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Identification of NSAIDs as inhibitors through highly sensitive chemiluminescence method, expression analysis in mononuclear cells and computational studies

Wardah Shahid, Syeda Abida Ejaz, Mariya al-Rashida, Muhammad Saleem, Maqsood Ahmed, Jameel Rahman, Naheed Riaz, Muhammad Ashraf

PII: S0045-2068(21)00195-4 DOI: https://doi.org/10.1016/j.bioorg.2021.104818 Reference: YBIOO 104818

To appear in: Bioorganic Chemistry

Received Date: 23 December 2020 Revised Date: 14 February 2021 Accepted Date: 6 March 2021

Please cite this article as: W. Shahid, S. Abida Ejaz, M. al-Rashida, M. Saleem, M. Ahmed, J. Rahman, N. Riaz, M. Ashraf, Identification of NSAIDs as lipoxygenase inhibitors through highly sensitive chemiluminescence method, expression analysis in mononuclear cells and computational studies, Bioorganic Chemistry (2021), doi: https://doi.org/10.1016/j.bioorg.2021.104818

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© 2021 Published by Elsevier Inc. Identification of NSAIDs as lipoxygenase inhibitors through highly sensitive chemiluminescence method, expression analysis in mononuclear cells and computational studies

Wardah Shahida, Syeda Abida Ejazb, Mariya al-Rashidac, Muhammad Saleema,

Maqsood Ahmeda, Jameel Rahmana, Naheed Riaza, Muhammad Ashraf a* a Department of Chemistry, The Islamia University of Bahawalpur, Bahawalpur-63100. Pakistan. b Department of Pharmaceutical Chemistry, Faculty of Pharmacy, The Islamia University of Bahawalpur, Bahawalpur-63100. Pakistan. c Department of Chemistry, Forman Christian College (A Chartered University), Ferozepur Road, Lahore-54600. Pakistan.

*Corresponding author: M. Ashraf [email protected]

Abstract Here we report the inhibitory effects of nine non-steroidal anti-inflammatory drugs (NSAIDs) on soybean 15-lipoxygenase (15-LOX) (EC 1.13.11.12) by three different methods; UV- absorbance, colorimetric and chemiluminescence methods. Only two drugs, Ibuprofen and Ketoprofen, exhibited enzyme inhibition by UV-absorbance method but none of the drug showed inhibition through colorimetric method. Chemiluminescence method was found highly sensitive for the identification of 15-LOX inhibitors and it was more sensitive and several fold faster than the other methods. All tested drugs showed 15-LOX-inhibition with IC50 values ranging from 3.52±0.08 to 62.6±2.15 µM by chemiluminescence method. Naproxen was the most active inhibitor (IC50 3.52 ± 0.08 µM) followed by Aspirin (IC50 4.62 ± 0.11 µM) and Acetaminophen (IC50 6.52 ± 0.14 µM). Ketoprofen, Diclofenac and Mefenamic acid showed moderate inhibitory profiles (IC50 24.8 ± 0.24 to 39.62 ± 0.27 µM). Piroxicam and Tenoxicam were the least active inhibitors with IC50 values of 62.6 ± 2.15 µM and 49.5 ± 1.13 µM, respectively. These findings are supported by expression analysis, molecular docking studies and density functional theory calculations. The expression analysis and flow cytometry apoptosis analysis were carried out using mononuclear cells (MNCs) which express both human 15-LOX and 5-LOX. Selected

NSAIDs did not affect the cytotoxic activity of MNCs at IC50 concentrations and the cell death showed dose dependent effect. However, MNCs apoptosis increased only at the higher concentrations, demonstrating that these drugs may not induce loss of immunity in septic and other inflammatory conditions at the acceptable inhibitory concentrations. The data collectively suggests that NSAIDs not only inhibit COX as reported in the literature but soybean 15- LOX and MNCs LOXs are also inhibited at differential values. A comparison of the metabolomics studies of pathway after inhibition of either COX or LOX enzymes may reconfirm these findings.

1 Key Words: Lipoxygenase inhibitors, Chemiluminescence assay, NSAIDs, Expression analysis, In Silico studies, DFT calculations, Mononuclear cells, Flow cytometry.

1. Introduction

Inflammation is a self-protective mechanism to cellular injuries caused by pathogenic infection or any physical damage. Persistent inflammatory conditions may lead to the development of pathophysiological states, including inflammatory bowel disease, arthritis, atherosclerosis, carcinogenesis and bronchial asthma [1, 2]. The arachidonic acid produced during these injuries plays a central role in the development and production of biologically active chemical mediators involved in inflammatory response [3]. The two separate mechanisms are operated by two separate enzyme systems; the cyclooxygenase (COX) pathway and the lipoxygenase (LOX) pathway [4]. The COX-I (EC 1.14.99.1) is a constitutive enzyme involved in homeostatic functions and COX-II (EC 1.14.99.2) is an inducible enzyme involved in inflammation and that both represent the rate-limiting steps in the metabolism of arachidonic acid which eventually results in the synthesis of prostaglandins and other biologically active molecules [5,6]. These enzymes are the targets of nonsteroidal anti-inflammatory drugs (NSAIDs). Several variants of COX are also reported and few NSAIDs also act by inhibiting the activity of recently identified COX-III enzyme such as Acetaminophen, Aspirin, Diclofenac and Ibuprofen [7,8].

Another group of enzymes, (LOXs; EC 1.13.11.12) are the most important class of non-heme iron enzymes that are expressed in different cell types including; endothelial, epithelial and immune cells where they are majorly responsible for various functions including cell differentiation, skin barrier formation and immunity [9, 10]. They contain dioxygenases and catalyze the regiospecific and stereospecific peroxidation of polyunsaturated fatty acids (PUFA) to their corresponding hydroperoxy derivatives [11]. The contains six functional LOX ; ALOX15, ALOX15B, ALOX12, ALOX12B, ALOX5, and ALOXE3 each encoding a distinct LOX enzyme depending upon the double bond being oxidized [12, 13]. The different types of mammalian LOXs enzymes exhibit different biological functions. For example, 15-LOX and 5-LOX are involved in the synthesis of (LXs), specifically LXA4. The 15-LOX produces 15S-HPETE (hydroperoxyeicosatetraenoic acid), which serves as a substrate for 5-LOX.

