Scheme-I)- Acid is converted into acid chloride followed by acid chloride treated with various amines and alcohols to obtained respective products. However, synthesis of acid chloride from acid has limitation in terms of product yield and lack of stability at RT. To overcome this difficulty, another synthetic route-II was established. By route-II method, (Scheme-II) acid is directly

Scheme-II: Synthesis of compound 1 to 9 by synthetic route-II

treated with amine and alcohols via steglich esterification process to yield amide and ester functionalized compounds 1 to 5. Compounds 6 to 9, hydroximic and hydrazide derivatives were synthesized as reported in the literature [40]. On the basis of comparison, we achieved 80% yield with high level of purity and stability of the compounds using route-II method compare to route-I pathway. It is noteworthy to mention that route-II method can be used for bulk synthesis of proposed compounds for advanced clinical development of the compounds as CVD therapeutics. Compounds 1, 2, 3, 4, 5, 6, 7, 8, 9 were synthesized with feasible chemical Routes-I and II. Purity of these compounds stands above 96% and it was determined by UPLC method. These compounds were fully characterized by 1H and 13C NMR techniques (Bruker Instrument 400 MHz); NMR data were provided in supplementary data as a Figure-S4. Molecular mass was determined by LC-ESI-Mass spectrometer; spectra were given in supplementary data Figure-S5. (The complete synthetic procedures and spectral data are provided in the supplementary data).

2.3 In vitro MPO Inhibition assay

i) Tetramethylbenzidine assay

The biological activity of hydroxyanisole derivatives was assessed by the inhibition of MPO activity in a cell-free system using commercially available MPO enzyme and TMB as a substrate based on the literature reported [41]. The IC50 value of inhibitors was determined by plotting absorbance of resulting solutions at 650 nm against inhibitor concentrations. The IC50 values of all synthesized compounds are presented in Table-2. Two lead compounds 7 (IC50= 0.82 µM) and 8 (IC50= 1.4 µM) showed maximum inhibition at minimum dosages. Similarly, the compounds 6 and 9 have been shown to inhibit MPO under comparable concentration.

2.4 Antioxidant assay

Antioxidant activity is assessed by observing the decrease in the DPPH radical absorbance at 517 nm. Results were reported as percentage inhibition of free radical generated during the course of reaction in presence of MPOIs. Similarly, ABTS antioxidant assay was performed without using any substrate. ABTS assay with an absorption maximum of 342 nm has excellent water solubility and chemical stability. Figure-1 provides the details of DPPH and ABTS radicals inhibition percentage by MPOIs. Our in vitro antioxidant study with MPOIs along with ascorbic acid as a positive control showed there is a correlation in antioxidant activity in comparison to a known antioxidant. Out of nine compounds, compound 6, 7, 8 and 9 seem to show moderate radical scavenging reactivity. On the other hand, compounds 7 and 8 showed the maximum inhibition.

Figure-1: % of DPPH and ABTS Inhibition by MPOIs

2.5 LDL oxidation in presence of MPO

LDL was subjected to oxidation in presence of 0.2 U MPO with and without MPOI. Presence of conjugated dienes has been examined by monitoring the UV absorption at 234 nm [42]. As represented in Figure-2A, MPO produced ox-LDL attained maximum increment in absorption at about 3.5 h. In the presence of MPOIs there was a rise in lag time, delineating that the compounds could extend the time of oxidation rate. Leukomethylene blue (LMB) and thiobarbituric acid reactive

Figure-2: MPOIs inhibit the oxidation of Lipoproteins

*Plasma from consented subjects is used to isolate LDL for MPO oxidation. 100μg LDL was treated with MPOIs (25μg) and then oxidation was carried out with 0.2U MPO in 1ml PBS and OD was measured at 234nm. As shown in Figure-2A all MPOIs delayed the initiation of LDL oxidation. After oxidation, the samples were used to determine peroxide content and thiobarbituric acid substances using LMB (Figure-2B) and TBARS (Figure-2C) assays. *P < .05.

substance (TBARS) assay values also corroborated the results with the oxidation peak as shown in Figure-2B & 2C.

