- Email: [email protected]
Phytochemicals as multi-target inhibitors of the inflammatory pathway- A modeling and experimental study
Accepted Manuscript Phytochemicals as multi-target inhibitors of the inflammatory pathway- A modeling and experimental study Nisha S. Devi, Meera Ramanan, Padmapriya Paragi-Vedanthi, Mukesh Doble PII:
S0006-291X(17)30071-2
DOI:
10.1016/j.bbrc.2017.01.046
Reference:
YBBRC 37108
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 22 November 2016 Revised Date:
8 January 2017
Accepted Date: 10 January 2017
Please cite this article as: N.S. Devi, M. Ramanan, P. Paragi-Vedanthi, M. Doble, Phytochemicals as multi-target inhibitors of the inflammatory pathway- A modeling and experimental study, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.01.046. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
ACCEPTED MANUSCRIPT
Phytochemicals as multi-target inhibitors of the inflammatory pathway- A modeling and experimental study Nisha S. Devi#, Meera Ramanan#, Padmapriya Paragi-Vedanthi, Mukesh Doble*
of Technology, Madras, Tamil Nadu, 600036, India. #
Both authors contributed equally.
*Email ID: [email protected]
RI PT
Bhupat&Jyoti Mehta School of Biosciences, Department of Biotechnology, Indian Institute
SC
Keywords: Inflammation; 5-Lipoxygenase; Microsomal Prostaglandin E2 Synthase 1;
AC C
EP
TE D
M AN U
Pharmacophore; Multitarget drugs
2
ACCEPTED MANUSCRIPT Abstract The arachidonic acid pathway consists of several enzymes and targeting them is favored for developing antiinflammatory drugs. However, till date the current drugs are generally active against a single target, leading to undesirable side-effects. Phytochemicals are known to inhibit multiple targets simultaneously and hence, an
RI PT
attempt is made here to investigate their suitability. A pharmacophore based study is performed with three sets of reported phytochemicals namely, dual 5-LOX/mPGES1, alkaloids and FLAP inhibitors. The analysis
indicated that phenylpropanoids (including ferulic acid) and benzoic acids derivatives, and berberine mapped onto these pharmacophores with three hydrophobic centroids and an acceptor feature. 2,4,5-trimethoxy (7) and
SC
3,4-dimethoxy cinnamic acids (8) mapped onto all the three pharmacophores. Experimental studies indicated that berberine inhibited 5-LOX and PGE2 production by 72.2 and 72.0 % and ferulic acid by 74.3 and 54.4 %
M AN U
respectively. This approach offers a promising theoretical combined with experimental strategy for designing novel molecules against inflammatory enzymes. Abbreviations:
LT: Leukotriene; PG: Prostaglandin;5-LOX: 5-Lipoxygenase;COX: Cyclooxygenase;FLAP: 5-Lipoxygenase
TE D
Activating Protein;PGE2: Prostaglandin E2;mPGES1: microsomal Prostaglandin E Synthase 1; NF-kB: Nuclear Factor-κ-light chain enhancer of activated B cells; STAT-3: Signal Transducer and Activator of Transcription 3.
EP
The arachidonic acid pathway is responsible for the production of the inflammatory mediators, leukotrienes (LT) and prostaglandins (PG), via the lipoxygenase (LOX) and cyclooxygenase (COX) and
AC C
pathways respectively. The LTs are synthesized mainly by the inflammatory cells including granulocytes, monocytes and mast cells and are responsible for the pathogenesis of asthma allergy and rheumatoid arthritis1-3. However, drugs targeting the enzymes in this pathway namely, 5-lipoxygenase (5LOX), are yet to enter the market. The only drug developed against this enzyme, Zileuton, is withdrawn in 2008 due to hepatotoxicity but still the extended release formulation of it is available in the market4. Several inhibitors for 5-lipoxyganase activating protein (FLAP), a helper protein of the leukotriene pathway, are under clinical trials but none have entered the market till date5. The prostaglandins are synthesized on being stimulated by injury or under the influence of proinflammatory cytokines and other stimuli1. They have been implicated in the development of both acute and
3
ACCEPTED MANUSCRIPT chronic inflammation and the most notable among them is prostaglandin E2 (PGE2), which is also involved in the progression of several cancers 6. Many of the drugs developed till date, including the non-steroidal antiinflammatory drugs (NSAIDs) and COX-2 inhibitors (Coxibs) which target COX-1/2 enzymes have been
associated with gastric toxicity and cardiovascular complications 7, 8. Hence, targeting microsomal prostaglandin E synthase1 (mPGES1) downstream of these two enzymes, for specifically inhibiting the production of PGE2 is
RI PT
being touted as a promising strategy for development of anti-inflammatory drugs 9.
Although the anti-inflammatory drugs, both currently available and under development, are mostly active against a single enzyme, studies have shown that inhibiting a single pathway results in the shunting of the
SC
substrate arachidonic acid to the other one, thus negating the therapeutic intervention 10. This has lead to interest in the development of dual inhibitors against 5-LOX/mPGES1 and COX/LOX 11. Studies have showed that for
M AN U
multi-factorial diseases such as inflammation, drugs active on multiple targets have better efficiency and safety profiles. Experimental studies have shown that 5-LOX/mPGES1 dual inhibitors such as a pirinixic acid derivative reduce vascular remodeling in a murine model of aortic aneurysm without significantly inhibiting COX1/212. Licofelone, a multi-targeted inhibitor which inhibits COX-1, mPGES1 and FLAP has completed Phase III clinical trials for arthritis treatment 13.
TE D
Phytochemicals (such as curcumin) have been shown to have multi-targeted action against COX-1, 5LOX and mPGES1 and signaling molecules such as NF-κB, stat-3 and P53 14-26(Supplementary table 1). Natural products are capable of interacting with a wide range of biological macromolecules, occupy a diverse chemical
EP
space and exhibit better drug-like properties when compared to synthetic molecules, making them a promising resource for identification of leads molecules for drug development 27, 28. A study by David J. Newman (2012)
AC C
found that in the period ranging from 1940s to 2010, of the 175 small molecules approved for cancer treatment, about half of them were either phytochemicals or their direct derivatives 29. More than 60% of the drugs in the market are derived from natural compounds 30.This points to the importance of these molecules as excellent sources for identifying novel scaffolds and leads for drug development. Several phytochemicals have been shown to have anti-inflammatory properties and have been in use for a long time in traditional medicines 31. Flavonoids such as quercetin have been shown to inhibit both acute and chronic phases of inflammation in rats 32. They purportedly act by downregulating the expressions and activities of the enzymes involved in the synthesis of inflammatory mediators such as nitric oxide, prostaglandins and leukotrienes 33. Dietary polyphenols have also been shown to attenuate atherosclerosis by
4
ACCEPTED MANUSCRIPT alleviating inflammation in mouse models of atherosclerosis 34. These findings suggest the possibility of utilising the chemical diversity of phytochemicals and their reported anti-inflammatory action in the design of multi-target drugs against inflammation. The important functional features of a biomolecule are represented by a pharmacophore which is a set
RI PT
of features including hydrogen bond donors, acceptors, hydrophobic and aromatic centers that identify their properties necessary for the ligand-enzyme interaction. Pharmacophore studies provide an insight on the
structural and chemical properties shared by active molecules and can be a powerful tool for zeroing in on
potential inhibitors. In the current study, this approach is used to identify phytochemicals which may serve as
SC
starting scaffolds for development of dual or multi-target inhibitors against these enzymes/proteins involved in inflammation. The possible candidates identified by this study are experimentally tested against the two
M AN U
enzymes, 5-LOX andmPGES1.The pharmacophore based approach for identifying leads coupled with experimental studies hold the promise of developing molecules which can exert a multi-pronged influence towards treating inflammation. Materials and Methods
TE D
All the phytochemicals were purchased from Sigma-Aldrich and Sisco Research Laboratories (SRL, Mumbai, India). HeLa cells were purchased from NCCS (Pune, India) and maintained in Dulbecco’s Minimal Essential Medium (DMEM) at 37°C with 5% CO2. The PGE2-EIA kit was obtained from Cayman chemicals (# 514010, Ann Arbor, MI). The leucocyte concentrates for the 5-LOX enzyme assay were obtained from healthy
EP
human volunteers with the necessary ethical clearance.
AC C
The overall scheme of this work has been outlined in Fig 1. More detailed information is given in supplementary section.
Pharmacophore elucidation of inhibitors of inflammatory enzymes The pharmacophoric features of a group of molecules are derived using the ‘Pharmacophore
elucidation’ module of Molecular Operating Environment (MOE) 2015.2001 software (Chemical Computing Group, Quebec, Canada).
5
ACCEPTED MANUSCRIPT Docking of hit molecules with inflammatory enzymes The representative hit molecules identified from the pharmacophore search were docked to the active sites of 5-LOX, mPGES1 and FLAP using Goldsuite 5.2.
RI PT
Extraction of 5-LOX protein from human blood: 5-LOX is isolated from the leukocyte concentrates (Buffy coats) of healthy human donors obtained from blood bank (Voluntary Health Services, Chennai) following the approved procedure after obtaining the necessary ethical clearance. The enzyme activity was determined following a standard protocol 50, 51.
SC
Estimation of PGE2 production:
The inhibition of the prostaglandin synthesis pathway was detected by measuring the amount of
M AN U
the COX-2/mPGES1 product, prostaglandin E2 (PGE2), as previously described 52.
