[3] Identification of anti-inflammatory compounds present in Nigella sativa and analyzing their effects on the inflammation pathway using in silico techniques ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE IV This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. IDENTIFICATION OF ANTI-INFLAMMATORY COMPOUNDS PRESENT IN NIGELLA SATIVA AND ANALYZING THEIR EFFECTS ON THE INFLAMMATION PATHWAY USING IN SILICO TECHNIQUES HABIBA ABDELHALIM SONIA ARORA, PHD (FACULTY ADVISOR) ✵ ABSTRACT Nigella sativa, also known as black cumin, is an herb native to Asia, the Middle East, and the Mediterranean. The chemical constituents found in Nigella sativa seeds have been known to have a wide array of pharmacological actions. Previous studies have primarily focused on identifying the structures of these compounds and their medicinal properties based on physiochemical analysis. There have been few studies that explain how these com- pounds interact with enzymes found in the human body. Hence, the objective of this study was to iden- tify anti-inflammatory compounds present in Nigella sativa from previous literature and see if these com- pounds can be used to target the cyclooxygenase- 2 inflammatory pathway. We compiled a list of twenty-three chemical compounds present in Ni- gella sativa and then constructed a comprehensive molecular database of their three-dimensional structures using chemical modeling approaches. We then docked these compounds into two differ- ent cyclooxygenase-2 molecules, using in silico techniques, to observe their conformation as well as binding affinity. Furthermore, we analyzed the inter- action patterns of the five most stable compounds to understand their effects on the cyclooxygenase binding pocket. In conclusion, using structural bio- informatics approaches, we have identified novel compounds derived from Nigella sativa that can be used as possible agents to target inflammation. 1 INTRODUCTION Ethnobotany is the study of traditional cus- toms observed by humans in relation to plants re- garding their medicinal, nutritional, and religious values1. Historically, plants and the natural com- pounds they produce have been used differently by different societies; thus ethnobotany has a wide ar- ray of applications ranging from plant dyes to guid- ing drug discovery and development. Previously, researchers have collected plant samples and per- formed phytochemistry studies that would later be used in clinical studies. Although synthetic bio- chemistry has greatly reduced the need to use nat- ural compounds derived from plants, ethnobotani- cal studies have been of extreme academic interest in recent years2. This interest stems from the fact that more plant-derived drug interactions have been ob- served as traditional and herbal medicines become more popular. As of 2019, the World Health Organ- ization reports that about 80% of the world popula- tion relies on traditional medicine3. Consequently, it is important to have clear regulations on these types of conventional medicine. Studying the possible in- teractions these natural compounds have on mole- cules and enzymes in the human body is the first step in establishing these guidelines. Nigella sativa, also known as black cumin or black seed, is a plant native to Eastern Europe and Western Asia. It has also naturalized over to North Africa and parts of the Middle East4. The seeds of Nigella sativa have been used in traditional Islamic and Ayurvedic medicine for generations5. Tradition- ally, it is used postpartum to aid with lactation and menstruation5. It is also thought to have a wide array of pharmacological actions including anti-microbial, antioxidant, anti-hyperlipidemic, and the focus of our research, anti-inflammatory action5. Several ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE IV chemical compounds present in Nigella sativa, namely thymoquinone, can reduce asthma symp- toms and treat rheumatoid arthritis by targeting the inflammatory pathway5. Inflammation is a biological immune re- sponse that can be triggered by many external fac- tors such as bacteria, viruses, and foreign bodies. Pain, swelling, redness and warmth are some of the symptoms associated with an inflammatory re- sponse. On the cellular