2.6 Reduction of peroxidase by MPOIs

Peroxides offer a major function in inducing inflammation and further progress of the illness. As shown in Figure-3, except compound 1, 4 and 5, all the compounds notably decrease the peroxide quantity of FFAOOH when incubated together. Similarly, all the other compounds were able to reduce H2O2 as shown in Figure-4, which is a substrate for MPO reactions.

2.7 Effect of MPOIs on LPS-mediated pro-inflammatory gene expression in RAW macrophages

LPS actively influenced the mRNA levels of pro-inflammatory genes in RAW macrophages. However, as seen in Figure-5A & 5B, in presence of different MPOIs, the pro-inflammatory genes tumor necrotic factor α (TNF-alpha) and interleukin 1β (IL-1β) were reduced differently in a dose dependent manner. Only MPOIs did not cause any inflammatory gene expression.

2.8 MTT cell viability assay

MTT assay (cell viability) protocol using RAW macrophage cells (RAW 264.7 from the ATCC) [43] method was adopted. The outcome of this study indicated that conjugates 1 and 2 are highly safe for human consumption and non-toxic even at higher concentration (data not reported).

2.9 Purity of the lead MPOIs

For the purpose of biological study, we have used compounds with more than 96% purity. Out of nine synthesized compounds, we have selected compound 7 and 8 based on the docking scoring function and initial inhibition screening. Based on UPLC-DAD purity check, purity of the compound 7 and 8 show 97.37% and 96.18% respectively. Figure-S7 (Supplementary Information) represent the chromatogram of UPLC-DAD purity graph.

2.10 Mechanism of Non-Suicidal MPO Inhibition

i) UV-Visible spectroscopic method:

MPO acts on a variety of substrates and oxidizes them, it is theoretically possible that our inhibitors were acting as substrates and competitively masking formation of product(s) from the added substrate. However, we concluded that this chemistry was unlikely as the substrate was added several

Figure-3: MPOIs in decomposition of HPODE:

*200 nmoles/mL of HPODE was carefully incubated with various concentrations of MPOIs (0-25 µg) for one hour at 37 °C. Lipid peroxide formed in the reaction mixture was evaluated by LMB assay. All the compounds (MPOIs) revealed remarkable reduction in the peroxide content of FFAOOH in a concentration dependent manner. *P < 0.05,

folds in excess. The spectroscopic method of evaluating reaction mixture failed to reveal any additional products in the presence of MPO. There was no colored product even when the reaction mixture contained only the inhibitors and not the TMB substrates. As can be seen from Figure-6A & 6B, there is no indication of chemical transformation that could have occurred when our lead inhibitors can covalently link at the MPO active site since UV-Visible spectrum of lead inhibitors with and without MPO showed same absorption bands.

ii) Thin Layer Chromatography method:

Formation of any product from inhibitor due to oxidation of MPO will be reflected on TLC by characteristic new spots when it is eluted with specific solvent systems (eluant). Based on this TLC experiment, we observed that the lead inhibitors 7 and 8 when they were reacted with MPO protein in a suitable buffer did not produce any characteristic oxidative products in presence of MPO. As can be seen from TLC in Figure-6C, spots correspond to before and after MPO treatment appear at same Rf value indicating there was no specific product formed between inhibitor and MPO delineating the reversible nature of the inhibitor. Assuming the lead compounds 7 and 8 inhibit MPO irreversibly (suicide inhibition), the nature of resultant product will be chemically modified. The modified structure can be easily observed in TLC when compare to parent compound. Similar pattern of reactivity was observed when MPO is reacted with lead inhibitors 7 and 8 in presence of protein substrate, tetramethylbenzidine, as given in Figure-6C.

Figure-4: MPOIs in decomposition of H2O2:

*200 nmoles/ml of H2O2 was incubated with variable concentrations of MPOIs (0-25 µg) for 1hr at 37°C. Peroxide decomposition was measured by LMB assay. All the compounds (MPOIs) showed significant reduction in the peroxide content of H2O2 in a concentration dependent manner. *P < 0.05.