2 The 5-LOX converts the 15-LOX product to LXA4 as chemical mediator which is involved in inflammation and other related pathophysiological conditions [14, 15]. Both 15-LOX and 5-LOX have been associated with several diseases, that is, they are involved in the chronic obstructive pulmonary disease and progression of certain types of cancer especially colon and pancreatic cancer [16, 17]. The expression of these enzymes has been implicated in blood mononuclear cells (MNCs) and, therefore, these enzymes are thought to be the emerging target for therapeutic intervention [18].

Identification of small molecules as LOX inhibitors may not only lead to the therapeutic treatment but may also provide invaluable research tools to study the potential role of LOX in diverse physiological and pathological conditions [19]. Soybean 15-LOX is the most easily accessible than human 5-LOX and therefore it was used for in vitro studies so that its results could be generalized and extendable to human 5-LOX. Further, a logical symmetry occurs among the two enzymes that become indistinguishable for more than 50% within 10 Å in the of the pocket [20, 21].

Several methods have been reported for the identification of LOX inhibitors [22]. UV- absorbance method involves the measurement of the diene product (produced via lipid peroxidation), 5(S)-hydroperoxy-6-trans-8,11,14-cis eicosatetraeinoic acid (HPETE) by oxidation of linoleic acid as substrate at 234 nm [23, 24]. The colorimetric method measures the formation of colored product with the oxidation of the substrate and its measurement. A ferric-xylenol orange assay was developed for the screening of LOX inhibitors but it gave unstable and variable absorbance values. Lu et al., (2013) developed a colorimetric method wherein LOX-derived lipid hydroperoxides oxidized ferrous ions to ferric ions which in turn bound thiocyanate ions to form a red ferrithiocyanate complex which was measured at 480 nm [25]. A fluorescence method has also been developed for inhibition studies of human 5-LOX [26, 27]. Moreover, an indirect method has been reported that involves the measurement of metabolites of arachidonic acid metabolism using supercritical fluid chromatography-tandem mass spectrometry instead of the direct measurement of enzyme inhibition [28]. Chemiluminescence assays have also been reported by using luminol which was a direct measurement of lipid hydroperoxide formed by LOX reaction and also improved the sensitivity of assay by adding cytochrome C in the luminol-

3 mediated reaction. The reaction involved the generation of hydroperoxide from unsaturated fatty acid substrate that reacts with cytochrome C to produce oxygen radicals, which oxidize luminol to its excited state to generate photon emissions measured by the machine [29, 30]. This method is highly sensitive and alternate method for the measurement of LOX activity. Previous studies revealed the inhibition of COX by different NSAIDS, but so far not a single study is reported on inhibition of 15-LOX by NSAIDs. So, we took initiative to explore the effect of already reported well-known NSAIDs against 15-LOX using three different methods. The development of dual inhibitors of COX/LOX is high priority which could increase the anti- inflammatory effects and reduce the side effects related to NSAIDs [12, 16]. The objective of the current study was to compare the chemiluminescence method with the conventional UV- absorbance and colorimetric methods to identify the LOX inhibitory potential of routinely used NSAIDs as COX inhibitors. Below are given the structures of NSAIDs used in this study.

OH

H3C O OH O

H3C N H O

1.Aspirin 2.Acetaminophen 3.Diclofenac 4.Ketoprofen

CH3

CH3 COOH

H3C

5.Piroxicam 6.Tenoxicam 7.Ibuprofen 8.Naproxen

9.Mefenamic acid 10.Quercetin 11. Baicalein

4 The results exhibited that the selected NSAIDs not only inhibited COX as reported earlier but were also proved as potent inhibitors of soybean 15-LOX. The binding interactions of the identified inhibitors within the active pocket of the enzyme were further confirmed by molecular docking studies and analyzed in terms of their relative strength by using the electrostatic potential surface maps obtained from standard DFT calculations. The effect of the identified inhibitors against 5-LOX was observed through expression analysis and apoptotic studies in MNCs.

2 Results and discussion

2.1 Enzyme inhibitory activities of NSAIDs

The commonly used nine NSAIDs were selected for the present studies along with Quercetin and Baicalein as standards. Inhibition studies of these drugs were determined by three methods as given in Table 1. Through colorimetric assay, none of the selected drugs exhibited 50% inhibition of 15-LOX even when tested at the final concentration of at 0.5 mM. When the drugs were tested through UV-absorbance method, only Diclofenac, Ibuprofen and Ketoprofen showed inhibition. Ibuprofen was the most effective 15-LOX inhibitor with IC50 91.7 ± 0.5 µM.

Ketoprofen and Diclofenac exhibited IC50 values of 215.2 ± 0.5 and 241.5 ± 0.3 µM, respectively. The obtained results were compared with Quercetin and Baicalein which exhibited

IC50 values of 2.3 ± 0.3 and 22.41 ± 1.2 µM, respectively. All other drugs showed < 50% inhibitory potential at the final concentration of 0.25 mM. Chemiluminescence method was optimized and employed in the determination of 15-LOX inhibition by NSAIDs. General screening was done at 0.25 mM and all nine drugs exhibited inhibition of over 82% at this concentration (Table 1). Our results suggest that this chemiluminescence assay is far better and sensitive and fast than other methods and is useful for chemical library screening. The most active inhibitor was Naproxen (IC50 3.52 ± 0.08 µM) followed by Aspirin (IC50 4.62 ± 0.11 µM) and Acetaminophen (IC50 6.52 ± 0.14 µM). The least active drugs were Piroxicam and

Tenoxicam with the inhibitory concentration value of IC50 62.6 ± 2.15 µM and 49.5 ± 1.13 µM, respectively. Ketoprofen, Diclofenac and Mefenamic acid showed moderate inhibitory profiles

(IC50 24.8 ± 0.24 to 39.62 ± 0.27 µM).