Results and Discussion
Several phytochemicals have been shown to be active against chronic inflammatory diseases including rheumatoid arthritis and inflammatory bowel disease53.They have also been found to aid in the
TE D
prevention and treatment of inflammation associated with malignancies such as colorectal cancer54. The identification of their target could shed light on the mechanism by which they exert their anti-inflammatory effects. For this study, we have chosen a range of phenylpropanoids, benzoic acids and alkaloids present as
EP
active components in several medicinal plants with previously documented anti-inflammatory activity. We have attempted to deduce the action of natural products on pro-inflammatory enzymes using both
AC C
computational and experimental strategies and explore their potential as the starting structure for the development of multi-target anti-inflammatory drugs.
Pharmacophore elucidation
The elucidation of pharmacophoric features common to a set of enzyme inhibitors provides
important information regarding the features essential for their interaction. In an attempt to study the features common to anti-inflammatory molecules across different targets, we collected phytochemicals belonging to three structurally diverse groups: phenolic 5-LOX/mPGES1 dual inhibitors, alkaloids with known anti-inflammatory functions and FLAP inhibitors (Supplementary fig 2).
6
ACCEPTED MANUSCRIPT Five phenolic phytochemicals which are known to have the 5-LOX/mPGES1 dual inhibitory activity were selected, namely, arzanol, garcinol, embelin, curcumin and trans-resveratrol (Supplementary fig 1A)14, 35-38. Arzanol has a phloroglucinylpyrone ring while Garcinol consists of polyprenylated chain
and is used in traditional medicine for the treatment of inflammation. Embelin has a benzoquinone moiety and a lipophilic tail and curcumin consists of a lipophilic linker and multiple phenolic groups and is a
RI PT
mainstay in the Indian traditional medicine system of Ayurveda against inflammation. Trans-resveratrol is a phenol which has a good anti-oxidant activity and is a prominent anti-inflammatory agent in folk medicine. A four-point pharmacophore (Ph-dual) elucidated from these five compounds has three
SC
hydrophobic centroids (Hyd) and one projected hydrogen bond acceptor (Acc2) (Fig 2A). This Ph-dual was set as a query against an in-house conformational database of natural products (mainly phenylpropanoids, benzoic acids and alkaloids) derived from active components of medicinal plants
M AN U
(Supplementary fig 2). It was found that eight compounds, consisting of phenylpropanoids including 2,4,5trimethoxy cinnamic acid (7) and benzoic acids such as 2,3-dimethoxy benzoic acid (2),map onto this pharmacophore.
Since alkaloids are an important constituent in phytochemicals having anti-inflammatory properties,
TE D
studying their common pharmacophore features will aid in the design of novel drugs. The second set of known anti-inflammatory alkaloid phytochemicals selected includes stylopine, glaucine, boldine and tylophorine (Supplementary fig 1B) 39-42. The salient structural features of the pharmacophore thus derived (Ph-alk) (Fig 2B)
EP
consists of fused aromatic and heterocyclic rings and presence of nitrogen as part of the ring. Stylopine is reported to inhibit several inflammatory molecules namely COX-2, PGE2, NO, iNOS and inflammatory
AC C
cytokines such as TNFα, IL-1β and IL-6. Glaucine is a potent inhibitor of phosphodiesterase-4 and is used in the treatment of asthma in traditional medicine. Boldine reduces the levels of TNFα, NF-κb and IL-6 while tylophorine is a phenanthraindolizidine with significant inhibition of the pro-inflammatory molecules IL-6, TNFα, IFNγ, NO. Hence these set of alkaloids exhibits their anti-inflammatory activity through different mechanisms. The elucidated pharmacophore (Ph-alk) (Fig 2B) for these compounds was found to consist of three hydrophobic centroids and one projected acceptor. In a pharmacophore search of our previously mentioned natural products database (Supplementary fig 2), berberine (17), ferulic acid (12) and other phenylpropanoids were found to share these pharmacophoric features.
7
ACCEPTED MANUSCRIPT FLAP is a carrier protein for 5-LOX, which makes it an important drug target and could be a site of action for multi-target drugs. A 4-point pharmacophore elucidated from eight reported FLAP inhibitors (Ph-FLAP)43-48(Supplementary fig 1C) including indole-sulphur compounds and quinolones (Fig 2C) consisted of three hydrophobic centroids and one projected acceptor. Ph-FLAP was able to identify
berberine (17), a few phenylpropanoids and benzoic acid derivatives from the database to possess the same
RI PT
pharmacophoric features.
A comparison of Ph-dual, Ph-alk and Ph-FLAP(Fig 4) indicated that the three hydrophobic
centroid groups (F1, F2 and F3)are approximately at the same distance from one another, namely, the
SC
distances between F1-F2, F2-F3 and F3-F1 ranged between 2.2-3.3, 3.2-3.4 and 5.2-5.7 respectively. 2,4,5trimethoxy cinnamic acid (7) and 3,4-dimethoxy cinnamic acid(8) were found to possess all the three pharmacophores. Berberine (17) mapped onto Ph-alk and Ph-FLAP; 2,3-dimethoxy benzoic acid (2)and
M AN U
2,5-dimethoxy benzoic acid (3) onto Ph-dual and Ph-FLAP and methyl eugenol (14) onto Ph-dual and Phalk. The structures of these phytochemicals were overlapped with the pharmacophores to determine the groups that contributed to these structural features (Fig 3). The common groups contributing to the hydrophobic centroids features could be attributed to three specific groups in the case of 2,4,5-trimethoxy (7) and 3,4-dimethoxy cinnamic acid (8). These included the C=C present in the propionic acid tail, the
TE D
benzene ring and the –CH3 group of the methoxy tail. The projected acceptor group is contributed by the phenolic oxygen of the propionic acid tail. In the case of berberine, the hydrophobic moieties are the tetracyclic skeleton while the acceptor feature is the oxygen atom in the azapentacyclo ring. The presence
EP
of three hydrophobic centroid features appears to be crucial for the anti-inflammatory activity and hence could be an important factor for designing novel drugs for this ailment. Based on these pharmacophore
AC C
searches, a group of nine phenyl propanoids, six benzoic acid derivatives, alkaloids and other diverse phytochemicals were shortlisted for in vitro testing of inhibition of 5-LOX activity and PGE2 production. The importance of these hydrophobic centroids in aiding the interaction of the compounds with the active site residues of the inflammatory enzymes was revealed by the docking analysis. Molecular docking of ligand with enzyme is used to study the binding interactions and the preferred pose of the ligand in the binding with the enzyme using various conformational search algorithms. In our case, 2,4,5- trimethoxy cinnamic acid was selected as the representative molecule since it mapped onto all the three pharmacophores and for docking studies against 5-LOX, FLAP and mPGES1 (Supplementary fig 3). The analysis revealed extensive Van der waals interactions with the hydrophobic amino acids of the 5-LOX
8
ACCEPTED MANUSCRIPT proteinnamelyPhe610, Leu607 and Val604 (Supplementary fig 3A). The compound also showed hydrophobic interactions with the residues such as Tyr112, Ile113 and Phe114 in FLAP protein (Supplementary fig 3B). Interaction of this compound with the hydrophobic residues such as Gly35 and Leu39 present in mPGES1 was also observed (Supplementary fig 3C). The hydrophobic centroids identified in the pharmacophore search appear to be crucial for the interaction with the various
Inhibition of 5-LOX and COX-2/mPGES1 by phytochemicals
RI PT
hydrophobic aminoacids that lines the active site of these inflammatory proteins.
Eugenol, ferulic acid and cinnamic acid inhibited 5-LOX by 86.5±0.6%, 74.4±4.2% and
SC
66.6±2.6% respectively at a concentration of 100 µ M (Fig 4). The alkaloid, berberine, also showed a high inhibition (72.2±3.5%) while flavonoid, quercetin, showed an inhibition of 55.2±2.4%. Of the benzoic acid derivatives tested, 4-hydroxy 3-methoxy benzoic acid (vanillic acid) showed the maximum inhibition
M AN U
(50.3±5.2%) of 5-LOX.
4-hydroxy 3-methoxy benzoic acid (vanillic acid) at a concentration of 50 µ Mshowed the maximum inhibition of PGE2 production(87.1±3.5%). Among the phenylpropanoids, 3,4dimethoxycinnamic acid exhibited the highest inhibition (86.7±11.3%). The alkaloid, berberine, was inhibited by 71.9±5.1% while the flavonoid quercetin was inhibited by 84.1±6.6%. Potent mPGES1
TE D
inhibitors including MK886 are shown to be ineffective in in vivo systems due to their plasma binding tendency(in vitro IC50- 1.6 µM; ~ 20% inhibition for 100 µM in whole blood) whereas our approach of inhibiting PGE2 in vivo in HeLa cells appear to be a better strategy to a priori address the above mentioned
EP
problem.
An elevated level of PGE2 is observed under inflammatory conditions and it functions by binding
AC C
to four EP receptors (EP1-4) and amplifying the inflammatory signals 55. It is also associated with the development of cancer as it participates in metastasis and angiogenesis. COX-2 and mPGES1 are found to be functionally coupled in the synthesis of PGE2, thus favoring the progression of inflammation 56.Thus, inhibiting PGE2 promises to attenuate inflammation and indeed, has been shown to halt the progression of leukaemia and a decrease of anti-apoptotic markers57. The above results indicate that compounds such as 2,4,5-trimethoxy cinnamic acid, berberine, vanillic acid and quercetin inhibit 5-LOX as well as the enzymes that are involved in the production of PGE2, namely, COX and mPGES1. The phenylpropanoid 2,4,5-cinnamic acid which mapped onto all 3 pharmacophores viz, Ph-dual, Ph-alk and Ph-FLAP, also showed good activity against 5-LOX (47.6%) and PGE2 production (79.9%).