2.11 Structure Activity Relationship

Critical in vitro assays have been used to identify two lead compounds, 7 and 8 wherein functional group modification is done to improve the efficacy of the final structures. Chemical linkage of anti-inflammatory drugs (1 to 5 starting materials) with hydroxyanisole moiety yielded compounds (1 to 5) which have moderately reduced MPO inhibition value up to 7-fold compare to parent structures. Conversion of acid functionality of hydroxyanisole (6 to 9) to esters groups (6a to 9a) has slightly increased inhibition value from ~55 µM to ~85 µM. Further, modification of ester functionality (6a- 9a) into more bio-active functional groups such as hydroximic acid and hydrazide functional group, there was dramatic decrease in the inhibition values. There was a 30-fold

decrease in IC50 value for hydroxamic acid and 22-fold decrease in IC50 value for hydrazide functionality. Hydroxyanisoles (1 to 5) need ~4-fold excess of drug to inhibit MPO (~7 µM) compare to hydroximic acid substituents (6 to 9) which require ~1.5 µM. With regard to substitution of anti-inflammatory agents (1 to 5) in the LDL oxidation reaction, there was 10-fold decrease in the inhibition when hydroximic acid substituents are used. For in HPODE and H2O2 decomposition assay, we observed that there was 90% efficacy in peroxide degradation for compounds 6-9 compare to compound 1-5. This observation is also applicable to multiple concentrations ranging from 1µg to 25µg. As a representative sample, Figure-7 provides comparative inhibition (IC50) value of basic building block and its modified structures.

Figure-5: LPS mediated pro-inflammatory gene expression in- RAW 264.7 cells in presence of MPOIs. RAW 264.7 cells have been incubated with LPS and MPOIs in serum free methods for 24 h. RNA was separated and qPCR analysis was executed for Figure-5A TNF-α and Figure-5B IL-1β genes in RAW cells treated with LPS in presence and absence of MPOIs of various concentrations using appropriate primers. Results are given as mean ± SD and significance considered as *P < 0.05.

5A)

Overall, the presence of hydroximic acid and hydrazides functional groups within the basic building blocks has significantly increased the MPO inhibition potentials and peroxide decomposition assays. Additionally, LDL and gene expression studies also correlated the above results. With these observations in hand, two lead molecules 7 and 8 are considered as prospective chemical agents for the treatment of MPO-mediated CVD.

Figure-7: Representative example for comparative inhibition (IC50) value of parent and modified parent to reveal SAR study.

3. Discussion

Based on the literature reports, there is a strong correlation exists between phenolic compounds and MPO inhibition [26]. Existence of methoxy group (anisole type molecules) can further strengthen the efficacy. A well-known example is curcumin wherein presence of hydroxyanisole type structures offers special properties to the parent molecules. Synthesis of these compounds was designed in such a way that methods can be adopted to prepare compounds for large scale production. The docking results as well as biochemical assays with our inhibitors revealed that MPO inactivation could occur either via active site blockade or rendering MPO inactive by compound II. This hypothesis is supported by literature reports that have shown phenols and anilines prevent MPO cascading reactions from producing

(5B)

Figure-6 A & B: The UV pattern of MPO reaction of lead inhibitors in presence and absence of MPO with compound 7 and 8. Figure-6C: The TLC pattern of MPO reaction of lead inhibitors in presence and absence of MPO with compound 7 and 8

hypochlorous acid by scavenging the enzyme in its dormant compound II form [44]. This type of inhibition is reversible because superoxide reduces compound II back to its native form. Therefore, at this point of time, we reasonably believe that compounds 1-9 may act as reducing substrate and may promote compound II formation and it could act as a substitute for hydrogen peroxide in compound I formation. It is also received our focus that the large binding cavity of MPO can be utilized to further optimize the hydroxyanisole derivatives that we have synthesized. Additional docking results with specific pharmacophores (data not shown) have provided very weak lead structures when it was docked with MPO [45].

Next, the results of specific MPO inhibition assay namely, TMB assay revealed that the proposed structures are moderate to high active inhibitors. Within the nine compounds, compound 7 and 8 showed inhibition in sub-micromolar to high nano molar concentration. The inhibition values of these two compounds correlate well with binding energy of these molecules within the MPO protein. To further substantiate these findings, DPPH and ABTS generic antioxidant assays were conducted with all these compounds and outcome of this data indicated all nine compounds are generic antioxidants. The degree of antioxidant potentials slightly varies for compounds 6, 7, 8 and 9 compare to other structures. However, difference in antioxidant activity of 7 and 8 with other compounds seems to be comparable.