5 Table 1 Comparative analysis of enzyme inhibition profiles of NSAIDs.

No. Name Inhibitory concentration values, IC50 (µM) UV-absorbance Colorimetric Chemiluminescence 1 Aspirin >250 >500 4.62±0.11 2 Acetaminophen >250 >500 6.52±0.14 3 Diclofenac 241±2.33 >500 31.7±0.32 4 Ketoprofen 215±2.15 >500 24.8±0.24 5 Piroxicam >250 >500 62.6±2.15 6 Tenoxicam >250 >500 49.5±1.13 7 Ibuprofen 91.7±0.5 >500 14.5±0.13 8 Naproxen >250 >500 3.52±0.08 9 Mefenamic acid >250 >500 39.6±0.27 Quercetin 2.31±0.32 >500 4.86±0.14 Baicalein 22.4±1.2 ND ND The initial screening of NSAIDs through UV-absorbance and Chemiluminescence method was carried out at 0.25 mM. Screening was done at 0.5 mM through Colorimetric method. Data is presented as the mean (mean ± S.E.M.) of three independent inhibitory concentration values (n =3). ND = not determined.

The inhibition profiles of these drugs against the soybean 15-LOX have not been reported earlier and are given here for the first time. To confirm the binding sites of these drugs, docking studies were performed. The data shows that these drugs bind the active site of the enzyme even though they bind COX enzyme system too wherein other groups or atoms may be involved in the interactions.

2.2 Molecular based studies

2.2.1 Relative quantification of mRNA by real time PCR

PCR is molecular biology technique that involves the formation of multiple copies of complementary DNA (cDNA). The First step was involved the isolation of total RNA from MNCs as both 5-LOX and 15-LOX activity is enhanced in peripheral blood MNCs. The purity and integrity of total RNA was checked by nanodrop method (ratio of RNA) and further confirmed by the appearance of sharp band on agarose Gel (data not shown). The good quality total RNA was further used for the synthesis of complementary DNA using Galaxy XP Thermal

6 Cycler (Bioer, PRC). Finally multiple copies were produced from cDNA using Mic PCR (Bio Molecular System). The effect of inhibitors on the expression level of LOX enzyme was observed through the Livak method [31]. The results showed that the potent NSAIDs also influenced the expression level of the targeted enzyme (Table 2). Naproxen was identified as the most potent moiety to down-regulate the targeted enzyme followed by Aspirin, Ibuprofen and Acetaminophen. The results were further confirmed by molecular docking studies.

Table 2 Relative expression of 5-LOX after treatment with different molecules.

MNCs Sr. No. Compound Total Expression (fold change) 1 Ibuprofen 0.00033

2 Aspirin 0.00028 3 Acetaminophen 0.00077

4 Naproxen 0.00013

Control Quercetin 0.00024

2.2.2 Flow cytometry analysis of treated MNCs The toxic effects of selected NSAIDs, i.e., Ibuprofen, Naproxen, Aspirin, Acetaminophen and Quercetin were evaluated on MNCs using FITC-Annexin V and PI method (FITC-Annexin V Apoptosis detection kit with PI-8230 from BioLegend). It facilitated the quantification of cell damage (quadrant 1) and the identification of late apoptotic cells/necrotic cells (quadrant 2), viable cells (quadrant 3) and early apoptotic cells (quadrant 4). The analysis was carried out at the two different concentrations, i.e., at IC50 values and at x 2 of IC50 values obtained through LOX enzyme inhibition assay (Fig. 1). These drugs showed differential effects on cell viability and results were compared with negative control. The total 10,000 events were observed for the determination of viability and death of MNCs.

7 Fig. 1. Cytotoxic effect of NSAIDs on MNCs. Typical dot plots showing apoptosis ratio comparing negative control (A, B) with Ibuprofen at 14 µM and 28 µM (C, D) Naproxen at 3 µM and 6 µM (E, F), Aspirin at 4 µM and 8 µM (G, H), Acetaminophen at 6 µM and 12 µM (I, J) and Quercetin at 4 µM and 8 µM (K, L) using PI and FITC-Annexin V. The quadrant 1 represents damaged cells (PI positive and Annexin negative), quadrant 2 represents cell that are in late apoptosis or already dead (both Annexin and PI positive), quadrant 3 represents viable cells (both Annexin and PI negative) and quadrant 4 represents cells in early apoptosis (cell apoptosis) Annexin positive and PI negative.

The obtained results supported the enzyme inhibition data and expression analysis as MNCs express both 15-LOX and 5-LOX and the cell death by the selected drugs showed dose dependent effect; low toxicity at low concentration and high toxicity at high concentration (Table 3). After differentiating between cells of MNCs by gating, it was found that apoptosis levels were different in lymphocytes after treatment with different drugs. Our results suggest that the selected

8 NSAIDs do not affect the cytotoxic activity towards MNCs at IC50 concentrations. MNCs apoptosis increased only at the higher concentrations. The percentage of dead cells (upper left quadrant) increased when Ibuprofen concentration was doubled from 14 µM (0.95) to 28 µM (21.99%) during the assay compared with other moieties. The percentage of living cells decreased more pronouncedly in Aspirin and Acetaminophen compared with Ibuprofen and Naproxen when

drug concentration was doubled from IC50 value concentration. Appreciable decrease in early apoptotic cells was noticed in Ibuprofen (from 6.15% to 1.33%), in Aspirin (7.48 to 20.40%) compared with Acetaminophen where no change was observed. However, increase in Naproxen resulted in increased percentage of number of early apoptotic cells (from 22.09 to 31.714%). When cells were monitored in upper right quadrant with late apoptotic stage, decrease in percentage of cells was observed on increased drug concentration only in Ibuprofen whilst in all other drugs increased drug concentrations resulted in increased percentage of cells in their late apoptotic stage. The exact mechanism of this differential behavior of drugs is not known but it is speculated that this data demonstrates that these drugs may not induce loss of immunity in septic as well as other inflammatory conditions, providing that these are acceptable inhibitory concentration values.

Table 3 Cytotoxic effects of NSAIDs on MNCs at two different concentrations.