9
ACCEPTED MANUSCRIPT Another phenylpropanoid, methyl eugenol, which mapped onto two pharmacophores, namely, Ph-dual and Ph-
alk, also inhibit 5-LOX (49.5%) and PGE2 production (78.3% resp.).The alkaloid, berberine which mapped onto Ph-alk and Ph-FLAP also exhibited significant inhibition of 5-LOX activity (72.19%) as well as PGE2 production (71.88%). Two benzoic acid derivatives, namely, 2,3- and 2,5-dimethoxy benzoic acid which possessed the pharmacophoric features of Ph-dual and Ph-FLAP, exhibited moderate inhibition of 5-LOX
RI PT
activity (33.7 and 42.0% resp.) and PGE2 production (18.2 and 80.1 % resp.). These studies indicate that
phenylpropanoids and alkaloids have the pharmacophoric features necessary for efficient anti-inflammatory action which is further validatedfrom our experimental studies, hence suggesting the possibility of developing
.
SC
these molecules as a multi-target inhibitor of the arachidonic acid pathway.
Inflammation has a complex pathology characterized by the interplay of several inflammatory
M AN U
enzymes, cytokines and mediators. For such multi-factorial diseases, the strategy of targeting single enzymes may not lead to effective treatment of the underlying condition, as seen in the case of NSAIDs and coxibs with unwanted side effects. Since an anti-inflammatory drug with minimal side-effects remains a distant possibility, a multi-prongedtargeting of the enzymes in the inflammatory pathway seems to be need of the hour.In this regard, we have attempted to use a systematic approach involving theoretical and experimental strategy towards
TE D
exploring the features of phytochemicals which may enable them to target several inflammatory enzymes such as 5-LOX, mPGES1 and FLAP simultaneously. Pharmacophore studies allowed us to identify chemical features which are prevalent in several reported anti-inflammatory molecules which target a range of protein/enzymes.
EP
This allowed us to rationally select phytochemicals to test against these inflammatory enzymes experimentally. The pharmacophore analysis showed that the inhibitors of 5-LOX, mPGES1 and FLAP had three hydrophobic
AC C
centroid features and a projected acceptor. This information could be an useful guide while designing drugs active on multiple points of the inflammatory pathway. Further studies using other inflammatory enzymes could add meaningful insights into the present findings. Our studies showed that phenylpropanoids such as 2,4,5trimethoxy cinnamic acid and berberine are privileged structures which mapped onto multiple pharmacophores and also showed significant inhibition of 5-LOX and COX-2/mPGES1 activities, thus underlining their importance as starting structures for multi-target drugs.
Acknowledgement & Funding sources
10
ACCEPTED MANUSCRIPT NSD acknowledges Council of Scientific and Industrial Research (CSIR) for the fellowship; MR and PPV acknowledge WOS-A, DST, India for the project funding and fellowship. Author contributions:
RI PT
MD conceived and supervised the study; NSD & MR designed & performed experiments; NSD & PV standardized 5-LOX extraction protocol; MD analyzed the data; NSD & MR wrote the manuscript which was revised by MD. References:
SC
[1] C. D. Funk, Prostaglandin and Leukotrienes: Advances in Eicosanoid Biology, Sci. Transl. Med. (2001) 294, 1871-1875. [2] A.S. Cowburn, K. Sladek, J. Soja, L. Adamek, E. Nizankowska, A. Szczeklik, B.K. Lam, J.F. Penrose, F.K. Austen, S.T. Holgate, A.P. Sampson,Overexpression of leukotriene C4 synthase in bronchial biopsies from patients with aspirin-intolerant asthma, The Journal of clinical investigation. 101(1998)834-846. [3] B. Yousefi, F. Jadidi-Niaragh, G. Azizi, F.Hajighasemi, A. Mirshafiey, The role of leukotrienes in immunopathogenesis of rheumatoid arthritis,Mod. Rheumatol. 24(2)(2014) 225-235.
M AN U
[4] E.M. Joshi, B.H. Heasley, M.D. Chordia, T.L. Macdonald, In Vitro Metabolism of 2-Acetylbenzothiophene: Relevance to Zileuton Hepatotoxicity,Chem. Res. Toxicol. 17 (2004) 137-143.
AC C
EP
TE D
[5] D. Pettersen, O. Davidsson, C. Whatling,Recent advances for FLAP inibitors, Bioorg. Med. Chem Lett. 25 (2015) 2607-2612. [6] E. Ricciotti, G.A. FitzGerald, Prostaglandins and inflammation, Arterioscler. Thromb. Vasc. Biol.31 (2011) 986-1000. [7] H. Matsui, O. Shimokawa,T. Kaneko, Y. Nagano, K. Rai, I.J. Hyodo, The pathophysiology of non-steroidal anti-inflammatory drug (NSAID)-induced mucosal injuries in stomach and small intestine, Clin. Biochem. Nutr. 48 (2011) 107-111. [8] C.P. Cannon, P.J. Cannon, COX-2 inhibitors and cardiovascular risk, Science. 2012, 336, 1386-1387. [9] A. Koeberle, O. Werz,Perspective of microsomal prostaglandin E2 synthase-1 as drug target in inflammation-related disorders, Biochem. Pharmacol.98 (2015) 1-15. [10] R. Ganesh, D.J. Marks, K. Sales, M.C. Winslet, A.M. Seifalian, Cyclooxygenase/lipoxygenase shunting lowers the anti-cancer effect of cyclooxygenase-2 inhibition in colorectal cancer cells, World journal of surgical oncology.10(2012) 200. [11] F. Celotti, S. Laufer, Anti-inflammatory drugs: new multitarget compounds to face an old problem. The dual inhibition concept, Pharmacol. Res.43 (2001) 429. [12] M. Revermann, A.Mieth, L. Popescu, A. Paulke, M. Wurglics, M. Pellowska, A.S. Fischer, R. Steri, T.J. Maier, R.T. Schermuly, G. Geisslinger,A pirinixic acid derivative (LP105) inhibits murine 5-lipoxygenase activity and attenuatesvascular remodelling in a murine model of aortic aneurysm, Br. J. Pharmacol.163 (2011) 1721. [13] J.N. Dulin, M.L. Moore, R.J. Grill, The dual cyclooxygenase/5-lipoxygenase inhibitor licofelone attenuates p-glycoprotein-mediated drug resistance in the injured spinal cord, Journal of neurotrauma. 30 (2013) 211-226. [14] A. Koeberle, O. Werz,Multi-target approach for natural products in inflammation, Drug discovery today. 19 (2014) 1871-1882. [15] H.H. Chang, E.J. Meuillet, Identification and development of mPGES-1 inhibitors: where we are at?Future Med. Chem. 3(2011) 1909-1934. [16] http://phenol-explorer.eu/. [17] M.C. Kim, S.J. Kim, D.S. Kim, Y.D. Jeon, S.J. Park, H.S. Lee, J.Y. Um, S.H. Hong, Vanillic acid inhibits inflammatory mediators by suppressing NF-kB in lipopolysaccharide-stimulated mouse peritoneal macrophages, Immunopharmacol. Immunotoxicol. 33 (2011) 525. [18] Q. Bian, H. Yang, C.O. Chan, D. Jin, D.K.W. Mok, S. Chen, Fingerprint analysis and simultaneous determination of phenolic compounds in extracts of curculiginisrhizoma by hplc-diode array detector, Chem. Pharm. Bull. 61 (2013) 802. [19] W.C. Chang, J.S.B. Wu, C.W. Chen, P.L. Kuo, H.M. Chien, Y.T. Wang, S.C. Shen, Protective effect of vanillic acid against hyperinsulinemia, hyperglycemia and hyperlipidemia via alleviating hepatic insulin resistance and inflammation in High-Fat Diet (HFD)-fed rats,Nutrients. 7 (2015) 9946-9959.