In view of implication of MPO in CVD, it is important to investigate on the effect of MPOI toward lipids oxidation and its prevention. Involvement of ox-LDL in atherosclerotic plaque development and characterization of MPO-mediated biomarker products within the lesion demonstrate that importance of lipids in CVD. LDL and HDL on oxidation can generate a characteristic diene which absorbs at 234 nm. Increase in the absorption at 234 nm indicates the formation of elevated level of diene due to oxidation of lipids. In vitro oxidation of isolated LDL with and without MPOIs has shown that there was an increase in lag time when oxidation was done in presence of MPOI suggesting that the proposed compounds can delay the oxidation of LDL thereby reduce the ox-LDL level as represented in Figure-2A. The oxidized samples are further studied by treating them with LMB and TBARS assays by measuring its characteristic absorption at 660 nm and 532 nm respectively. Once again, based on the data provided in the Figure-2B and 2C, it is reasonably assumed that compound 7 and 8 can control the oxidation of LDL. This can offer an explanation that these compounds can be considered as powerful MPOI and efficient cardioprotective agents.

Next, the effect of MPOI on peroxide decomposition was investigated. Peroxides can create havoc by inducing inflammation and further progression of the disease. Hence, the decomposition of H2O2 and HPODE was studied in presence of MPOI and it was estimated by LMB assay. In line with activity prediction, compound 7 and 8 seem to decompose peroxides more efficiently compare to other compounds. By establishing good activity response under in vitro condition,

We turned our attention to cell line study to mimic physiological condition. If there is any correlation exist between in vitro and cell line study, there is a greater chance of identifying lead compounds within the proposed structures. Several diseases including cardiovascular, diabetes, inflammatory bowel disease, cancer, and different types of neurological disorders are associated with oxidative stress and inflammation. At stage these two factors will play a key role and impacts disease progression is not yet completely known. In this methodology, we studied the effect of MPOIs on LPS induced inflammation. Indeed, each compound showed different levels of attenuation to LPS induced inflammation, however all the compounds were significantly attenuated LPS mediated pro-inflammatory cytokines TNF-α and IL-1β as shown in Figure-5A and 5B. Also, enzymatic and non-enzymatic LDL oxidation mechanism is different and various MPOIs can inhibit or delayed the enzymatic (MPO) oxidation of LDL. Also, all anti-inflammatory molecule may not be antioxidant molecules and vice versa; further, even some of the MPOIs might need high doses for their beneficial action either in oxidative stress/inflammation.

These compounds sometimes had divergent effects on multiple measures such as conjugated dienes assay, methylene blue assay etc. These effects are congruent with the effects on in vitro assays suggesting that the effects on measurements of lipid functionality can sometimes diverge, necessitating comprehensive evaluation of a number of parameters. However, the molecules are non-toxic even at high concentrations using viability assays. Purity of the synthesized compound was determined by UPLC method and it was established to be above 96%.

Further, the mechanism of inhibition of MPO is important in terms of understanding the interaction between inhibitor and protein. Essentially, the proposed inhibitors deactivate the MPO protein through reversible pathway. The evidence for this reversible mechanism (non-suicidal inhibition) is ascertained from UV-Visible study and TLC study. Irreversible inhibitors change chemically due to covalent linking with protein which can be inferred by observing change in absorption pattern in UV studies. Similarly, formation of chemically modified product can be observed in thin layer chromatographic studies. Lack of characteristic change in UV spectrum and TLC studies point to the fact that proposed lead structures inhibit MPO in a reversible fashion.

Existing literature reports suggest that the hydroxylanisole derivatives are more potent anti-inflammatory agents and can efficiently inhibit various inflammatory signaling pathways such as TNF alpha, TLR4 and NFkB [46,47]. In addition, recent studies from our collaborator laboratory have demonstrated the whole sesame oil aqueous extract (SOAE: enriched with methoxy phenolic compounds) efficiently attenuated the NFkB mediated inflammatory mechanism [48]. All these results strengthen our data that the most of the compounds used in this study were methoxy phenols, which seem to have therapeutic potential. Further, it might provide a regulatory component in several anti-inflammatory pathways and possess numerous beneficial effects.