Ibuprofen Naproxen Aspirin Acetaminophen Quercetin Percentage Negative per control quadrant (% cells) (% cells) (% cells) (% cells) (% cells) (% cells) 14 µM 28 µM 3 µM 6 µM 4 µM 8 µM 6 µM 12 µM 4 µM 8 µM Upper Left 0.90 21.99 0.10 0.19 6.55 7.12 1.04 4.68 3.98 5.81 0.80 dead cells Upper Right late 27.48 18.82 1.01 3.05 10.40 45.45 1.05 32.30 10.9 28.59 1.23 apoptotic cells Lower Left 65.39 56.04 76.73 67.15 75.57 27.43 67.35 36.36 80.82 39.83 94.85 living cells Lower Right early 6.15 1.33 22. 09 31.74 7.48 20.40 26.06 26.67 4.26 25.77 3.12 apoptotic cells

9 2.3 Molecular docking studies

Baicalein is a standard LOX inhibitor; therefore, its docking studies were also carried out. It was found to bind in major binding pocket (having 51 amino acid residues; Fig. 2).

Fig. 2. Baicalein of soybean 15-LOX (PDB ID: 3pzw).

All compounds were docked in the Baicalein binding site (Fig. 3). For each compound, a total of 10 docking poses were generated. Each of the docked pose was then scored using HYDE utility of LeadIT software suit (HYDE gives an estimate of binding free energy ΔG, by considering bound and unbound states of the ligand), after which the most stable docked conformation was selected (Table 4-5).

10 Fig. 3. Overlap of all docked compounds, standard inhibitor Baicalein is displayed in pink color; Fe is displayed as a sphere.

Table 4 Estimate of binding free energy (ΔG) by HYDE affinity, by considering bound and unbound states of the ligand.

No. Name Free binding energy (ΔG) (kJ mol-1) 1 Naproxen -24 2 Aspirin -19 3 Quercetin -27 4 Acetaminophen -15 5 Piroxicam 2 6 Ibuprofen -16 7 Ketoprofen -20 8 Diclofenac -11

11 9 Mefenamic acid -6 10 Tenoxicam -8

Table 5 Hydrogen bonded interactions of 15-LOX inhibitors.

15-LOX Interacting amino acid residues Inhibitors H-Bond donors H-Bond acceptors

Acetaminophen Gly247 (NH), Arg533 (NH2) Asp243 (-C=O) Aspirin Asp768 (NH), Asn769 (NH), Asn128 - (NH2) Ibuprofen Arg533 (NH2) -

Diclofenac Lys110 (NH2), Asn769 (NH2), Asn128 Asp768 (-COO) (NH2) Ketoprofen Asp768 (NH), Asn769 (NH), Arg533 - (NH2) Mefenamic acid Arg533 (NH) Gly247 (-C=O) Naproxen Asp768 (NH), Asn534 (NH) -

Tenoxicam Asn128 (NH2), Tyr525 (OH) Asp768 (-COO), Asn128 (- C=O) Piroxicam Asn128 (NH2), Tyr525 (OH) Asp768 (-COO)

Quercetin Arg533 (NH2), His515 (NH) Asp768 (-COO), Glu244 (- COO), Asp243 (-C=O)

Fig. 4 shows binding site interactions of the most stable docked conformations for all 15- LOX inhibitors studied herein. Some of the common interacting amino acid residues were Arg533, Asp768, Asn128, Asn769 and Gly247. The carboxylate groups of Ibuprofen, Ketoprofen, and Mefenamic acid exhibited hydrogen bond formation with Arg533, where Arg533 was acting as a hydrogen bond donor. Acetaminophen and Quercetin also exhibited hydrogen bond formation with Arg533 though they did not contain carboxylic acid groups, wherein these molecules the oxygen atom of the –OH group acted as a hydrogen bond acceptor towards Arg533 (which was again found to be acting as a hydrogen bond donor).

The interaction with Asp768 appears to be of particular significance for 15-LOX inhibitors. Aspirin, Diclofenac, Ketoprofen, Naproxen, Quercetin, Piroxicam and Tenoxicam all indicated hydrogen bond formation with Asp768. For Aspirin, Ketoprofen and Naproxen Asp768

12 acted as a hydrogen bond donor towards carboxylate group of Aspirin, carbonyl oxygen of Ketoprofen, and methoxy group of Naproxen, whereas, in Diclofenac, Piroxicam and Tenoxicam, Asp768 acted as a hydrogen bond acceptor. The carboxylate group of Asp768 made hydrogen bonded interaction with NH group of Diclofenac, Piroxicam and Tenoxicam. Asp768 acted both as a hydrogen bond acceptor (via its carboxylate group) and as a hydrogen bond donor (via its NH group), towards OH groups substituted at the phenyl ring of Quercetin.

Piroxicam and Tenoxicam were the only sulfonamide containing drugs studied that indicated similar binding orientations, the latter being the least active 15-LOX inhibitor. For both these molecules, the sulfonamide oxygen atom was found to be acting as a hydrogen bond acceptor towards Tyr525. It is interesting to note that Tyr525 did not exhibit any interaction with all other 15-LOX inhibitors studied herein. The NH and OH groups of Piroxicam and Tenoxicam were making hydrogen bonded interactions with Asp768 and Asn128. It is suggested that the least inhibitory activity of Tenoxicam may be due to the hydrogen bond donor (via –NH group) as well as hydrogen bond acceptor (via –C=O) properties of Asn128.

13 Fig. 4. Binding site interactions of docked conformations of 15-LOX inhibitors.

To find a plausible reason for binding affinity of 15-LOX inhibitors studied herein, SeeSAR analysis of the most active Naproxen and the least active Tenoxicam along with Piroxicam was carried out. SeeSAR provides visual display of binding affinity. The structural features of the ligand that are contributing positively to the overall binding affinity are indicated with green colored coronas; greater the contribution, larger is the size of the corona. Similarly, the structural elements that are not contributing favorably to the overall binding are indicated with red colored coronas, whereas the structural features with no contribution are not colored. Fig. 5 shows SeeSAR visualization of the most effective inhibitor, Naproxen. As can be seen, most of the atoms in the molecule are contributing favorably to overall binding (indicated by green colored coronas), only two structural elements, i) a carboxylate oxygen, and ii) methyl carbon are not contributing favorably (indicated by red colored coronas) because of high desolvation energy.