level, cytokines, small cell- signaling proteins, are released to recruit other im- mune cells to the site of infection. Vasodilation and increased permeability allow other signaling mole- cules to diffuse across to address the inflammation. In normal circumstances, an acute defense mecha- nism is employed to protect cells and eliminate en- dogenous compounds. However, uncontrolled in- flammatory responses may become chronic leading to extreme pain and, in extreme cases, tissue death6. There are typically two routes by which in- flammation can proceed. One includes cytokine se- cretion due to the recognition of pathogen-associ- ated molecular patterns (PAMPS) by toll-like recep- tors (TLRs); the other is bradykinin synthesis, which triggers prostaglandin formation and leads to vaso- dilation7. We focus on the latter pathway here. Ara- chidonic acid found in poultry and meat is con- verted by cyclooxygenase-1 and 2 (COX-1 and COX-2) into prostaglandins. These two enzymes can be targeted by traditional non-steroidal anti-inflam- matory drugs (NSAIDs). Since COX-1 is an important enzyme for gastric protection and platelet function, selective NSAIDs need to target only COX-2, which is responsible for pain and inflammation8. The ob- jective of this research is to create a comprehensive database of chemical constituents present in Nigella sativa and analyze their interaction with COX-2 us- ing in silico docking methods. 2 METHODOLOGY CONSTRUCTION OF MOLECULAR DATABASE FOR NIGELLA SATIVA CONSTITUENTS PubChem and PubMed were used to com- pile the database. PubChem is a database that pro- vides information on chemicals and their activity. PubChem Compound, one of PubChem’s inter- linked databases, shows a wide array of compounds and uses another database, PubChem Bioassay, to compile each chemical’s information9. PubMed houses an extensive number of biomedical journals and peer-reviewed literature. In addition, it provides access to related entries in other National Center for Biotechnology Information (NCBI) databases10. We used PubMed to retrieve the structure of the chem- ical compounds from scientific literature and com- pile them into an Excel spreadsheet. After recording twenty-three different chemical compounds, their chemical structures were downloaded as Standard Delay Format (SDF) files from PubChem. The Pro- tein Data Bank (PDB), which is a database containing 3D structures of biological molecules, was then used to obtain three different COX-2 files with inhib- itors in their binding pockets. The three targets were 5F1A, 5IKT, and 5KIR with salicylate acid, tolfenamic acid, and Vioxx as the inhibitors, respectively. The three files used differed only in the type of inhibitor that was bound to the COX-2 molecule. Since the focus of the paper is on surrounding the anti-inflam- matory properties of these novel compounds, the three files were specifically selected, as all three in- hibitors are used in a variety of NSAIDs. Salicylate acid and tolfenamic acid are nonselective, while Vi- oxx is selective to COX-2. MOLECULAR MODELING STUDIES WITH AUTODOCK VINA AND UCSF CHIMERA We then performed molecular docking us- ing UCSF Chimera to study the ligand conformation and binding affinity of the different compounds in the COX-2 binding pocket11. The twenty-three lig- ands (chemical constituents) were then docked into the three targets (COX-2) using Autodock Vina inte- grated into UCSF Chimera. These two programs vis- ualize interactions and molecular structures by providing high-quality images and docking trajecto- ries11. After obtaining the delta G values from the docking, the five most stable ligands were further analyzed in Pymol to study the amino acid interac- tions in the binding pocket. PyMOL is a cross-plat- ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE IV form tool that allows for 3D visualization of macro- molecular analysis, protein-ligand docking, and mo- lecular docking simulations among other features12. 