Even though several pharmacological as well as nutritional approaches exist, still the mortality rate is continued to rise due to various oxidative stress and inflammation associated disease. This emphasizes the vital requirement of new therapeutic agents to address this malady. The current study evaluated the potential therapeutic nature of several MPOIs against oxidative stress and inflammation. This will shed light on the role of these MPOIs as therapeutic agents and open up new avenues for clinicians and pharmaceutical companies to explore further in this line of investigation.

Conclusion

Revelation of anisole moiety chemically combined with anti-inflammatory agents as MPOI is demonstrated to operate exceedingly well based on the outcome of in vitro and cell line studies. Blockade of MPO active site is the basis for its inhibition which is substantiated by computation docking study and Lipinski rule. Based on the literature reports that have demonstrated that the phenols and anilines inhibit MPO from generating hypochlorous acid by scavenging the enzyme in its dormant form. Reversible form of inhibition is postulated since superoxide reduces compound II back to its native form. Compound 7 and 8 may act as reducing substrate and can support compound II generation and it could behave as alternate for H2O2 in compound I formation. Results based on in vitro and cell line-centered assay and effect of compound 7 and 8 on LDL revealed that compound 7 and 8 are efficient MPO inhibitors and can provide promising therapeutic properties against CVD.

Future Perspective

The discernment of MPO-mediated pathways has improved dramatically, and it is therefore very essential to suitably earmark interventions and resources, for the scope bringing out an inhibitor with a minimum risk-benefits ratio. In order to develop MPO inhibitors as crucial therapeutic agents, it is necessary to include numerous multiple intricate parameters that are involved to impact the outcome of the study. This is not only for CVD, but other illness of which MPO is implicated. The hydroxyl anisole analogs as MPOIs are expected to show their effect on illness target, but at the same time offer minimum efficacy on immune suppression addressing the involvement on the progress of infections due to lack of MPO. This study undoubtedly would pave the way for developing cardioprotective agents that are bio-compatible and non-toxic due to natural origin of the chemical entities.

Summary points

Design and synthesis of new class of hydroxyanisole conjugates as MPOI for effective cardioprotective agents.

Based on in-silico docking, enzyme inhibition assay and cell line-based assay to filter out lead compounds from hit analogs.

Two lead compounds 7 and 8 exhibited favorable properties in critical bio-assays like LDL oxidation, H2O2 and lipid peroxide reduction assay and reduction in pro-inflammatory gene expression.

Outcome of this study points to a fact that hydroxylanisole conjugates can be used as a potential building block against CVD.

4. Experimental Section

4.1 Materials

Starting materials were purchased from Sigma Aldrich, Bangalore, India. All reagents were purchased from SD Fine chemicals, India. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI), dimethyl aminopyridine (DMAP), were purchased from Alfa Aesar chemicals (Germany). CDCl3 and DMSO-d6 was purchased from Merck Pvt. Ltd, India. All biological proteins and reagents were purchased from Sigma Aldrich Pvt. Ltd; Solvents were purchased from SD Fine chemicals India Ltd. polymerase chain reaction (PCR) primers and TrizolTM testing agent were purchased from Invitrogen, Carlsbad, CA. Human leukocyte MPO (myeloperoxidase), Lipopolysaccharide (LPS) (E. Coli) and regular reagents and chemicals were purchased from Sigma, St. Louis, MO, USA.

4.2 Methods

4.2.1 In silico studies

Lipinski rule was invoked to predict the feasibility of the proposed structures to ensure increase the success rate of proposed compound library.