14 Fig. 5. SeeSAR analysis of Naproxen. The structural elements that are contributing favorably to the overall binding affinity are represented with green colored coronas, structural elements that with unfavorable contribution are represented with red colored coronas, neutral elements are not colored.

Binding site interactions of Naproxen are presented in Fig. 6. The methoxy oxygen was making a hydrogen bond with Asp768, and the carboxylate group was making hydrogen bond with Asn534. Other non-bonded interactions such as pi-anion and pi-alkyl interactions were also observed with amino acids Asp768, Arg533 and Pro530.

Fig. 6. Binding site interactions of Naproxen.

15 Piroxicam and Tenoxicam have identical structures, with only one difference; the phenyl ring in Piroxicam is replaced with a thiophene ring in Tenoxicam. The detailed SeeSAR analyses attempts to elaborate why this seemingly small change has a profound effect on the binding affinities of the two molecules was carried out. The HYDE binding affinity for Piroxicam is quite poor (2 kJ mol-1) indicating that it is not an effective 15-LOX inhibitor, whereas for Tenoxicam, it is comparatively better (-8 kJ mol-1) though still not as good as that observed for other inhibitors. Red colored coronas (indicating unfavorable interactions) were observed for the nitrogen atom of pyridine ring, the oxygen atom of the amide carbonyl group and two carbon atoms of phenyl ring. As can be seen from Fig. 6-7, there is very high desolvation energy for all above mentioned atoms, the desolvation penalty is not compensated in the form of any hydrogen bonded interaction, even though there are at least three unsatisfied non-bonded interactions (Fig. 7-8).

Fig. 7. SeeSAR Analysis of Piroxicam. The structural elements that are contributing favorably to the overall binding affinity are represented with green colored coronas, structural elements that with unfavorable contribution are represented with red colored coronas, neutral elements are not colored.

16 Fig. 8. Binding site interactions of Piroxicam. Three unsatisfied non-bonded interactions (encircled in black color) can be seen for the nitrogen atom of pyridine ring, the oxygen atom of the amide carbonyl and sulfonamide oxygen atom.

When we compared the SeeSAR analysis of Piroxicam with Tenoxicam, the nitrogen atom of the pyridine ring had high desolvation energy. The replacement of phenyl ring of Piroxicam with thiophene in Tenoxicam resulted in alleviation of high desolvation energy penalty which was previously observed for Piroxicam, only one of the ring carbon atoms now showed (slight) desolvation energy of 0.7 kJ mol-1 (Fig. 9).

Fig. 9. SeeSAR Analysis of Tenoxicam. The structural elements that are contributing favorably to the overall binding affinity are represented with green colored coronas, structural elements that

17 with unfavorable contribution are represented with red colored coronas, neutral elements are not colored. Binding site interactions of Tenoxicam revealed two unsatisfied non-bonded interactions (encircled in black color) that can be seen for the nitrogen atom of pyridine ring, the oxygen atom of the amide carbonyl (Fig. 10); these observations are similar to those observed for Piroxicam.

Fig. 10. Binding site interactions of Tenoxicam. Two unsatisfied non-bonded interactions (encircled in black color) can be seen for the nitrogen atom of pyridine ring, the oxygen atom of the amide carbonyl. 2.4 Electrostatic potential (ESP) analysis

To further analyze and relatively rank the electrostatic interactions responsible for the binding of each ligand in the binding pocket, ESP maps were obtained from DFT calculations. Fig. 11 shows that the deprotonated carboxylic group of Aspirin is the most negative part of the molecule. The plot further indicates that the carboxylate group is significantly more negatively charged than the ester oxygens, which establishes that the H-bonding of former with Asp768 -NH group is the primary interaction that holds Aspirin ion in the binding pocket of LOX. Similarly, Ibuprofen has only one strong interaction site associated with the deprotonated carboxylic group. However, ESP plot indicates that terminal aliphatic hydrogens are positively charged, which can also form non-covalent interactions with Pro530. All the NSAIDs calculated below have a common feature in them. Their ESP surface is polarized and contains a positive and a negative binding site.

18 Fig. 11. Optimized geometries and corresponding ESP maps plotted on 0.001 e/bohr3 electron density isosurface obtained from the B3LYP/6-311+g(d,p) level of DFT calculations. 2.5 Frontier molecular orbital studies DFT calculations were performed with an aim to have a graphical representation of the highest occupied molecular orbitals (HOMO), lowest unoccupied molecular orbitals (LUMO) and other structural features of drugs (Fig. 12, Table 6). The HOMO and LUMO frontier orbitals are associated to the molecule’s reactivity while their energies are related to ionization potential and electron affinity, respectively [32]. HOMO energy is closely related to susceptibility to electrophilic attack while LUMO energy is closely related to susceptibility to nucleophilic attack. The graphical representation of HOMO and LUMO is given in Fig. 10 while the energies and the energy gap has been tabulated in Table 6. The HOMO of Piroxicam, Tenoxicam and Quercetin are spread over the whole molecule surface while the LUMO in Piroxicam and Tenoxicam are delocalized around the sulfonamide ring section while in Quercetin it is delocalized over the whole molecular surface. In the next group, Ibuprofen, Mefenamic acid, Aspirin, Diclofenac, and

19 Ketoprofen, HOMO is delocalized over the carboxylate moiety and the neighboring aromatic ring while their LUMO are delocalized over aliphatic side chains. The LUMO in Naproxen is mainly spread over the aromatic rings. The delocalization of frontier molecular orbitals and their mutual energy gaps conform to their electrostatic potential surfaces and their proposed binding affinities.