11
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
[20] W.C. Lin, Y.F. Peng, C.W. Hou, Ferulic acid protects PC12 neurons against hypoxia by inhibiting the pMAPKs and COX-2 pathways, Iran. J. Basic Med. Sci. 18 (2015) 478. [21] W.P. Chen, L.D. Wu, Chlorogenic acid suppresses interleukin-1β-induced inflammatory mediators in human chondrocytes,Int. J. Clin. Exp. Pathol.7(2014) 8797-8801. [22] S. Roy, S.K. Metya, S. Sannigrahi, N. Rahaman, F. Ahmed, Treatment with ferulic acid to rats with streptozotocin-induced diabetes: effects on oxidative stress, pro-inflammatory cytokines, and apoptosis in the pancreatic β cell, Endocrine, 44 (2013) 369-379. [23] E. Fuentes, J. Caballero, M. Alarcón, A. Rojas, I. Palomo, Chlorogenic acid inhibits human platelet activation and thrombus formation, PloS one. 9 (2014) e90699. [24] K.W. Kim, K.T. Ha, C.S. Park, U.H. Jin, H.W. Chang, I.S. Lee, C.H. Kim, Polygonumcuspidatum, compared with baicalin and berberine, inhibits inducible nitric oxide synthase and cyclooxygenase-2 gene expressions in RAW 264.7 macrophages, Vasc. Pharmacol. 47 (2007) 99-107. [25] J. Li, C. Tang, L. Li, R. Li, Y. Fan, Quercetin sensitizes glioblastoma to t-AUCB by dual inhibition of Hsp27 and COX-2 in vitro and in vivo, J. Exp. Clin. Cancer Res. 35(2016) 1. [26] Z. Li, J. Zheng, N. Zhang, Berberine improves airway inflammation and inhibits NF-κB signaling pathway in an ovalbumin-induced rat model of asthma J. Asthma. 2016. [27] E.K. Schmitt, C.M. Moore, P. Krastel, F. Petersen, Natural products as catalysts for innovation: a pharmaceutical industry perspective, Curr. Opin. Chem. Biol.15 (2011) 497-504. [28] A.L. Harvey, Natural products as a screening resource, Curr. Opin. Chem. Biol.11 (2007) 480-484. [29] D.J. Newman, G.M. Cragg, Natural products as sources of new drugs over the 30 years from 1981 to 2010, J. Nat. Prod. 75(2012) 311-335. [30] G. Molinari, Natural products in drug discovery: present status and perspectives Adv. Exp. Med. Biol. 655 (2009) 13-27. [31] C. Gonçalves, T. Dinis, M.T. Batista, Antioxidant properties of proanthocyanidins of Uncariatomentosa bark decoction: a mechanism for anti-inflammatory activity, Phytochemistry. 66 (2005) 89-98. [32] T. Guardia, A.E. Rotelli, A.O. Juarez, L.E. Pelzer, Anti-inflammatory properties of plant favonoids. Effects of rutin, quercitin and hesperidin on adjuvant arthritis in rat II Farmaco. 56 (2001) 683-687. [33] J. Gonzalez-Gallego, M.V. Garcia-Mediavilla, S.Sanchez-Campos, M.J.Tunon, Fruit polyphenols, immunity and inflammation, Br. J. Nutr.104(2010), 104 Suppl 3, S15-27. [34] W.M. Loke, J.M. Proudfoot, J.M.Hodgson, A.J.McKinley, N.Hime, M.Magat, R. Stocker, K.D.Croft,Specific dietary polyphenols attenuate atherosclerosis in apolipoprotein E-knockout mice by alleviating inflammation and endothelial dysfunction, Arterioscler Thromb Vasc Biol.30 (2010) 749-757. [35] J. Bauer, A. Koeberle, F. Dehm, F. Pollastro, G.Appendino, H.Northoff, A.Rossi, L. Sautebin, O.Werz, Arzanol, a prenylated heterodimeric phloroglucinyl pyrone, inhibits eicosanoid biosynthesis and exhibits antiinflammatory efficacy in vivo, Biochem. Pharmacol. 81 (2011) 259-268. [36] A. Koeberle, H. Northoff, O. Werz, Identification of 5-lipoxygenase and microsomal prostaglandin E2 synthase-1 as functional targets of the anti-inflammatory and anti-carcinogenic garcinol, Biochem. Pharmacol.77(9) (2009) 1513-1521. [37] A.M. Schaible, H. Traber, V.Temml, S.M.Noha, R.Filosa, A. Peduto, C.Weinigel, D.Barz, D.Schuster, O. Werz, Potent inhibition of human 5-lipoxygenase and microsomal prostaglandin E 2 synthase-1 by the anticarcinogenic and anti-inflammatory agent embelin, Biochem. Pharmacol. 86 (2013) 476-486. [38] Y. Moon, W.C.Glasgow, T.E.J. Eling, Curcumin suppresses interleukin 1β-mediated microsomal prostaglandin E synthase 1 by altering early growth response gene 1 and other signaling pathways, Pharmacol. Exp. Ther.315 (2005) 788-795. [39] S. Jang, B.H.Kim, W.-Y. Lee, S.J.An, H.G.Choi, B.H.Jeon, H.-T. Chung, J.-R.Rho, Y.-J. Kim,K.-Y. Chai, Stylopine fromChelidonium majus inhibits LPS-Induced inflammatory mediators in RAW 264.7 cells, Arch. Pharm. Res. 27(2004) 923-929. [40] J. Cortijo, V.Villagrasa, R.Pons, L. Berto, M. Martí‐Cabrera, M. Martinez‐Losa, T. Domenech, J.Beleta, E. Morcillo, Bronchodilator and anti‐inflammatory activities of glaucine: In vitro studies in human airway smooth muscle and polymorphonuclear leukocytes, Br. J. Pharmacol. 127(1999) 1641-1651. [41] A.K. Pandurangan, N.Mohebali, M.Hasanpourghadi, C.Y. Looi, M.R.Mustafa, N.Mohd Esa, Boldine suppresses dextran sulfate sodium‐induced mouse experimental colitis: NF‐κB and IL‐6/STAT3 as potential targets, BioFactors. 2016. [42] C.-W.Yang,W.-L. Chen, P.-L.Wu, H.-Y.Tseng,S.-J.Lee,Anti-inflammatory mechanisms of phenanthroindolizidine alkaloids,Mol. Pharmacol. 69(2006) 749-758. [43] J.F. Evans, C. Leville, J.A.Mancini, P.Prasit, M. Thérien, R. Zamboni, J.Y. Gauthier, R. Fortin,P. Charleson, D.E. MacIntyre, 5-Lipoxygenase Activating Protein is the target of a quinoline class of leukotriene synthesis inhibitors, Mol. Pharmacol. 40 (1991) 22-27. [44] J.F. Evans, A.D. Ferguson, R.T. Mosley, J.H.Hutchinson,What's all the FLAP about? 5-lipoxygenaseactivating protein inhibitors for inflammatory diseases,Trends Pharmacol. Sci. 29 (2008) 72-78.
12
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
[45] M. Lemurell, J. Ulander, S. Winiwarter, C. Whatling, Discovery od AZD6642, an inhibitor of 5Lipoxygenase Activating Protein (FLAP) for the Treatment of Inflammatory Diseases, J. Med. Chem. 58 (2015) 897-911. [46] J.H. Hutchinson, S.Charleson, J.F.Evans, J.-P. Falgueyret, K. Hoogsteen, T.R.Jones, S. Kargman, D. Macdonald, C.S. McFarlane, D.W.Nicholson, H.Piechuta, H.D.Riendeau, J.Scheigetz, M.Therien, Y. Girard, Thiopyranol[2,3,4-c,d]indoles as Inhibitors of 5-Lipoxygenase, 5-Lipoxygenase-Activating Protein, and Leukotriene C4 Synthase, J. Med. Chem. 38 (1995) 4538-4547. [47] R. De Simone, M.G. Chini, I. Bruno, R.Riccio, D.Mueller, O.Werz, G.Bifulco, Structure-based discovery of inhibitors of microsomal prostaglandin E2 synthase-1, 5-lipoxygenase and 5-lipoxygenase-activating protein: promising hits for the development of new anti-inflammatory agents,J. Med Chem.54 (2011) 1565-1575. [48] K.R. Fandrick, J.A.Mulder, N.D. Patel, J. Gao, M.Konrad, E.Archer, F.G. Buono, A.Duran, R.Schmid, J. Daeubler,Development of an Asymmetric Synthesis of a Chiral Quaternary FLAP Inhibitor,J. Org. Chem. 80 (2015) 1651-1660. [49] N.S. Prasad, R. Raghavendra, B.R. Lokesh, K.A. Naidu, Spice phenolics inhibit human PMNL 5lipoxygenase,Prostaglandins Leukotrienes Essent Fatty Acids. 70 (2004) 521-528. [50] H. Raghavenra, B.T. Diwakr, B.R.Lokesh, K.A. Naidu, Eugenol-the active principle from cloves inhibits 5lipoxygenase activity and leukotriene C4 in human PMNL cells, Prostaglandins Leukotrienes Essent Fatty Acids.74(2006) 23-27. [51] D. Aharony, R.L. Stein,Kinetic mechanism of guinea pig neutrophil 5-lipoxygenase,J Biol. Chem. 261(25)(1986) 11512-11519. [52] J. Zhou, D.G. Joplin, J.V. Cross, D.J. Templeton, Sulforaphane Inhibits Prostaglandin E2 Synthesis by Suppressing Microsomal Prostaglandin E Synthase 1, PLOS One. 7(11) (2012) [53] C.M. Recio, I. Andujar, L.J. Rios,Anti-Inflammatory Agents from Plants: Progress and Potential, Curr. Med. Chem. 19(14) (2012) 2088-2103. [54] V. Madka, V.C. Rao, Anti-inflammatory Phytochemicals for Chemoprevention of Colon Cancer,Curr. Cancer Drug Targets. 13(5) (2013) 542-557. [55] M.A. Hull, S.C. Ko, G. Hawcroft, Prostaglandin EP receptors: targets for treatment and prevention of colorectal cancer?, Mol.Cancer Ther. 3 (2004) 1031-1039. [56] M. Murakami, H. Naraba, T. Tanioka, N. Semmyo, Y. Nakatani, F. Kojima, T. Ikeda, M. Fueki, A. Ueno, A. Oh-ishi,Regulation of prostaglandin E2 biosynthesis by inducible membrane-associated prostaglandin E2 synthase that acts in concert with cyclooxygenase-2,J. Biol. Chem. 275(2000) 32783-32792. [57] Y. Li, S. Yin, D. Nie, S. Xie, L. Ma, X. Wang, Y. Wu, J. Xiao, MK886 inhibits the proliferation of HL-60 leukemia cells by suppressing the expression of mPGES-1 and reducing prostaglandin E2 synthesis, Int. J.Hematol. 94 (2011) 472-478.