Molecular docking: An AutoDock-1.5.6 docking tool was used for molecular modeling studies [49] using protein-heme complex from the RCSB-PDB (Protein Data Bank – https://www.rcsb.org) crystal structure entry by using PDB ID: 5FIW. The original X-ray crystal structure resolution was 1.7 Å used. This protein was employed as input structure for protein preparation. Out of four chains, A, B, C and D; chain A and C was considered for docking while the crystal symmetry-related B, D chains were removed. Hydrogen atoms were added to the myeloperoxidase-heme followed by the addition of Gasteiger and Kollman charges. It may be noted that the PDB entry did not contain any crystallographic water molecules in the protein-heme complex. The ligands were drawn using Chemdraw Professional-15.0 and the grid box were prepared to cover active site pocket. Then the grid run was performed followed by docking protocol was evaluated. After the completion of docking procedure, critical analysis of various parameters was performed for protein ligand complex. The analyzed complex was visualized using Discovery Studio Visualizer-2019-R2 and the images were taken in 2D and 3D format. None of the MPO structures in the PDB contained a natural ligand and we have created lead entities based on the hydroxyanisole pharmacophore building block.

ADMET: admetSAR database is used to derive Adsorption Distribution Metabolism Excretion and Toxicity (ADMET) Profile prediction and regression have been calculated for the all designed compounds [50].

4.2.2 Chemical Synthesis of MPOI

The synthesis of proposed hydroxyanisole derivatives was achieved by adopting two different chemical routes, in order to synthesize target compounds with high yield and purity. Accordingly, for the synthetic route-I (Scheme-I), we utilized various chemical methods for the conversion of acid to acid chloride followed by coupling reaction with substituted hydroxyanisole-based amine and alcohol moieties. In case of synthetic route-II (Scheme-II), we used substituted acid derivatives directly coupled with amine and alcohol moieties by steglich esterification for Method-I (compound 1 to 5). And utilizing method-II to synthesize compound 6 to 9 by acid to ester followed by treatment with amine by condensation reaction. The synthetic route-II is a feasible approach in terms of yield, cost effectiveness, with easy-handle methodology. The complete details on synthetic Route-I and synthetic route-II are provided in supplementary data file.

4.2.3 MPO inhibition assay

Tetramethylbenzidine (TMB) assay:

MPO inhibition assay was performed based on the literature report using TMB as an enzyme substrate [51]. Salicylhydroxamic acid, a known MPO inhibitor, was used as a positive control. Other MPO co-substrate; H2O2, NaCl, with variable concentration of hydroxyanisole derivatives were used to prepare a dose-response curve and finally determine the IC50 values [41].

4.2.4 In vitro antioxidant evaluation

Briefly, 100 µM DPPH solution in methanol was made and 1000 µL of this DPPH solution was mixed to a total volume of 3 mL of the total reaction mixture containing MPOIs at different concentration. The solution was shaken thoroughly and kept it ⁓27 șC (RT) for 30 minutes. Absorbance was then determined at 517 nm. By spectroscopically monitoring the colour change will provide clue on antioxidant properties of the drug. Similar study was performed using ABTS method of antioxidant study to scavenge 2,2’ azinobis (ethylbenzthiozoline 6-sulfonic acid) (ABTS+) radical cation to identify antioxidant potentials of the drugs. Briefly, by reacting 7 mM ABTS in water and 2.45 mM K2S2O8 (1:1) in the absence of light (dark) at ⁓27 șC (RT) for a period of 16 h, ABTS·+ cation radical was generated. Appropriate dilution of ABTS·+ species with methanol is done and finally absorbance at 734 nm was measured. Combination of MPOIs at different concentration to a required volume of diluted ABTS·+ solution, the absorbance was determined at 30 min after the initial mixing. An applicable background / solvent blank was determined in each measurement. To compare the efficiency, ascorbic acid was used as a positive control. Measurement was also done in the absence of inhibitor.

