In vitro and In Silico Studies on Curcumin and Its Analogues as Dual Inhibitors for cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2).doc

ITB J. Sci., Vol. 44 A, No. 1, 2021, 51-66 51In vitro and In Silico Studies on Curcumin andIts Analogues as Dual Inhibitors for cyclooxygenase-1(COX-1) and cyclooxygenase-2 (COX-2)Nunung Yuniarti , Perdana Adhi Nugroho , Aditya Asyhar , Sardjiman , 1 2 2 31 4Zullies Ikawati& Enade Perdana Istyastono1Department of Pharmacology and Clinical Pharmacy, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta, Indonesia2Curcumin Research Centre, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta, Indonesia3Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta, Indonesia4Leiden/Amsterdam Center for Drug Research (LACDR), Division of Medicinal Chemistry, Department of Pharmacochemistry, Faculty of Exact Sciences, Vrije University Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The NetherlandsEmail: nungki_fa97yahoo Abstract. Curcumin has been widely reported as an anti-inflammatory agent isolated from the plant Curcuma longa L. (turmeric). This anti-inflammatory activity was associated with the ability of this compound to inhibit the activity of both cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) in arachidonic acid metabolism. Dual COX-1 and COX-2 inhibitors are preferred to be employed in the therapy of chronic inflammatory diseases compared to selective inhibitors, since it was reported that the use of selective inhibitors led to severe adverse side effect. In the present study, in vitro and in silico assays on curcuminand its analogues as dual inhibitors for both COX-1 and COX-2 were performed. The results provide theoretical contribution in understanding the ligand-protein interactions at the molecular level to develop new curcumin analogues which possess better anti-inflammatory activity as well as to avoid unsolicited side effects.Keywords: curcumin; cyclooxygenase-1; cyclooxygenase-2; in silico docking; in vitro inhibition assay; dual inhibitor.1IntroductionCurcumin (Figure 1; compound 1a and 1b), the yellow pigment isolated fromthe plant Curcuma longa L. (turmeric), has been reported to have anti-inflammatory activity, as well as antiviral, antioxidant and anti-infectiousactivities 1,2. Moreover, in several studies, curcumin caused suppression,retardation, or inversion of carcinogenesis 3-6. The ability of curcumin toinhibit prostaglandins (PGs) biosynthesis in the arachidonic acid cascade hasReceived March 5 , 2021, Revised November 3 , 2021, Accepted for publication November 21 , 2021.th rd st 52Nunung Yuniarti, et al.been suggested as an important factor for its anti-inflammatory activity and itsanticarcinogenic action 2-7. Enzyme cyclooxygenases (COXs), which consistof at least two isoforms, i.e., cyclooxygenase-1 (COX-1) and cyclooxygenase-2(COX-2), catalyze the conversion of arachidonic acid to PGH2 in the first twosteps 3,7,8. The first isoform, COX-1, has been suggested as a constitutivelyexpressed enzyme, which is responsible for maintaining normal physiologicalfunction 3,8. On the other hand, the second isoform, COX-2 is inducible andthe expression is stimulated in the inflammation process 7-9. At the beginningof the discovery of COX-2, therefore, the selectivity towards COX-2 wasconsidered as beneficial 10. However, the withdrawal of Rofecoxib, aselective COX-2 inhibitor, due to its increased risk of cardiovascular events,raises an issue that the development of the dual inhibitor of the COX-1 andCOX-2 is of considerable interest 8. Even more, the COX-1 and COX-2 dualinhibitors are suggested to be more appropriate for treating chronicinflammation 11.Figure 1 Structures of curcumin (1) in the keto (1a) and the enol (1b) forms, 2,5-bis(4-hydroxy-3-methoxybenzilidene)-cyclopentanone (2), 1,5-bis(4-hydroxy-3-methoxyphenyl)penta-1,4-dien-3-one (3), and flurbiprofen (4).Curcumin inhibits the PGs biosynthesis by inhibiting the activity ofcyclooxygenases(COXs),bothcyclooxygenase-1(COX-1)andcyclooxygenase-2 (COX-2) 2-7. The anti-inflammatory activity of curcuminwas related to its ability in inhibiting COX-2 as well as lipoxygenase (LOX) 7,12. However, a recent study by Hong et al. 4 suggested that curcumin is moreselective to ovine COX-1 compared to ovine COX-2. This result led Handler etal. 3 to synthesize some curcumin