ACCEPTED MANUSCRIPT
M AN U
SC
RI PT
1
AC C
EP
TE D
Figure 1: Identification of multi-targeted anti-inflammatory molecules; Known anti-inflammatory compounds were used to derive three pharmacophores, viz, Ph-dual, Ph-alk and Ph-FLAP. The query molecules were searched for these pharmacophores and the hits documented. * represents hits molecules present in all the three pharmacophores; # represents those that are mapped onto Ph-dual and Ph-FLAP; ^ shows the compound present in both Ph-dual and Ph-alk; $ represents the molecules mapped onto Ph-alk and Ph-FLAP
ACCEPTED MANUSCRIPT
TE D
M AN U
SC
RI PT
2
AC C
EP
Figure 2: Positioning of the pharmacophoric features of A) Ph-dual B) Ph-alk C) Ph-FLAP as deduced by the pharmacophore elucidation module of MOE2015.1001; Hyd- Hydrophobic centroids & Acc2- Projected acceptor; The distances between the pharmacophores are given in A⁰.
ACCEPTED MANUSCRIPT
SC
RI PT
3
M AN U
Figure 3: Structure of 2,4,5 trimethoxycinnamic acid mapped onto Ph-dual (A), Ph-alk (B) and Ph-FLAP (C); The hydrophobic centroids are depicted in blue spheres and the projected acceptor is shown as red sphere; denotes the element that has hydrophobic property and denotes the atom that possess acceptor property.
Eugenol
100
50
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
TA
0
EP
150
3,5 dimethoxycinnamicacid
2,4,5 trimethoxycinnamicacid
100
AC C
% Inhibition of PGE2
Berberine
Ferulic acid
TE D
% Inhibition of 5-LOX
150
Berberine
50
19
16 17 18
13 14 15
9 10 11 12
7 8
5 6
4
2 3
1
TA
0
Figure 4: Percentage inhibition of (a) 5-LOX extracted from PMNL by natural products at 100 µM concentration and (b) mPGES1 in HeLa cells by natural products at 50 µM concentration; Zileuton(2 µM- 90% inhibition for 5LOX) and Licofelone (6 µM- 55% inhibition for PGE2) are included as reference molecules; TA-Total activity of the enzyme in the absence of test compounds ( Highlights the three best compounds); Compounds 7&8 appear in all the three pharmacophores.
ACCEPTED MANUSCRIPT
EP
TE D
M AN U
SC
RI PT
Pharmacophores were elucidated using three sets of anti-inflammatory molecules. All pharmacophores had three hydrophobic centroids and a projected acceptor feature. Two methoxy derivatives of cinnamic acid mapped onto all the three pharmacophores. Berberine showed good inhibition of 5-LOX activity and PGE2 production.
AC C
1) 2) 3) 4)
S0006-291X(17)30071-2
DOI:
10.1016/j.bbrc.2017.01.046
Reference:
YBBRC 37108
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 22 November 2016 Revised Date:
8 January 2017
Accepted Date: 10 January 2017
Please cite this article as: N.S. Devi, M. Ramanan, P. Paragi-Vedanthi, M. Doble, Phytochemicals as multi-target inhibitors of the inflammatory pathway- A modeling and experimental study, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.01.046. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
ACCEPTED MANUSCRIPT
Phytochemicals as multi-target inhibitors of the inflammatory pathway- A modeling and experimental study Nisha S. Devi#, Meera Ramanan#, Padmapriya Paragi-Vedanthi, Mukesh Doble*
of Technology, Madras, Tamil Nadu, 600036, India. #
Both authors contributed equally.
*Email ID: [email protected]
RI PT
Bhupat&Jyoti Mehta School of Biosciences, Department of Biotechnology, Indian Institute
SC
Keywords: Inflammation; 5-Lipoxygenase; Microsomal Prostaglandin E2 Synthase 1;
AC C
EP
TE D
M AN U
Pharmacophore; Multitarget drugs
2
ACCEPTED MANUSCRIPT Abstract The arachidonic acid pathway consists of several enzymes and targeting them is favored for developing antiinflammatory drugs. However, till date the current drugs are generally active against a single target, leading to undesirable side-effects. Phytochemicals are known to inhibit multiple targets simultaneously and hence, an
RI PT
attempt is made here to investigate their suitability. A pharmacophore based study is performed with three sets of reported phytochemicals namely, dual 5-LOX/mPGES1, alkaloids and FLAP inhibitors. The analysis
indicated that phenylpropanoids (including ferulic acid) and benzoic acids derivatives, and berberine mapped onto these pharmacophores with three hydrophobic centroids and an acceptor feature. 2,4,5-trimethoxy (7) and
SC
3,4-dimethoxy cinnamic acids (8) mapped onto all the three pharmacophores. Experimental studies indicated that berberine inhibited 5-LOX and PGE2 production by 72.2 and 72.0 % and ferulic acid by 74.3 and 54.4 %
M AN U
respectively. This approach offers a promising theoretical combined with experimental strategy for designing novel molecules against inflammatory enzymes. Abbreviations:
LT: Leukotriene; PG: Prostaglandin;5-LOX: 5-Lipoxygenase;COX: Cyclooxygenase;FLAP: 5-Lipoxygenase
TE D
Activating Protein;PGE2: Prostaglandin E2;mPGES1: microsomal Prostaglandin E Synthase 1; NF-kB: Nuclear Factor-κ-light chain enhancer of activated B cells; STAT-3: Signal Transducer and Activator of Transcription 3.
EP
The arachidonic acid pathway is responsible for the production of the inflammatory mediators, leukotrienes (LT) and prostaglandins (PG), via the lipoxygenase (LOX) and cyclooxygenase (COX) and
AC C
pathways respectively. The LTs are synthesized mainly by the inflammatory cells including granulocytes, monocytes and mast cells and are responsible for the pathogenesis of asthma allergy and rheumatoid arthritis1-3. However, drugs targeting the enzymes in this pathway namely, 5-lipoxygenase (5LOX), are yet to enter the market. The only drug developed against this enzyme, Zileuton, is withdrawn in 2008 due to hepatotoxicity but still the extended release formulation of it is available in the market4. Several inhibitors for 5-lipoxyganase activating protein (FLAP), a helper protein of the leukotriene pathway, are under clinical trials but none have entered the market till date5. The prostaglandins are synthesized on being stimulated by injury or under the influence of proinflammatory cytokines and other stimuli1. They have been implicated in the development of both acute and
3
ACCEPTED MANUSCRIPT chronic inflammation and the most notable among them is prostaglandin E2 (PGE2), which is also involved in the progression of several cancers 6. Many of the drugs developed till date, including the non-steroidal antiinflammatory drugs (NSAIDs) and COX-2 inhibitors (Coxibs) which target COX-1/2 enzymes have been
associated with gastric toxicity and cardiovascular complications 7, 8. Hence, targeting microsomal prostaglandin E synthase1 (mPGES1) downstream of these two enzymes, for specifically inhibiting the production of PGE2 is
RI PT
being touted as a promising strategy for development of anti-inflammatory drugs 9.
Although the anti-inflammatory drugs, both currently available and under development, are mostly active against a single enzyme, studies have shown that inhibiting a single pathway results in the shunting of the
SC
substrate arachidonic acid to the other one, thus negating the therapeutic intervention 10. This has lead to interest in the development of dual inhibitors against 5-LOX/mPGES1 and COX/LOX 11. Studies have showed that for
M AN U
multi-factorial diseases such as inflammation, drugs active on multiple targets have better efficiency and safety profiles. Experimental studies have shown that 5-LOX/mPGES1 dual inhibitors such as a pirinixic acid derivative reduce vascular remodeling in a murine model of aortic aneurysm without significantly inhibiting COX1/212. Licofelone, a multi-targeted inhibitor which inhibits COX-1, mPGES1 and FLAP has completed Phase III clinical trials for arthritis treatment 13.
TE D
Phytochemicals (such as curcumin) have been shown to have multi-targeted action against COX-1, 5LOX and mPGES1 and signaling molecules such as NF-κB, stat-3 and P53 14-26(Supplementary table 1). Natural products are capable of interacting with a wide range of biological macromolecules, occupy a diverse chemical
EP
space and exhibit better drug-like properties when compared to synthetic molecules, making them a promising resource for identification of leads molecules for drug development 27, 28. A study by David J. Newman (2012)
AC C
found that in the period ranging from 1940s to 2010, of the 175 small molecules approved for cancer treatment, about half of them were either phytochemicals or their direct derivatives 29. More than 60% of the drugs in the market are derived from natural compounds 30.This points to the importance of these molecules as excellent sources for identifying novel scaffolds and leads for drug development. Several phytochemicals have been shown to have anti-inflammatory properties and have been in use for a long time in traditional medicines 31. Flavonoids such as quercetin have been shown to inhibit both acute and chronic phases of inflammation in rats 32. They purportedly act by downregulating the expressions and activities of the enzymes involved in the synthesis of inflammatory mediators such as nitric oxide, prostaglandins and leukotrienes 33. Dietary polyphenols have also been shown to attenuate atherosclerosis by
4
ACCEPTED MANUSCRIPT alleviating inflammation in mouse models of atherosclerosis 34. These findings suggest the possibility of utilising the chemical diversity of phytochemicals and their reported anti-inflammatory action in the design of multi-target drugs against inflammation. The important functional features of a biomolecule are represented by a pharmacophore which is a set
RI PT
of features including hydrogen bond donors, acceptors, hydrophobic and aromatic centers that identify their properties necessary for the ligand-enzyme interaction. Pharmacophore studies provide an insight on the
structural and chemical properties shared by active molecules and can be a powerful tool for zeroing in on
potential inhibitors. In the current study, this approach is used to identify phytochemicals which may serve as
SC
starting scaffolds for development of dual or multi-target inhibitors against these enzymes/proteins involved in inflammation. The possible candidates identified by this study are experimentally tested against the two
M AN U
enzymes, 5-LOX andmPGES1.The pharmacophore based approach for identifying leads coupled with experimental studies hold the promise of developing molecules which can exert a multi-pronged influence towards treating inflammation. Materials and Methods
TE D
All the phytochemicals were purchased from Sigma-Aldrich and Sisco Research Laboratories (SRL, Mumbai, India). HeLa cells were purchased from NCCS (Pune, India) and maintained in Dulbecco’s Minimal Essential Medium (DMEM) at 37°C with 5% CO2. The PGE2-EIA kit was obtained from Cayman chemicals (# 514010, Ann Arbor, MI). The leucocyte concentrates for the 5-LOX enzyme assay were obtained from healthy
EP
human volunteers with the necessary ethical clearance.