3 RESULTS TABLE 1 was compiled from all the different compounds found in the literature. Twenty-three compounds were recorded along with their loca- tions within the plant, pharmacological activity, ex- tract type, and percent yield present in the overall composition of the plant. TABLE 2 shows the x, y, and z calculations of where the original ligands for both targets can be found. These calculations were used to re-dock the original ligands, salicylate acid and Vioxx, in their respective targets by defining the size of the binding pocket. Tolfenamic acid (5KIT) was excluded from the findings of the paper as there were some issues with the docking of the chemical ligands into that file: appropriate x, y, and z calcula- tions could not be obtained for that target. The delta G values and root mean squared deviation (RMSD) values were also recorded in this table. The RMSD values signify the deviation between the original and redocked ligands. The RMSD value is used as a control to highlight that the original ligand can be redocked into the same target. The best confor- mation, denoted by the highest delta G value and RMSD < 2.5, was used to compare the remaining 25 ligands and obtain the delta G values, measured in kcal/mol. The delta G values, along with the confor- mation of each docking for targets, were recorded in TABLE 3. The average delta G value for both targets was also recorded in these tables. The interactions between the five most stable ligands and the bind- ing pockets of both targets were analyzed and rec- orded in TABLE 4. Visualization of these interactions was also recorded to observe the hydrophobicity and hydrophilicity of each interaction. Visualization of these interactions can be observed in FIGURE 2 and FIGURE 3. 4 DISCUSSION Chronic inflammation occurs when an infec- tion remains unresolved. This could lead to a wide array of problems including tissue death and in- creased risk of cancer18. Non-steroidal anti-inflam- matory drugs (NSAIDs) have proven to be effective against many types of inflammation. NSAIDs work by inhibiting cyclooxygenase (COX) enzymes and preventing the conversion of arachidonic acid to prostaglandins. Most of the NSAIDs currently on the market are not selective and work by inhibiting both COX-1 and COX-2 enzymes. This can lead to many adverse effects such as decreased platelet for- mation, which can cause bleeding and gastrointes- tinal complications19. Thus, it is crucial to find a more selective inhibitor for the COX-2 enzyme. Nigella sa- tiva has been proven to reduce inflammation along with many other pharmacological actions, but mechanisms by which these natural constituents in- teract with the inflammatory pathway remain un- clear. Identification of the ligand interactions that mediate the anti-inflammatory effect of Nigella sa- tiva will facilitate the development of more selective COX-2 inhibitors and help create more comprehen- sive guidelines on traditional medicine usage. In this study, we researched the interactions between the chemical constituents and the binding pocket of COX-2 molecules to identify the best compounds for inhibition. Through a combination of in silico docking techniques, we identified five dif- ferent ligands for each COX-2 molecule that have a more stable interaction than the original inhibitors. Salicylate acid, the inhibitor found in the 5F1A target, is the active constituent present in as- pirin. Although a low dosage of aspirin has been proven to help with age-related diseases, extensive usage has been shown to cause gastrointestinal damage and compromise kidney function20. This is due to salicylate acid inhibiting COX-1, which is im- portant in the production of prostaglandin-1, a hor- mone responsible for maintaining homeostasis and platelet function. Thus, it is important to find alterna- tives that do not inhibit COX-1 and have stable bind- ing with COX-2. The five compounds observed to have a better binding affinity are thymoquinone, ni- gellicimine, 4-terpineol, thymol, and carvacrol (TABLE 4). Vioxx, the inhibitor of the other COX-2 tar- get, is the active constituent present in Rofecoxib. ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE IV Rofecoxib was a drug marketed as a safer alterna- tive to NSAIDs for treating rheumatoid arthritis and migraines. The drug was later withdrawn from the market due to studies showing increased probabil- ity of cardiovascular disease in users21. Although TABLE 1: Chemical constituents present in Nigella sativa. The part of the plant, pharmacological activity, extract, and percent- age yield for each compound are also noted. Chemical Constituent Part of Plant Activity Extract Percent yield (%) Thymoquinone14 seed anti-inflammatory + anti- fungal + anticancer methanol 48 Carvacrol15 seed antioxidant + anti-inflam- matory ethanol 12 t-anethole15 seed antioxidant + anti-inflam- matory ethanol 4 4-terpineol15 seed antioxidant + anti-inflam- matory ethanol 7 sesquiterpene longifo- lene15 seed antibacterial + anti-inflam- matory ethanol 8 α-pinene16 seed anti-inflammatory ethanol trace amounts Thymol16 seed anti-inflammatory ethanol trace amounts Nigellicimine16 seed antifungal methanol 2 nigellicimine-N-oxide16 seed antifungal methanol 2 Nigellidine16 seed anti-inflammatory aqueous trace amounts Nigellicine16 seed anti-inflammatory aqueous trace amounts alpha-hederin16 seed antidiabetic aqueous trace amounts Longifolene16 leaves/seed antibacterial + anti-inflam- matory aqueous trace amounts Thujene16 leaves/seed antibacterial + anti-inflam- matory aqueous trace amounts Sabinene17 leaves/seed antibacterial + anti-inflam- matory aqueous trace amounts beta-sitosterol17 leaves/seed antibacterial + anti-inflam- matory aqueous trace amounts Phellandrene17 leaves/seed antibacterial + anti-inflam- matory aqueous trace amounts α-hederin17 leaves/seed antibacterial + anti-inflam- matory aqueous trace amounts Carvone17 leaves/seed antibacterial + anti-inflam- matory aqueous trace amounts Limonene17 leaves/seed antibacterial + anti-inflam- matory aqueous trace amounts β-pinene17 leaves/seed antibacterial + anti-inflam- matory aqueous trace amounts d-citronellol17 leaves/seed antibacterial + anti-inflam- matory aqueous trace amounts Saponin17 leaves/seed antibacterial + anti-inflam- matory aqueous trace amounts ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE IV TABLE 2: XYZ calculations of the original binding site for the two COX-2 targets. These calculations were used to dock the chemical ligands obtained. Redocking of the original ligands and RMSD associated with the ligands were noted. Target PDB file X Y Z Delta G (kcal/mol) RMSD 5KIR 23.38 x 1.34 x 34.57 -7.9 1.7 5F1A 1.60 x 24.07 x 240.24 -6.3 2.114 FIGURE 1A. FIGURE 1B. FIGURE 1. A) Crystal structure of 5F1A (COX-2) with redocked salicylate acid ligand. B) Crystal structure of 5KIR (COX-2) with redocked Vioxx ligand. TABLE 3: Delta G values and of the conformation obtained from the docking of chemical ligands obtained. Average of all dockings was also recorded. Ligand Delta G value for 5F1A(kcal/mol) Delta G value for 5KIR(kcal/mol) Thymoquinone -7 -6.6 carvacrol -6.3 -6.8 t-anethole -6.3 -6.4 4-terpineol -6.6 -6.4 sesquiterpene longifolene -2.8 -6 α-pinene -6.2 -6.1 thymol -6.4 -6.3 nigellicimine -6.8 -7 nigellicimine-N-oxide -5 -6 nigellidine -4 -8.8 nigellicine -5.2 -8.3 alpha-hederin -6 -7 Longifolene -6.3 -6 thujene -6 -6.1 sabinene -6.5 -6.1 beta-sitosterol -2.1 -5.5 α-hederin -5.7 -6.9 Phellandrene -6.3 -6.3 carvone -6.3 -6.8 limonene -6 -6.3 β-pinene -5.4 -5 d-citronellol -5.5 -6 saponin -5 -5 Average -5.6 -6.4 ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE IV FIGURE 2: Visualization of the amino acid interactions for the five ligands and the original ligand associated with the 5F1A target. Red refers to interactions that are extremely hydrophobic while blue refers to interactions that are extremely hydro- philic. The range of colors determines the level of hydrophobicity or hydrophilicity. a-f) Amino acid interactions for salicylate acid, thymoquinone, nigellicimine, 4-terpineol, thymol and carvacrol respectively. FIGURE 3: Visualization of the amino acid interactions for the five ligands and the original ligand associated with the 5KIR target. Red refers to interactions that are extremely hydrophobic while blue refers to interactions that are extremely hydrophilic. The range of colors determines the level of hydrophobicity or hydrophilicity. A-F) Amino acid interactions for Vioxx, nigellidine, nigellicine, nigellicimine, alpha hederin, and carvacrol respectively. FIGURE 2A FIGURE 2D FIGURE 2B FIGURE 2C FIGURE 2E FIGURE 2F FIGURE 3A FIGURE 3B FIGURE 3C FIGURE 3D FIGURE 3E FIGURE 3F ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE IV TABLE 4: Amino acid interactions of the five