4.2.5 Isolation and Oxidation of Lipoproteins

Subsequent Institutional Review Panel approval, blood sample was collected in a heparinized vial from healthy volunteers after receiving their consent and stored on cold condition. Plasma was separated by centrifuging the blood at 3000 rpm for 20 min. Low-density lipoprotein was separated from ordinary blood plasma by consecutive ultra-centrifugation using a Bekman TL-100 tabletop ultracentrifuge (Beckman, Palo Alto, CA) [52]. The separated lipoprotein was dialyzed against 0.3mM ethylenediaminetetraacetic acid (EDTA) in 1x phosphate buffer saline (PBS) of pH 7.4 at 12 h afterward filter sterilized. The amount of protein was quantified using the Bio-Rad DC protein assay (Hercules, CA). LDL sample was made to undergo oxidation rapidly / immediately after dialysis. Oxidation of LDL (100μg/mL) was performed with 0.2U MPO and 100μM hydrogen peroxidase (H2O2) in 1000 µL of phosphate buffer saline at 37°C both in presence and absence of 25μgm of the MPO inhibitors. The formation of conjugated dienes has been observed at an optical density of 234nm for 4 h using Jenway DB-6500 spectrophotometer furnished with an 8-chamber cuvette changer. Using LMB [53] and TBARS assays determine peroxide content by the degree of LDL oxidation processes.

4.2.6 Preparation of HPODE and Incubation with MPOIs

13-Hydroperoxylinoleic acid (13-HPODE of 200 nmol/mL was developed as already discussed [54] and used to measure the effect of MPOI on FFAOOH (free fatty acid peroxides). HPODE was incubated with increasing concentrations of compounds (0-25μg) for 1 h at 37șC. Lipid peroxide produced in this reaction method was tested by leucomethylene blue (LMB) assay. Compounds also were incubated with H2O2 to determine whether it has similar effect as seen with HPODE.

4.2.7 Cell Culture

RAW 264.7 macrophages were purchased from American Type Culture Collection (ATCC). Cells have been grown as a single-layer in dishes and flasks and maintained in DMEM medium supplemented with 10% fetal bovine serum (Sigma, St Louis, MO, USA), L-glutamine 2mM, and 1x antibiotic penicillin-streptomycin solution. Cells have been maintained in a 5% carbondioxide (CO2) atmosphere at 37°C. For experimental studies, the cells were incubated in serum-free condition.

i) Incubation of RAW 264.7 cells with LPS and MPOIs

To detect the variation in gene expressions of TNF-α and IL-1β, RAW 264.7 cells (2×105 cells/well) were preincubated in serum-free DMEM for 3 h. Cells were incubated with 10ng/ml LPS either in presence and absence of 5 or 25μg of compounds for 24 h. At the endpoint of one day incubation, cells have been collected in TrizolTM for RNA separation (isolation) [55].

ii) cDNA Synthesis and RT-PCR reaction

Total RNA from cells was separated by using the TrizolTM testing agent. 1μg of RNA was reverse transcribed into cDNA by using SuperscriptTMIII First Strand-Synthesis system (Invitrogen, Carlsbad, CA). cDNA (50ng) compound used to execute Quantitative RT-PCR (real-time polymerase chain reaction) by CFX96 iCycler Multicolor RT-PCR Detective System (Bio-Rad, Hercules, CA) with SYBR Green (Invitrogen Carlsbad, CA). PCR was performed with IL-1β and TNF-α particular primers for a mouse Table-S6 (available in supplementary file), consequential in 200-bp pieces. For the purpose of comparison in gene we used glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primers, consequential in a 200-bp fragment. PCR was executed with an initial method of denaturation at 50șC for 2 minutes, 95șC for 10 minutes followed by 40 cycles of 95șC for 20 secs and 60șC for 20 sec. Melt were constituted for the reactions. Normalized fold aspects were measured by a 2-ΔΔCt methodology.

4.2.8 Purity of Lead MPOIs

In order to confirm the highest purity level of the synthesized compound for in vitro testing, we established purity determination with UPLC-DAD method (Ultra Performance Liquid Chromatography- Waters model) and purity of the lead compound was found to be >96%.

4.2.9 Mechanism of Non-Suicide Inhibition

Irreversible inhibitors are suicidal compounds. They react with the active site of the enzyme forming stable covalent bonds. These bonds may be formed between the inhibitor and the heme group or between the inhibitor and the amino acids located beside the heme group. Two families of compounds were characterized as irreversible inhibitors including aminobenzoic acid hydrazide and thioxanthene’s. Most of the reported compounds in the literature are reversible competitive inhibitors of MPO. They react reversibly with the active site of the enzyme. We established the reversible nature of the MPOI by performing two relevant experiments as given below viz. UV-Visible spectroscopic method and thin layer chromatographic method.