AC C
The overall scheme of this work has been outlined in Fig 1. More detailed information is given in supplementary section.
Pharmacophore elucidation of inhibitors of inflammatory enzymes The pharmacophoric features of a group of molecules are derived using the ‘Pharmacophore
elucidation’ module of Molecular Operating Environment (MOE) 2015.2001 software (Chemical Computing Group, Quebec, Canada).
5
ACCEPTED MANUSCRIPT Docking of hit molecules with inflammatory enzymes The representative hit molecules identified from the pharmacophore search were docked to the active sites of 5-LOX, mPGES1 and FLAP using Goldsuite 5.2.
RI PT
Extraction of 5-LOX protein from human blood: 5-LOX is isolated from the leukocyte concentrates (Buffy coats) of healthy human donors obtained from blood bank (Voluntary Health Services, Chennai) following the approved procedure after obtaining the necessary ethical clearance. The enzyme activity was determined following a standard protocol 50, 51.
SC
Estimation of PGE2 production:
The inhibition of the prostaglandin synthesis pathway was detected by measuring the amount of
M AN U
the COX-2/mPGES1 product, prostaglandin E2 (PGE2), as previously described 52.
Results and Discussion
Several phytochemicals have been shown to be active against chronic inflammatory diseases including rheumatoid arthritis and inflammatory bowel disease53.They have also been found to aid in the
TE D
prevention and treatment of inflammation associated with malignancies such as colorectal cancer54. The identification of their target could shed light on the mechanism by which they exert their anti-inflammatory effects. For this study, we have chosen a range of phenylpropanoids, benzoic acids and alkaloids present as
EP
active components in several medicinal plants with previously documented anti-inflammatory activity. We have attempted to deduce the action of natural products on pro-inflammatory enzymes using both
AC C
computational and experimental strategies and explore their potential as the starting structure for the development of multi-target anti-inflammatory drugs.
Pharmacophore elucidation
The elucidation of pharmacophoric features common to a set of enzyme inhibitors provides
important information regarding the features essential for their interaction. In an attempt to study the features common to anti-inflammatory molecules across different targets, we collected phytochemicals belonging to three structurally diverse groups: phenolic 5-LOX/mPGES1 dual inhibitors, alkaloids with known anti-inflammatory functions and FLAP inhibitors (Supplementary fig 2).
6
ACCEPTED MANUSCRIPT Five phenolic phytochemicals which are known to have the 5-LOX/mPGES1 dual inhibitory activity were selected, namely, arzanol, garcinol, embelin, curcumin and trans-resveratrol (Supplementary fig 1A)14, 35-38. Arzanol has a phloroglucinylpyrone ring while Garcinol consists of polyprenylated chain
and is used in traditional medicine for the treatment of inflammation. Embelin has a benzoquinone moiety and a lipophilic tail and curcumin consists of a lipophilic linker and multiple phenolic groups and is a
RI PT
mainstay in the Indian traditional medicine system of Ayurveda against inflammation. Trans-resveratrol is a phenol which has a good anti-oxidant activity and is a prominent anti-inflammatory agent in folk medicine. A four-point pharmacophore (Ph-dual) elucidated from these five compounds has three
SC
hydrophobic centroids (Hyd) and one projected hydrogen bond acceptor (Acc2) (Fig 2A). This Ph-dual was set as a query against an in-house conformational database of natural products (mainly phenylpropanoids, benzoic acids and alkaloids) derived from active components of medicinal plants
M AN U
(Supplementary fig 2). It was found that eight compounds, consisting of phenylpropanoids including 2,4,5trimethoxy cinnamic acid (7) and benzoic acids such as 2,3-dimethoxy benzoic acid (2),map onto this pharmacophore.
Since alkaloids are an important constituent in phytochemicals having anti-inflammatory properties,
TE D
studying their common pharmacophore features will aid in the design of novel drugs. The second set of known anti-inflammatory alkaloid phytochemicals selected includes stylopine, glaucine, boldine and tylophorine (Supplementary fig 1B) 39-42. The salient structural features of the pharmacophore thus derived (Ph-alk) (Fig 2B)
EP
consists of fused aromatic and heterocyclic rings and presence of nitrogen as part of the ring. Stylopine is reported to inhibit several inflammatory molecules namely COX-2, PGE2, NO, iNOS and inflammatory
AC C
cytokines such as TNFα, IL-1β and IL-6. Glaucine is a potent inhibitor of phosphodiesterase-4 and is used in the treatment of asthma in traditional medicine. Boldine reduces the levels of TNFα, NF-κb and IL-6 while tylophorine is a phenanthraindolizidine with significant inhibition of the pro-inflammatory molecules IL-6, TNFα, IFNγ, NO. Hence these set of alkaloids exhibits their anti-inflammatory activity through different mechanisms. The elucidated pharmacophore (Ph-alk) (Fig 2B) for these compounds was found to consist of three hydrophobic centroids and one projected acceptor. In a pharmacophore search of our previously mentioned natural products database (Supplementary fig 2), berberine (17), ferulic acid (12) and other phenylpropanoids were found to share these pharmacophoric features.
7
ACCEPTED MANUSCRIPT FLAP is a carrier protein for 5-LOX, which makes it an important drug target and could be a site of action for multi-target drugs. A 4-point pharmacophore elucidated from eight reported FLAP inhibitors (Ph-FLAP)43-48(Supplementary fig 1C) including indole-sulphur compounds and quinolones (Fig 2C) consisted of three hydrophobic centroids and one projected acceptor. Ph-FLAP was able to identify
berberine (17), a few phenylpropanoids and benzoic acid derivatives from the database to possess the same
RI PT
pharmacophoric features.
A comparison of Ph-dual, Ph-alk and Ph-FLAP(Fig 4) indicated that the three hydrophobic
centroid groups (F1, F2 and F3)are approximately at the same distance from one another, namely, the
SC
distances between F1-F2, F2-F3 and F3-F1 ranged between 2.2-3.3, 3.2-3.4 and 5.2-5.7 respectively. 2,4,5trimethoxy cinnamic acid (7) and 3,4-dimethoxy cinnamic acid(8) were found to possess all the three pharmacophores. Berberine (17) mapped onto Ph-alk and Ph-FLAP; 2,3-dimethoxy benzoic acid (2)and
M AN U
2,5-dimethoxy benzoic acid (3) onto Ph-dual and Ph-FLAP and methyl eugenol (14) onto Ph-dual and Phalk. The structures of these phytochemicals were overlapped with the pharmacophores to determine the groups that contributed to these structural features (Fig 3). The common groups contributing to the hydrophobic centroids features could be attributed to three specific groups in the case of 2,4,5-trimethoxy (7) and 3,4-dimethoxy cinnamic acid (8). These included the C=C present in the propionic acid tail, the
TE D
benzene ring and the –CH3 group of the methoxy tail. The projected acceptor group is contributed by the phenolic oxygen of the propionic acid tail. In the case of berberine, the hydrophobic moieties are the tetracyclic skeleton while the acceptor feature is the oxygen atom in the azapentacyclo ring. The presence
EP
of three hydrophobic centroid features appears to be crucial for the anti-inflammatory activity and hence could be an important factor for designing novel drugs for this ailment. Based on these pharmacophore
AC C
searches, a group of nine phenyl propanoids, six benzoic acid derivatives, alkaloids and other diverse phytochemicals were shortlisted for in vitro testing of inhibition of 5-LOX activity and PGE2 production. The importance of these hydrophobic centroids in aiding the interaction of the compounds with the active site residues of the inflammatory enzymes was revealed by the docking analysis. Molecular docking of ligand with enzyme is used to study the binding interactions and the preferred pose of the ligand in the binding with the enzyme using various conformational search algorithms. In our case, 2,4,5- trimethoxy cinnamic acid was selected as the representative molecule since it mapped onto all the three pharmacophores and for docking studies against 5-LOX, FLAP and mPGES1 (Supplementary fig 3). The analysis revealed extensive Van der waals interactions with the hydrophobic amino acids of the 5-LOX
8
ACCEPTED MANUSCRIPT proteinnamelyPhe610, Leu607 and Val604 (Supplementary fig 3A). The compound also showed hydrophobic interactions with the residues such as Tyr112, Ile113 and Phe114 in FLAP protein (Supplementary fig 3B). Interaction of this compound with the hydrophobic residues such as Gly35 and Leu39 present in mPGES1 was also observed (Supplementary fig 3C). The hydrophobic centroids identified in the pharmacophore search appear to be crucial for the interaction with the various
Inhibition of 5-LOX and COX-2/mPGES1 by phytochemicals
RI PT
hydrophobic aminoacids that lines the active site of these inflammatory proteins.