most stable chemical ligands as well as the original ligands docked into the two COX-2 targets. These interactions include hydrophobic, hydrophilic, ionic, and hydrogen bonds. Ligand Interaction pattern in 5F1A Ligand Interaction pattern in 5KIR Original ligand (salicylate acid) PHE487 TRP356 TYR354. TYR317. LEU321. VAL318. VAL492. PHE 350 SER499 Original ligand (Vioxx) PHE529. TRP360. TYR466. LEU508. VAL434. PHE463 Thymoquinone PRO335 PHE487. CYS340. PRO320. LYS470. VAL318. PHE350. SER499 Nigellidine TYR460. ARG456. GLU380. ARG150. TYR466. LEU503. GLU502 Nigellicimine TYR348. PHE198. LEU390. TRP387. PHE518. LEU352. PHE529. SER530 Nigellicine LEU508 PRO389. ALA435. VAL434. LYS436. LEU507. 4-terpineol PHE335 TRP345 PRO335. CYS340. LEU321. TRP387. LYS470. PHE350 Nigellicimine TYR466 PHE529 ASN382. LEU508. TYR466. ARG150 Thymol TYR385. TRP387. PHE198. TYR348. PHE 381. PHE529. LEU390. GLY526 Alpha hederin ARG150. TYR460. TYR466. LEU503. LEU507. VAL525. PHE529. ASN382 Carvacrol PHE357 ASN110. ALA111. TRP340. PRO335. LEU321. TRP387. PHE350 Carvacrol PHE463. TYR466. LEU384. TYR504. LEU507. GLU510. TYR475 Rofecoxib was successful in selectively inhibiting COX-2, its risks outweighed its benefits. The five compounds observed to have a better binding af- finity are nigellidine, nigellicine, nigellicimine, alpha hederin, and carvacrol (TABLE 4). THYMOQUINONE Thymoquinone, the most abundant com- pound in the Nigella seed (TABLE 1), has been proven to decrease nitric oxide synthesis as well as inhibit prostaglandin formation. Therefore, it could treat asthma and rheumatoid arthritis22. Thymoquinone had a higher delta G value than salicylate acid, prov- ing it has the stability to potentially be used as an inhibitor to target the COX-2 enzyme (TABLE 3). The delta G value denotes the stability of the interaction between the ligand and the target. A higher delta G value indicates that the ligand binds strongly to the target, thus acting as an effective inhibitor. In addi- tion, the interaction of serine and cysteine in the binding pocket shows strong hydrogen bonding between thymoquinone and COX-2 (TABLE 4). The presence of these hydrophilic amino acids stabilizes thymoquinone in the binding pocket. However, the presence of proline in the set of interactions shows a possibility that thymoquinone might not be stable enough for prolonged interactions, as the cyclic ring in proline can distort the structure. Overall, thymo- quinone appears to have numerous hydrophilic in- teractions that are required for sufficient protein-lig- and interactions. NIGELLICIMINE Nigellicimine, a novel compound found mainly in the Nigella seed, also appears to have a higher delta G value compared to the original lig- and of one of the COX-2 targets (5F1A) but not the other (5KIR). Serine, cysteine, and tyrosine appear to be the main amino acids in the interactions between nigellicimine and the 5F1A binding pocket (TABLE 4). Like thymoquinone, nigellicimine has a stable inter- action in the binding pocket. Although there are hy- drophilic interactions between nigellicimine and 5KIR, the presence of multiple amino acids (phenyl- alanine) with aromatic rings may destabilize the structure. Nigellicimine appears to have a higher delta G value compared to some of the other natural compounds present in Nigella sativa. The difference ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE IV in interactions between the two targets could be at- tributed to the original ligands (salicylate acid and Vioxx) not sharing the exact same binding pocket. This can be observed through the XYZ calculations denoting that there could be different amino acid interactions involved in each docking (TABLE 2). 