analogues as selective COX-1 inhibitors.On the other hand, those results also indicated that curcumin binds to COX-2 aswell as to COX-1 and results in a significant inhibition 3,4. Moreover, Padhyeet al. 13 has reported recently that fluorocurcumin is a COX-2 inhibitor.Curcumin, therefore, can serve as a lead compound to develop new inhibitorsfor both COX-1 and COX-2. In vitro and In Silico Studies on Curcumin and Its Analogues53In silico study in drug design and discovery has flourished together with theadvance of computer technology 14,15. The method is promising to increasethe efficiency and effectiveness of the drug and design processes 14. Anumber of in silico studies on curcumin have also been reported recently 3,13,16,17. Handler et al. 3 constructed models by using docking method toexplain the activity of curcumin and its analogue as selective COX-1 inhibitors.Other models have been constructed by Padhye et al. 13 to explain the activityof curcumin and its fluoro analogues as selective COX-2 inhibitors. Besidesdocking method, another in silico approach named quantitative structure-activity relationship (QSAR) has been employed as well 16,17. Appiah-Oponget al. 16 generated QSAR models of some curcumin analogues as inhibitors ofhuman cytochrome P450 (CYP450) and glutathione S-transferases (GSTs).Moreover, Fujisawa et al. 17 performed a QSAR study on a series of 2-methoxyphenols as COX-2 inhibitors with curcumin as one of the studiedcompound. These studies suggest that in silico study on curcumin and itsanalogues can perform as a complimentary method in the discovery of newdrugs.This article describes in vitro and in silico studies of some analogues ofcurcumin(Figure1),i.e.,2,5-bis(4-hydroxy-3-methoxybenzilidene)-cyclopentanone (compound 2), and 1,5-bis(4-hydroxy-3-methoxyphenyl)penta-1,4-dien-3-one (compound 3) as well as the parent curcumin molecule, both inketo tautomer (compound 1a) and enol tautomer (compound 1b) as dualinhibitors for COX-1 and COX-2. Compounds 2 and 3 were selected as therepresentatives of the curcumin analogues among those in house analoguessynthesized by Sardjiman et al. 19 since both have been tested in extensivepreclinical studies and have the most similar pharmacophore features withcurcumin 18,19.2ExperimentalMaterialsCurcumin and its analogues were synthesized by Sardjiman et al. 19. Theinhibitory activity of curcumin and its analogues to both COX-1 and COX-2were measured using Colorimetric COX (ovine) Inhibitor Screening assaypurchased from Cayman Chemicals (Cayman Chemical Co., Cat. No. 760111,Ann Arbor, MI, USA).The COX-1 and COX-2 (ovine) Inhibitory AssayThe assay was carried out using a colorimetric COX (ovine) Inhibitor ScreeningAssay Kit (Cayman Chemical Co., Cat. No. 760111) as previously described in 54Nunung Yuniarti, et al.Yang, et al. 20. The final concentrations of both COX-1 and COX-2 in theassay are 1.45 % /v (14.5 mL/L).vMolecular Modeling: Hardware and SoftwareThe docking protocols were performed using Molecular Operating Environment(MOE), version 2021 10, developed by Chemical Computing Group, Inc.(Montreal, Canada). Interaction fingerprints (IFP) application developed byMarcou and Rognan 21 were performed as the post docking processing. TheOpenEyes OEChem1.3 library was used in the IFP computation 21,22. Aslong as no further explanation is stated, the default setting was used. Allsimulations were performed on a Linux (Ubuntu 8.04 LTS Hardy Heron)machine with Intel Core 2 Duo ( 2.5 GHz) as the processors and 3.00 GB ofRAM.Preparation of Ligand StructuresCurcumin (both in keto (1a) and enol form (1b), its analogues (compound 2-4)as well as flurbiprofen (compound 4) as the reference molecule in the dockingsimulations were built in the 2D model in an MOE database viewer.Conformational search was then performed on the database using theConformational Import module. The results were then washed and minimized.Docking SimulationsThe 3D coordinates of COX-1 and COX-2 were obtained from the protein databank ( :/ pdb.org/; PDB code: 1eqh and 3pgh, respectively) 23, 24.Both proteins contain the reference molecule flurbiprofen (compound 4), a dualinhibitor for COX-1 and COX-2 23. The binding sites of both proteins weredefined by employing the Pocket Selection module in MOE and using thebinding sites of flurbiprofen in the crystal structures as the