Eugenol, ferulic acid and cinnamic acid inhibited 5-LOX by 86.5±0.6%, 74.4±4.2% and
SC
66.6±2.6% respectively at a concentration of 100 µ M (Fig 4). The alkaloid, berberine, also showed a high inhibition (72.2±3.5%) while flavonoid, quercetin, showed an inhibition of 55.2±2.4%. Of the benzoic acid derivatives tested, 4-hydroxy 3-methoxy benzoic acid (vanillic acid) showed the maximum inhibition
M AN U
(50.3±5.2%) of 5-LOX.
4-hydroxy 3-methoxy benzoic acid (vanillic acid) at a concentration of 50 µ Mshowed the maximum inhibition of PGE2 production(87.1±3.5%). Among the phenylpropanoids, 3,4dimethoxycinnamic acid exhibited the highest inhibition (86.7±11.3%). The alkaloid, berberine, was inhibited by 71.9±5.1% while the flavonoid quercetin was inhibited by 84.1±6.6%. Potent mPGES1
TE D
inhibitors including MK886 are shown to be ineffective in in vivo systems due to their plasma binding tendency(in vitro IC50- 1.6 µM; ~ 20% inhibition for 100 µM in whole blood) whereas our approach of inhibiting PGE2 in vivo in HeLa cells appear to be a better strategy to a priori address the above mentioned
EP
problem.
An elevated level of PGE2 is observed under inflammatory conditions and it functions by binding
AC C
to four EP receptors (EP1-4) and amplifying the inflammatory signals 55. It is also associated with the development of cancer as it participates in metastasis and angiogenesis. COX-2 and mPGES1 are found to be functionally coupled in the synthesis of PGE2, thus favoring the progression of inflammation 56.Thus, inhibiting PGE2 promises to attenuate inflammation and indeed, has been shown to halt the progression of leukaemia and a decrease of anti-apoptotic markers57. The above results indicate that compounds such as 2,4,5-trimethoxy cinnamic acid, berberine, vanillic acid and quercetin inhibit 5-LOX as well as the enzymes that are involved in the production of PGE2, namely, COX and mPGES1. The phenylpropanoid 2,4,5-cinnamic acid which mapped onto all 3 pharmacophores viz, Ph-dual, Ph-alk and Ph-FLAP, also showed good activity against 5-LOX (47.6%) and PGE2 production (79.9%).
9
ACCEPTED MANUSCRIPT Another phenylpropanoid, methyl eugenol, which mapped onto two pharmacophores, namely, Ph-dual and Ph-
alk, also inhibit 5-LOX (49.5%) and PGE2 production (78.3% resp.).The alkaloid, berberine which mapped onto Ph-alk and Ph-FLAP also exhibited significant inhibition of 5-LOX activity (72.19%) as well as PGE2 production (71.88%). Two benzoic acid derivatives, namely, 2,3- and 2,5-dimethoxy benzoic acid which possessed the pharmacophoric features of Ph-dual and Ph-FLAP, exhibited moderate inhibition of 5-LOX
RI PT
activity (33.7 and 42.0% resp.) and PGE2 production (18.2 and 80.1 % resp.). These studies indicate that
phenylpropanoids and alkaloids have the pharmacophoric features necessary for efficient anti-inflammatory action which is further validatedfrom our experimental studies, hence suggesting the possibility of developing
.
SC
these molecules as a multi-target inhibitor of the arachidonic acid pathway.
Inflammation has a complex pathology characterized by the interplay of several inflammatory
M AN U
enzymes, cytokines and mediators. For such multi-factorial diseases, the strategy of targeting single enzymes may not lead to effective treatment of the underlying condition, as seen in the case of NSAIDs and coxibs with unwanted side effects. Since an anti-inflammatory drug with minimal side-effects remains a distant possibility, a multi-prongedtargeting of the enzymes in the inflammatory pathway seems to be need of the hour.In this regard, we have attempted to use a systematic approach involving theoretical and experimental strategy towards
TE D
exploring the features of phytochemicals which may enable them to target several inflammatory enzymes such as 5-LOX, mPGES1 and FLAP simultaneously. Pharmacophore studies allowed us to identify chemical features which are prevalent in several reported anti-inflammatory molecules which target a range of protein/enzymes.
EP
This allowed us to rationally select phytochemicals to test against these inflammatory enzymes experimentally. The pharmacophore analysis showed that the inhibitors of 5-LOX, mPGES1 and FLAP had three hydrophobic
AC C
centroid features and a projected acceptor. This information could be an useful guide while designing drugs active on multiple points of the inflammatory pathway. Further studies using other inflammatory enzymes could add meaningful insights into the present findings. Our studies showed that phenylpropanoids such as 2,4,5trimethoxy cinnamic acid and berberine are privileged structures which mapped onto multiple pharmacophores and also showed significant inhibition of 5-LOX and COX-2/mPGES1 activities, thus underlining their importance as starting structures for multi-target drugs.
Acknowledgement & Funding sources
10
ACCEPTED MANUSCRIPT NSD acknowledges Council of Scientific and Industrial Research (CSIR) for the fellowship; MR and PPV acknowledge WOS-A, DST, India for the project funding and fellowship. Author contributions:
RI PT
MD conceived and supervised the study; NSD & MR designed & performed experiments; NSD & PV standardized 5-LOX extraction protocol; MD analyzed the data; NSD & MR wrote the manuscript which was revised by MD. References:
SC
[1] C. D. Funk, Prostaglandin and Leukotrienes: Advances in Eicosanoid Biology, Sci. Transl. Med. (2001) 294, 1871-1875. [2] A.S. Cowburn, K. Sladek, J. Soja, L. Adamek, E. Nizankowska, A. Szczeklik, B.K. Lam, J.F. Penrose, F.K. Austen, S.T. Holgate, A.P. Sampson,Overexpression of leukotriene C4 synthase in bronchial biopsies from patients with aspirin-intolerant asthma, The Journal of clinical investigation. 101(1998)834-846. [3] B. Yousefi, F. Jadidi-Niaragh, G. Azizi, F.Hajighasemi, A. Mirshafiey, The role of leukotrienes in immunopathogenesis of rheumatoid arthritis,Mod. Rheumatol. 24(2)(2014) 225-235.
M AN U
[4] E.M. Joshi, B.H. Heasley, M.D. Chordia, T.L. Macdonald, In Vitro Metabolism of 2-Acetylbenzothiophene: Relevance to Zileuton Hepatotoxicity,Chem. Res. Toxicol. 17 (2004) 137-143.
AC C
EP
TE D
[5] D. Pettersen, O. Davidsson, C. Whatling,Recent advances for FLAP inibitors, Bioorg. Med. Chem Lett. 25 (2015) 2607-2612. [6] E. Ricciotti, G.A. FitzGerald, Prostaglandins and inflammation, Arterioscler. Thromb. Vasc. Biol.31 (2011) 986-1000. [7] H. Matsui, O. Shimokawa,T. Kaneko, Y. Nagano, K. Rai, I.J. Hyodo, The pathophysiology of non-steroidal anti-inflammatory drug (NSAID)-induced mucosal injuries in stomach and small intestine, Clin. Biochem. Nutr. 48 (2011) 107-111. [8] C.P. Cannon, P.J. Cannon, COX-2 inhibitors and cardiovascular risk, Science. 2012, 336, 1386-1387. [9] A. Koeberle, O. Werz,Perspective of microsomal prostaglandin E2 synthase-1 as drug target in inflammation-related disorders, Biochem. Pharmacol.98 (2015) 1-15. [10] R. Ganesh, D.J. Marks, K. Sales, M.C. Winslet, A.M. Seifalian, Cyclooxygenase/lipoxygenase shunting lowers the anti-cancer effect of cyclooxygenase-2 inhibition in colorectal cancer cells, World journal of surgical oncology.10(2012) 200. [11] F. Celotti, S. Laufer, Anti-inflammatory drugs: new multitarget compounds to face an old problem. The dual inhibition concept, Pharmacol. Res.43 (2001) 429. [12] M. Revermann, A.Mieth, L. Popescu, A. Paulke, M. Wurglics, M. Pellowska, A.S. Fischer, R. Steri, T.J. Maier, R.T. Schermuly, G. Geisslinger,A pirinixic acid derivative (LP105) inhibits murine 5-lipoxygenase activity and attenuatesvascular remodelling in a murine model of aortic aneurysm, Br. J. Pharmacol.163 (2011) 1721. [13] J.N. Dulin, M.L. Moore, R.J. Grill, The dual cyclooxygenase/5-lipoxygenase inhibitor licofelone attenuates p-glycoprotein-mediated drug resistance in the injured spinal cord, Journal of neurotrauma. 30 (2013) 211-226. [14] A. Koeberle, O. Werz,Multi-target approach for natural products in inflammation, Drug discovery today. 19 (2014) 1871-1882. [15] H.H. Chang, E.J. Meuillet, Identification and development of mPGES-1 inhibitors: where we are at?Future Med. Chem. 3(2011) 1909-1934. [16] http://phenol-explorer.eu/. [17] M.C. Kim, S.J. Kim, D.S. Kim, Y.D. Jeon, S.J. Park, H.S. Lee, J.Y. Um, S.H. Hong, Vanillic acid inhibits inflammatory mediators by suppressing NF-kB in lipopolysaccharide-stimulated mouse peritoneal macrophages, Immunopharmacol. Immunotoxicol. 33 (2011) 525. [18] Q. Bian, H. Yang, C.O. Chan, D. Jin, D.K.W. Mok, S. Chen, Fingerprint analysis and simultaneous determination of phenolic compounds in extracts of curculiginisrhizoma by hplc-diode array detector, Chem. Pharm. Bull. 61 (2013) 802. [19] W.C. Chang, J.S.B. Wu, C.W. Chen, P.L. Kuo, H.M. Chien, Y.T. Wang, S.C. Shen, Protective effect of vanillic acid against hyperinsulinemia, hyperglycemia and hyperlipidemia via alleviating hepatic insulin resistance and inflammation in High-Fat Diet (HFD)-fed rats,Nutrients. 7 (2015) 9946-9959.