4-TERPINEOL AND THYMOL 4-terpineol and thymol are compounds that are present in low or trace amounts in the Nigella seed (TABLE 1). It was quite unexpected to observe that they have a higher delta G value compared to salicylate acid (TABLE 3). The presence of tyrosine and cysteine supports the delta G value regarding the stability of the interaction. Yet, the presence of mul- tiple phenylalanine and tryptophan amino acids in these interactions suggests a temporary binding that cannot be used for extended inhibition of COX- 2. CARVACROL Carvacrol, the second most abundant com- pound in the Nigella seed (TABLE 1), is known to re- duce inflammation but the mechanism by which it does so remains unclear. Some researchers have hy- pothesized that its anti-inflammatory activity could be caused by induction of interleukin-10 which in turn reduces other inflammatory cytokines23. Car- vacrol appears to have a stable interaction with both COX-2 targets, but it does not have a higher delta G value than salicylate acid and Vioxx (TABLE 3). This could be due to the presence of phenylalanine and tryptophan which cause distortions in the binding structure due to their large side chains that give them a bulky nature. Interactions with 5F1A appear to be more polar compared to interactions with 5KIR (TABLE 4). Further analysis would be required to know the effects of prolonged interactions of carvacrol on the COX-2 targets in question as the data regarding this compound appear to be inconclusive. NIGELLIDINE AND NIGELLICINE Nigellidine and nigellicine were observed to have a higher delta G value compared to Vioxx. Both compounds have been found to reduce in- flammation related to viral infections. Most notably, nigellidine and nigellicimine have been found to help reduce COVID-19-related inflammation and in- hibit interleukins involved in “cytokine storm”24. The presence of asparagine and leucine in the binding pocket interactions suggests that ionic bonds are present which stabilize the structure (TABLE 4). Similar to other compounds, serine, cystine, and tyrosine are involved as polar contacts for nigellidine and ni- gellicine (TABLE 4). The presence of these residues stabilizes the ligand in the pocket of the COX-2 pro- tein. In addition, nigellidine appears to be more sta- ble compared to nigellicine. This could be at- tributed to the extra aromatic ring present in nigel- lidine, which is known to increase ligand-receptor binding and lead to increased effectiveness of the compound25. ALPHA-HEDERIN Alpha-hederin is found in trace amounts in the Nigella seed. Additionally, it has anti-cancer and anti-diabetic pharmacological action (TABLE 1). Sur- prisingly, it was one of the five constituents that had a stable interaction with 5KIR. Alpha-hederin ap- peared to have a high delta G value compared to other constituents but did not exceed that of Vioxx (TABLE 3). This decrease in delta G could be at- tributed to the presence of multiple phenylalanine residues as well as basic residues (like lysine) that can cause distortions in the structure. The presence of multiple tyrosine residues in this interaction de- notes that there are polar contacts that can stabilize the structure. In conclusion, thymoquinone and nigelli- dine were found to have the most stable interaction with COX-2 targets. This research indicates that tra- ditional medicine has the potential to aid in the first step of the drug discovery process: identifying chemical compounds. Using naturally occurring compounds can greatly reduce adverse effects that result from using drugs like NSAIDs. However, fur- ther analysis and studies are needed to understand if these compounds can interact unfavorably with other drugs or molecules in the human body∎ ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE IV 5 REFERENCES [1] Jain, S. K. (1986). Ethnobotany. 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Journal of Drug Targeting just accepted (2020): 1-23. [24] Ritchie, Timothy J., and Simon JF Macdonald. "The impact of aromatic ring count on compound devel- opability–are too many aromatic rings a liability in drug design?" Drug discovery today 14.21-22 (2009): 1011-1020. ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE IV Habiba Abdelhalim is a senior at Rutgers University majoring in biotechnology and minoring in Biochemistry. Habiba’s research focused on the anti-inflamma- tory effects of Nigella sativa, a herb that is commonly used in her culture. Working under Dr. Sonia Arora, Habiba was able to learn about various in silico techniques used in the drug discovery process. Currently, Habiba is part of the Ahmed Lab at Rutgers University working on implementing Artificial Intelligence and Ma- chine Learning in the field of Precision Medicine. Habiba can be reached at: ha375@scarletmail.rutgers.edu.