Table 6. Energies of HOMO and LUMO orbitals and their gap calculated in gas phase at B3LYP/6-311+G(d,p) level of DFT calculations. Name HOMO (eV) LUMO (eV) Gap (eV) Acetaminophen -5.919 -0.688 5.231 Aspirin -2.041 2.623 4.665 Diclofenac -2.182 1.661 3.843 Ibuprofen -1.594 1.937 3.531 Ketoprofen -2.116 0.688 2.804 Mefenamic Acid -2.063 2.027 4.090 Naproxen -1.672 1.545 3.217 Piroxicam -6.449 -2.401 4.047 Quercetin -5.994 -1.784 4.211 Tenoxicam -6.592 -2.644 3.948

20 Fig. 12. The alpha HOMO and LUMO orbitals diagrams at B3LYP/6-311+G(d,p) level of DFT calculations (Isovalue = 0.02 e/bohr3).

21 3 Conclusion

The present study reveals that many LOX active compounds might be routinely missed during the screening assays by the less sensitive screening methods as NSAIDs have shown their potential as LOX inhibitors. These studies are confirmed by expression and apoptotic analyses supported by the docking studies. The in vitro inhibitory potential of the most active Naproxen and the least active Piroxicam and Tenoxicam is in agreement with the docking studies. These findings are further complemented by the electrostatic potential maps obtained from DFT calculations. ESP maps reveal that on all structures, there existed two potential binding sites dominated by most positive and most negative regions. In conclusion, this study demonstrated that the selected molecules, at the identified inhibitory concentrations against soybean 15-LOX exhibited down regulation of 5-LOX/15-LOX in MNCs. The flow cytometric results suggested that these drugs did not alter the extent of apoptosis of MNCs and maintained their cytotoxic potencies and therefore may not affect the normal cells of immune system. These results suggest the strong indications that these molecules should be explored for their further pharmacological studies.

4 Experimental

4.1 Chemicals and reagents

Lipoxygenase (15-LOX, EC 1.13.11.12) type 1-B from Glycine max having 66 mg solid; 221700 units/mg solid with activity ≥ 50,000 units/mg, Cat No. 7395, was purchased from Sigma- Aldrich Co. St. Louis, Mo. USA. Similarly, Tween-20, Linoleic acid, Arachidonic acid, Quercetin, Baicalein, Luminol and Cytochrome C were also purchased from Sigma-Aldrich. Assay kits were purchased from Cayman Chemicals, USA, Cat No. 760700. All NSAIDs were 97.5 – 98.5% pure except Tenoxicam that was 96.5% pure and these drugs were a kind gift from Punjab Drug Testing Laboratory, Lahore, Pakistan. All the chemicals and solvents were of analytical grade and were used without further purification.

4.2 Enzyme inhibition assays

4.2.1 UV-absorbance method

Lipoxygenase assay was carried out by slight modifications in the previously reported method [23]. The assay was based on the principle that in the end of reaction, the absorbance was

22 due to the formation of 5(S)-hydroperoxy-6-trans-8,11,14-cis-ecosatetraenoic acid (HPETE) by the reaction of enzyme with linoleic acid used as substrate. Linoleic acid was used as substrate with Tween-20 in 2:1 ratio instead of 1:1 [33, 34]. Stock solution of the substrate was prepared by the addition of 257 µL (280 mg) of Tween-20 and 155 µL (140 mg) of linoleic acid in 5 mL of deionized water. The mixture was emulsified by drawing back and forth and clarified by the addition of 0.6 mL 1N NaOH. This solution was further diluted to the final volume of 20 mL which was flushed with nitrogen gas to avoid the substrate from oxidation. Substrate was stored at -20°C. A total of 200 µL assay volume was used for the determination of LOX activity. To each well of 96 UV plate, 160 µL of 100 mM, pH 8.0 potassium phosphate buffer, 10 µL of test compound or solvent and 10 µL (210 units, after optimization of enzyme concentration) of LOX enzyme. The contents were mixed and reaction mixture was pre-incubated for 5 min at 25°C and then pre-read at 234 nm by BioTek HTX 96-well plate reader. Then to each well, 20 µL of substrate solution was added to initiate the reaction. The change in the absorbance was monitored after 10 min of incubation. All the reactions were carried out in triplicates and mean of these values was reported with standard error of mean in all assays. Quercetin / Baicalein were used as positive controls. The percentage inhibition was calculated using following formula. Inhibition (%) = (Abs of control – Abs of test comp / Abs of control) x 100 The active compounds were serially diluted and their percentage inhibitions were determined. This data was used for the computation of IC50 using Ez-Fit software (Perrella Scientific Inc. Amherst, USA).

4.2.2 Colorimetric method

Colorimetric method was used for the determination of LOX inhibitory activity as per instructions of the manufacturer (Cayman Chemical Cat No. 760700; Ann Arbor, Michigan 48108 USA). The reaction mixture of 100 µL contained 100 mM Tris HCl, pH 8.0, 10 µL test compound or solvent and 10 µL of soybean LOX enzyme. The contents were incubated for 5 min, shaken and pre-read at 500 nm. Substrate solution of linoleic acid or arachidonic acid 25 µL was added and contents incubated for further 10 min followed by addition of 40 µL given chromogen solution. The color was read after 5 min. Percent inhibition was calculated as mentioned above in UV absorbance method [35].

23 4.2.3 Chemiluminescence method

A previously reported chemiluminescence method [29] was slightly modified, optimized and used for the identification of inhibitors. A total volume of 100 µL contained 60 µL of borate buffer (200 mM, pH 9.0), 10 µL test compound or solvent and 10 µL soybean LOX enzyme. This mixture was incubated at 25oC in dark for 5 min. Then 10 µL solution of luminol (3 nM) containing cytochrome C (1 nM) was added to each well and luminescence was measured by BioTek HTX 96-well plate reader in luminescence mode. The reaction was initiated by the addition of 10 µL substrate solution (prepared as mentioned above in UV-absorbance method). Chemiluminescence was measured for 100 to 300 seconds. All experiments were performed in triplicates. The positive and negative controls were also included in the assay. The percentage inhibition and IC50 values were calculated as mentioned above for UV-absorbance method.