i) By UV-Visible spectrophotometer

Typically, the reaction mixture contains 100 mU human MPO (Sigma-Aldrich, St. Louis, MO), 2 µmols H2O2, 8 µmols of TMB, followed by the addition of lead molecules. The final volume was made to 1 mL with 50 mM sodium acetate (CH3COONa) buffer at pH 5.6. The reaction was induced by adding MPO. Wavelength scan between 200 to 800 nm was used to detect any products formation and it was reflected in the spectrum. Generated products were identified by comparing with a spectrum without MPO. This experiment was carried out with two controls, one without MPO enzyme and another with MPO enzyme.

ii) By Thin Layer Chromatography (TLC) method

Initially, a suitable mobile phase (eluant) was identified to determine the precise Rf value for each inhibitor on a pre-coated UV active silica gel plate (EMD, TLC Silica gel 60 F254). MPO mediated reaction was carried out by reaction mixture contains 100 mU human MPO (Sigma-Aldrich, St. Louis, MO), 2 µmols H2O2, 8 µmols of TMB, followed by the addition of lead molecules. The final volume was made to 1 mL with 50 mM sodium acetate buffer at pH 5.6. The reaction was induced by adding MPO. The organic compound present in the mixture was extracted with minimum amount of organic solvent and concentrated followed loaded on a pre-coated TLC plate and eluted with a known mobile phase. Appearance of any new products apart from the inhibitors was determined either by exposing the plate to UV light or iodine vapor to get a bright spot on the TLC plate.

Statistical Analysis

Statistical values are mentioned as mean ± SD, and statistical calculations have been carried out by way of Student t-test at the significance of P < 0.05.

Accession Code

The PDB code for MPO protein is 5IFW.

Corresponding Authors

* Rajagopal Desikan, e-mail: rajagopal.desikan@vit.ac.in

** Chandrakala Aluganti, e-mail: Chandrakala.Aluganti@ucf.edu

Author Contributions

PJ has done in silico studies and synthesis of anisole derivatives coupled with anti-inflammatory agents. DR and CA have contributed in terms of in vitro studies including enzyme based MPO assay, cell line-based assay and LDL assays. DR and CA have contributed in manuscript preparation. SP and SR have provided direction in terms of biochemical aspects of the study and manuscript revision. RD has supervised the in silico, synthesis, in vitro studies.

Acknowledgment

Authors are grateful to VIT-RGEMS for the financial support. We also thank DST-VIT-FIST for NMR, VIT-SIF for GC-MS and other instrumentation facilities.

Abbreviations

MPO, myeloperoxidase; LDL, low density lipoprotein; HDL, high density lipoprotein; HOCl, hypochlorous acid; HOBr, hypobromos; H2O2, hydrogen peroxide; Br, bromide; Cl, chloride; SCN, thiocyanate; 3-ClTyr, 3-Chlorotyrosine; Di-Tyr, dityrosine; ROS, reactive oxygen species; ox-LDL, oxidized LDL; apo A-1, apolipoprotein A-1; RTC, reverse cholesterol transport; ox-HDL, oxidized HDL; 3-NO2Tyr, 3-nitro tyrosine; SMC, smooth muscle cells; CVD, cardiovascular disease; EDCI, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; DMAP, dimethyl aminopyridine; PCR, polymerase chain reaction; LPS, lipopolysaccharide; ADMET, adsorption distribution metabolism excretion and toxicity; PDB, protein data bank; TLC, thin layer chromatography; EDTA, ethylenediaminetetraacetic acid; PBS, phosphate buffer saline; LMB, leucomethylene blue; MPOI, MPO inhibitors; TBARS, thiobarbituric acid reactive substances; 13-HPODE, 13-hydroperoxylinoleic acid; FFAOOH, free fatty acid peroxides; ATCC, american type culture collection; CO2, carbon dioxide; TNF-α, tumor necrosis factor alpha; IL-1β, interleukin 1-beta; RNA, ribonucleic acid; cDNA, complementary deoxyribonucleic acid; RT-PCR, real-time PCR; GAPDH, 3-phosphate dehydrogenase; UPLC, ultra-performance liquid chromatography; mRNA, micro RNA;

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