references.Sequences alignments of these binding sites were performed using ClustalW225. Hydrogens were added using the Protonate 3D module in MOE, and onlyligand and protein in chain A were used from the crystal structures. The proteinswere subsequently aligned using the Align module in MOE. For each protein,Docking Simulation module in MOE was performed using the databasecontaining prepared ligands as the input file. The results of the dockingsimulations were subsequently subjected for post docking processing byemploying IFP guided visual inspection. The protein-ligand interactionsfingerprint of flurbiprofen in the crystal structures were used as the references23,24. In vitro and In Silico Studies on Curcumin and Its Analogues553Result and DiscussionThe COX-1 and COX-2 (ovine) Inhibitory AssayIn the present study, the inhibitory activity of curcumin (1) and its analogues (2and 3) to COX-1 and COX-2 were tested in vitro and in silico. We found thatthe compounds are potent inhibitors for both proteins since they could inhibitthe activity of the proteins more than 50% in the concentration of 15 mM. Someselectivity trends were observed; however the statistical analysis showed thatthe differences were not significant. At the beginning of the discovery of COX-2, which was believed to be more responsible in the inflammatory processcompared to COX-1, curcumin was believed to be a more selective COX-2inhibitor than COX-1 inhibitor 2. Some previous studies also reported thatcurcumin is highly related to the COX-2 expression and also binds directly toCOX-2 7. However, a recent study by Hong et al. 4 suggested that curcuminis more selective to COX-1, which subsequently was supported by Handler etal. 3 by synthesizing some curcumin analogues as selective COX-1 inhibitors.These previous results underlined that curcumin is inhibitor for both COX-1 andCOX-2, which was supported by our finding in this study. Moreover, weindicated here that compounds 1-3 can be potent dual inhibitors for both COX-1and COX-2. Dual COX-1 and COX-2 inhibitors are preferred to be employed inthe therapy of chronic inflammatory diseases compared to selective inhibitors11, since it was reported that the use of selective inhibitors led to severeadverse side effect 8, 9, 11.10075COX-150COX-2250123CompoundFigure 2 The inhibitory activity of curcumin and its analogues (15 mM) to COX-1 and COX-2. The final concentrations of both COX-1 and COX-2 in the assay are 1.45 % v/v (14.5 mL/L). Data shown are mean ± SEM of three independent experiments. 56Nunung Yuniarti, et al.Figure 2 shows the inhibitory activity of compounds 1, 2 and 3 to both COX-1and COX-2. The activities range from 68.4-77.8% for COX-1 and 59.3-81.9%for COX-2. Compound 2 shows the highest inhibitory activity for both proteins.Compound 2 and 3 are more selective towards COX-2, while compound 1 ismore selective towards COX-1. Statistical analysis using Students t-test,however, shows that the differences between the inhibitory activities to COX-1and to COX-2 are not significant at 95% level of confidence intervals for eachcompound. Moreover, the ANOVA tests show that the differences between theinhibitory activities of compounds 1, 2, and 3 are also not significant at 95%level of confidence intervals for both COX-1 and COX-2.Docking StudiesThe sequences alignments of binding sites using ClustalW2 (Figure 3) have ascore of 80. When only residues that perform direct interaction with thereference compound (flurbiprofen; compound 4) as indicated in IFP analysiswere taken into account in the sequence alignments, the score is 94 and 16 outof 17 residues are identical. The residues with residue number 523 (ILE inCOX-1 and VAL in COX-2) are found to be the only ones that differentiates thebinding site of both enzymes, but still observed as “conserved in ClustalW2.The cross IFP calculation between the crystal structures gives the Tanimotometric Interaction Fingerprint (Tc-IFP) of 1.00 for both enzymes. Table 1presents Tc-IFP of the selected pose for each compound. The Tc-IFPs of thereference compound docking poses are more than 0.6, i.e. 1.00 and 0.95 in theCOX-1 and COX-2, respectively. Figure 4 shows the overlay of the crystalstructure conformation and the docking pose of compound 4 in the binding siteof COX-1 (Figure 4A) and COX-2 (Figure 4B). Curcumin (compound 1) in theketo form (1a)