4.3 Polymerase chain reaction based assays

4.3.1 Isolation of MNCs

Blood (3 mL) was collected in an EDTA tube from the healthy volunteer donors with their consent, which was diluted with PBS (phosphate buffer saline, 50 mM, pH 7.4) at 1:1 ratio. An equal volume of lymphocyte separation medium (density 1.077 g/mL, at 20˚C, Cat. No. L0560 Lymphosep, Biowest, USA) was added in 15 mL Falcon tube. The diluted blood was then loaded onto the surface of the medium slowly to avoid the mixing of blood in the medium. The contents were centrifuged at 1200 × g for 20 min at 20˚C using a swing rotor. The ring of MNCs was collected at the interface of lymphocyte separation medium and plasma by a Pasteur pipette. Collected cells were washed twice with 10 mL PBS 20˚C at 300 × g. The final pellet obtained was re-suspended in 0.5 mL PBS. RBCs and platelet pellets were discarded and MNCs were washed with PBS and their total volume was maintained to the required concentration.

4.3.2 Extraction of total RNA from MNCs

The total RNA was extracted from by trizol method, with slight modification in the previously reported method [36]. The cells (0.5×106) after treatment with inhibitors at 3 folds of their respective inhibitory concentration values obtained from chemiluminescent assay were used in the assay. The cells were suspended in phosphate buffer saline (PBS) and 1 mL of trizol was added in each tube to rupture the cell membrane surface and homogenate was made by gently

24 inverting tubes for several times. After incubation of 5 min at room temperature with occasional pipetting up and down, the trizol solution was removed and transferred to another RNase free eppendorf tube. Then 400 µL of chloroform was added to the same eppendorf tube and incubated for 3 min at room temperature with occasional mixing. After incubation, tubes were centrifuged at 12,000 rpm for 10 min at 4oC for phase separation. During this centrifugation step, new microfuge tubes were labeled and placed in ice box. Three layers were formed in the tube with upper aqueous layer containing RNA and lower pink layer containing protein. Between these two layers, DNA was present. The aqueous upper layer was transferred in new 1.5 mL tube placed on ice and isopropanol was added in equal ratio. The tubes were incubated on ice for 10 minutes in horizontal position to precipitate down RNA. After that, the tubes were centrifuged for 10 min (4oC) at 12,000 rpm. Finally the supernatant was removed from RNA pellet and 1 mL of ethanol was added along with incubation of 5 min at 4oC, to remove the impurities. Then the tubes were centrifuged at 7500 rpm for 5 min, followed by the careful removal of ethanol without disturbing RNA pellet. The RNA pellet was completely dried by placing in heat block at 70oC for 2 min. Finally 40 µL of RNase free water was added and again place in heat block at 60oC for 5 min, to completely dissolve the pellet. The total RNA was placed at -80°C until downstream application.

4.3.3 Quantification of total RNA

RNA quality and quantity were assessed by using Nanodrop plate (Skanit RE 4.1, Thermo Scientific). Absorbance was measured at 260, 280 and 320 nm. The 260/280 ratio was ranging from 1.9 to 2.2 confirming high quality RNA whereas RNA quantity was nearly 800 to 1200 ng/µL. The 260/280 ratio and RNA concentration of each sample is given in the supplementary information STable 1.

4.3.4 Synthesis of cDNA The complementarity DNA (cDNA) was synthesized from total RNA using cDNA synthesis kit (Vivantis cDSK01-050). The 7 µL of total RNA (depending upon concentration of RNA for each sample) was used for the synthesis of cDNA. The procedure involved the mixing of total RNA (7 µL), primer (1 µL; 4 µM), dNTPs (1 µL; 1 mM) and NF H2O (1 µL) in a PCR tube. The tube was short spinned in microfuge machine and placed in PCR machine at 65oC for 5 min. Then the tubes were chilled on ice for 2 min. Then the addition of M-MuLV Reverse

Transcriptase (0.2 µL; 100 U), M-MuLV buffer (2 µL; 2X) and NF H2O (7.8 µL) was carried

25 out. Finally the tubes were placed in PCR machine at 42oC for 60 min and then termination of reaction was carried out by placing tubes at 85oC for 5 min. At the end, the tubes were chilled on ice and were centrifuged briefly. The synthesized cDNA can be directly used for downstream application or stored at -20oC.

4.3.5 Primers design

Four primer sets were used to optimize the annealing temperature (Ta). Sequences of forward and reverse primers are as given in the supplementary information STable 2.

4.3.6 PCR reaction mixture

The chemicals including template cDNA, forward & reverse primers, taq polymerse enzyme 5U/ µL (Thermo Scientific), PCR buffer, dNTPs, MgCl2 and PCR water were used at available concentrations as mentioned in the supplementary information STable 3.

4.3.7 Amplification of cDNA by Galaxy XP thermal cycler

Polymerase chain reactions were performed on a Galaxy XP Thermal Cycler (Bioer, PRC). Optimized PCR conditions were used and were shown in the supplementary information STable 4.

4.3.8 Real Time - PCR

RT-PCR is a molecular biology technique which is carried out in a thermal cycler with the capacity to illuminate each sample with a beam of light of at least one specified wavelength and detect the fluorescence emitted by the excited fluorophore. The chemicals; template cDNA, forward and reverse primer, 2X Syber Green (intercalating dye) (Bilrt, Germany) and nuclease free water were used for the reaction. All of the optimized concentrations are shown below in supplementary information STable 5.

Real time polymerase chain reactions were performed on Mic PCR (Bio Molecular System) with the conditions given in supplementary information STable 6.

26 4.4 Flow cytometry analysis

The cytotoxic behavior of the selected NSAIDs on MNCs was carried out by flow cytometric analysis using Annexin V-FITC and Propidium Iodide (PI). The suitable dilutions of the NSAIDs were made at concentration of their respective IC50 values. The two different concentrations (IC50 value and x3 of IC50 value) of each selected drug was incubated with MNCs for 45 minutes along with the negative controls. The staining MNCs (1× 106 cells) were transferred to FACS tubes. The Annexin V-FITC (10 µL) was added to the tubes followed by the addition of 5 µL PI and contents were vortexed for few seconds. Then cells were incubated at room temperature for 15 min and Annexin V binding buffer (400 µL) was added. The stained cells were analyzed on BD FAC Scan (USA) flow cytometer. The data was analyzed using the BD FAC scan software and through Microsoft Excel. The experiments were performed in triplicate [37].

4.5 Molecular docking studies

The crystal structure of LOX enzyme from soybean (Glycine max, PDB id: 3pzw, 1.4 Å) was downloaded from the PDB and was prepared using DockPrep utility of Chimera [38], whereby hydrogen atoms are added, all solvent (water) molecules are deleted, and selenomethionine moieties (if present) are converted to methionine. Similarly, incomplete side chains (if any) are fixed or replaced using Dunbrack Rotamer Library (as embedded in Chimera), and charges are added using AMBER ff14SB. BioSolveIT’s LeadIT software was used for docking studies [39]. The iron atom in LOX enzyme is high-spin Fe (II) in in-active form, whereas, in catalytically active form, it is high-spin Fe(III). The Fe atom is highly speculated to be in (distorted) octahedral coordination environment, with five sites occupied by amino acid chains, and water or a hydroxide ion occupies the sixth site. BioSolveIT’s SeeSAR software was used to identify all unoccupied binding site pockets in the crystal structure of LOX [40].

4.6 Density functional theory (DFT) calculations

All gas-phase geometry optimization calculations were carried out using the Gaussian 16 [41] suite of programs at B3LYP/6-311+g(d,p) level. The energy of frontier molecular orbitals

(EHOMO and ELUMO) and the Mullikan charge distribution on the molecular structures was determined using this method. Tight convergence criteria were adopted to ensure the global

27 minimum upon structure optimization. The Gaussian formatted checkpoint files were used to obtain electron density and electrostatic potential (ESP) grid data in the cube file format using the Multiwfn program [42]. The same program was used to perform the quantitative analysis of the molecular surfaces. The obtained ESP maps were used to color the electron density isosurface to distinguish the acidic and basic regions on the surface of molecules using the GaussView 6 program.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

The work was funded to M.A. by Higher Education Commission, Pakistan vide NRPU Project 4950. W.S. worked as Research Assistant for her PhD studies. Thanks are due to Dr Munawar Hayat, Punjab Drug Testing Laboratory Lahore, Pakistan, who provided these drugs for the present studies.

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The Islamia University of Bahawalpur Department of Chemistry Dated: 24.12.2020

The Editor Bioorganic Chemistry

Subject: Declaration of Interest Statement

It is certified that all the authors related to submission of Manuscript entitled, ‘Identification of NSAIDs as lipoxygenase inhibitors through highly sensitive chemiluminescence method, expression analysis in mononuclear cells and computational studies’ are agreed with the contents, the work included in the manuscript, have read the whole manuscript. They declare no financial interests.

Prof Dr Muhammad Ashraf Department of Chemistry The Islamia University of Bahawalpur Bahawalpur-63100, Pakistan. Cell: 0301-773-6059 Email: [email protected]

Identification of NSAIDs as lipoxygenase inhibitors through highly sensitive chemiluminescence method, expression analysis in mononuclear cells and computational studies

Wardah Shahida, Syeda Abida Ejazb, Mariya al-Rashidac, Muhammad Saleema,

32 Maqsood Ahmeda, Jameel Rahmana, Naheed Riaza, Muhammad Ashraf a* a Department of Chemistry, The Islamia University of Bahawalpur, Bahawalpur-63100. Pakistan. b Department of Pharmaceutical Chemistry, Faculty of Pharmacy, The Islamia University of Bahawalpur, Bahawalpur-63100. Pakistan. c Department of Chemistry, Forman Christian College (A Chartered University), Ferozepur Road, Lahore-54600. Pakistan.

NSAIDS as lipoxygenase inhibitors: Overlap of all docked compounds, standard inhibitor baicalein is displayed in pink color and Fe as a sphere.

Reply to Reviewers Comments

Reviewer #2: Manuscript # BIOORG-D-20-01600 by Shahid et al. is an interesting work that might reflect on the selection of the proper biological evaluation method of anti-lipoxygenases. The success of chemiluminescence method to report inhibitory activity which is supported by expression analysis while UV and colorimetric methods failed, is an important outcome. The conducted in silico studies provide further insights. In my humble opinion, this manuscript might be accepted for publication after revising the following points: Comments

Query 1. According to the manuscript, baicalein is a standard inhibitor that was also used to

33 define the binding site during the docking study. However, in Table 1, it was not included in colorimetric and chemiluminescent assays but only in the insensitive UV assay. It should be included also in other assays. Please conduct in colorimetric and chemiluminescent assays for the standard baicalein and include in the Table. Response: Due to Covid-19 crises, Baicalein could not be purchased from Sigma or other companies because of shipment problems and its IC50 values could not be determined by colorimetry and chemiluminescence methods. However, its IC50 value by UV-method was previously determined in our laboratory and reproduced here and the same is confirmed by molecular docking studies and included in the present work. Because of comparable results, quercetin has been used as positive control. As soon as Baicalein is received from Sigma, we will include its inhibition data in our upcoming publications.

Query 2. The experimental section is NOT the appropriate section for a figure of chemical structures of compounds. Please move the figure to appropriate section such as the introduction. Response: Corrected as suggested.

Query 3. Also the experimental section is NOT the appropriate section for a figure and discussion of how the binding site was identified. Only practical steps should be included while the figure and the discussion should be moved to results and discussion sections. Response: Corrections have been made in the revised manuscript as suggested.

Query 4. Table 4, it is wrong to write "HYDE affinity" it is in fact "free binding energy (ΔG)" which was calculated by HYDE. Please correct. Response: Corrected as suggested.

Query 5. No Tables should be in experimental section. Move all of these tables to supporting information. Response: All experimental tables have been shifted to the Supplementary Work file as suggested.

Query 6. Overlayed ligands in Fig. 2 are very small and unclear. Please modify the figure and enlarge the region showing the overlayed docked ligands. Response: Modified figure as per suggestion.

If there are further queries, I would be happy to reply. Thanks.

34 Prof Dr Muhammad Ashraf

35