11
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
[20] W.C. Lin, Y.F. Peng, C.W. Hou, Ferulic acid protects PC12 neurons against hypoxia by inhibiting the pMAPKs and COX-2 pathways, Iran. J. Basic Med. Sci. 18 (2015) 478. [21] W.P. Chen, L.D. Wu, Chlorogenic acid suppresses interleukin-1β-induced inflammatory mediators in human chondrocytes,Int. J. Clin. Exp. Pathol.7(2014) 8797-8801. [22] S. Roy, S.K. Metya, S. Sannigrahi, N. Rahaman, F. Ahmed, Treatment with ferulic acid to rats with streptozotocin-induced diabetes: effects on oxidative stress, pro-inflammatory cytokines, and apoptosis in the pancreatic β cell, Endocrine, 44 (2013) 369-379. [23] E. Fuentes, J. Caballero, M. Alarcón, A. Rojas, I. Palomo, Chlorogenic acid inhibits human platelet activation and thrombus formation, PloS one. 9 (2014) e90699. [24] K.W. Kim, K.T. Ha, C.S. Park, U.H. Jin, H.W. Chang, I.S. Lee, C.H. Kim, Polygonumcuspidatum, compared with baicalin and berberine, inhibits inducible nitric oxide synthase and cyclooxygenase-2 gene expressions in RAW 264.7 macrophages, Vasc. Pharmacol. 47 (2007) 99-107. [25] J. Li, C. Tang, L. Li, R. Li, Y. Fan, Quercetin sensitizes glioblastoma to t-AUCB by dual inhibition of Hsp27 and COX-2 in vitro and in vivo, J. Exp. Clin. Cancer Res. 35(2016) 1. [26] Z. Li, J. Zheng, N. Zhang, Berberine improves airway inflammation and inhibits NF-κB signaling pathway in an ovalbumin-induced rat model of asthma J. Asthma. 2016. [27] E.K. Schmitt, C.M. Moore, P. Krastel, F. Petersen, Natural products as catalysts for innovation: a pharmaceutical industry perspective, Curr. Opin. Chem. Biol.15 (2011) 497-504. [28] A.L. Harvey, Natural products as a screening resource, Curr. Opin. Chem. Biol.11 (2007) 480-484. [29] D.J. Newman, G.M. Cragg, Natural products as sources of new drugs over the 30 years from 1981 to 2010, J. Nat. Prod. 75(2012) 311-335. [30] G. Molinari, Natural products in drug discovery: present status and perspectives Adv. Exp. Med. Biol. 655 (2009) 13-27. [31] C. Gonçalves, T. Dinis, M.T. Batista, Antioxidant properties of proanthocyanidins of Uncariatomentosa bark decoction: a mechanism for anti-inflammatory activity, Phytochemistry. 66 (2005) 89-98. [32] T. Guardia, A.E. Rotelli, A.O. Juarez, L.E. Pelzer, Anti-inflammatory properties of plant favonoids. Effects of rutin, quercitin and hesperidin on adjuvant arthritis in rat II Farmaco. 56 (2001) 683-687. [33] J. Gonzalez-Gallego, M.V. Garcia-Mediavilla, S.Sanchez-Campos, M.J.Tunon, Fruit polyphenols, immunity and inflammation, Br. J. Nutr.104(2010), 104 Suppl 3, S15-27. [34] W.M. Loke, J.M. Proudfoot, J.M.Hodgson, A.J.McKinley, N.Hime, M.Magat, R. Stocker, K.D.Croft,Specific dietary polyphenols attenuate atherosclerosis in apolipoprotein E-knockout mice by alleviating inflammation and endothelial dysfunction, Arterioscler Thromb Vasc Biol.30 (2010) 749-757. [35] J. Bauer, A. Koeberle, F. Dehm, F. Pollastro, G.Appendino, H.Northoff, A.Rossi, L. Sautebin, O.Werz, Arzanol, a prenylated heterodimeric phloroglucinyl pyrone, inhibits eicosanoid biosynthesis and exhibits antiinflammatory efficacy in vivo, Biochem. Pharmacol. 81 (2011) 259-268. [36] A. Koeberle, H. Northoff, O. Werz, Identification of 5-lipoxygenase and microsomal prostaglandin E2 synthase-1 as functional targets of the anti-inflammatory and anti-carcinogenic garcinol, Biochem. Pharmacol.77(9) (2009) 1513-1521. [37] A.M. Schaible, H. Traber, V.Temml, S.M.Noha, R.Filosa, A. Peduto, C.Weinigel, D.Barz, D.Schuster, O. Werz, Potent inhibition of human 5-lipoxygenase and microsomal prostaglandin E 2 synthase-1 by the anticarcinogenic and anti-inflammatory agent embelin, Biochem. Pharmacol. 86 (2013) 476-486. [38] Y. Moon, W.C.Glasgow, T.E.J. Eling, Curcumin suppresses interleukin 1β-mediated microsomal prostaglandin E synthase 1 by altering early growth response gene 1 and other signaling pathways, Pharmacol. Exp. Ther.315 (2005) 788-795. [39] S. Jang, B.H.Kim, W.-Y. Lee, S.J.An, H.G.Choi, B.H.Jeon, H.-T. Chung, J.-R.Rho, Y.-J. Kim,K.-Y. Chai, Stylopine fromChelidonium majus inhibits LPS-Induced inflammatory mediators in RAW 264.7 cells, Arch. Pharm. Res. 27(2004) 923-929. [40] J. Cortijo, V.Villagrasa, R.Pons, L. Berto, M. Martí‐Cabrera, M. Martinez‐Losa, T. Domenech, J.Beleta, E. Morcillo, Bronchodilator and anti‐inflammatory activities of glaucine: In vitro studies in human airway smooth muscle and polymorphonuclear leukocytes, Br. J. Pharmacol. 127(1999) 1641-1651. [41] A.K. Pandurangan, N.Mohebali, M.Hasanpourghadi, C.Y. Looi, M.R.Mustafa, N.Mohd Esa, Boldine suppresses dextran sulfate sodium‐induced mouse experimental colitis: NF‐κB and IL‐6/STAT3 as potential targets, BioFactors. 2016. [42] C.-W.Yang,W.-L. Chen, P.-L.Wu, H.-Y.Tseng,S.-J.Lee,Anti-inflammatory mechanisms of phenanthroindolizidine alkaloids,Mol. Pharmacol. 69(2006) 749-758. [43] J.F. Evans, C. Leville, J.A.Mancini, P.Prasit, M. Thérien, R. Zamboni, J.Y. Gauthier, R. Fortin,P. Charleson, D.E. MacIntyre, 5-Lipoxygenase Activating Protein is the target of a quinoline class of leukotriene synthesis inhibitors, Mol. Pharmacol. 40 (1991) 22-27. [44] J.F. Evans, A.D. Ferguson, R.T. Mosley, J.H.Hutchinson,What's all the FLAP about? 5-lipoxygenaseactivating protein inhibitors for inflammatory diseases,Trends Pharmacol. Sci. 29 (2008) 72-78.
12
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
[45] M. Lemurell, J. Ulander, S. Winiwarter, C. Whatling, Discovery od AZD6642, an inhibitor of 5Lipoxygenase Activating Protein (FLAP) for the Treatment of Inflammatory Diseases, J. Med. Chem. 58 (2015) 897-911. [46] J.H. Hutchinson, S.Charleson, J.F.Evans, J.-P. Falgueyret, K. Hoogsteen, T.R.Jones, S. Kargman, D. Macdonald, C.S. McFarlane, D.W.Nicholson, H.Piechuta, H.D.Riendeau, J.Scheigetz, M.Therien, Y. Girard, Thiopyranol[2,3,4-c,d]indoles as Inhibitors of 5-Lipoxygenase, 5-Lipoxygenase-Activating Protein, and Leukotriene C4 Synthase, J. Med. Chem. 38 (1995) 4538-4547. [47] R. De Simone, M.G. Chini, I. Bruno, R.Riccio, D.Mueller, O.Werz, G.Bifulco, Structure-based discovery of inhibitors of microsomal prostaglandin E2 synthase-1, 5-lipoxygenase and 5-lipoxygenase-activating protein: promising hits for the development of new anti-inflammatory agents,J. Med Chem.54 (2011) 1565-1575. [48] K.R. Fandrick, J.A.Mulder, N.D. Patel, J. Gao, M.Konrad, E.Archer, F.G. Buono, A.Duran, R.Schmid, J. Daeubler,Development of an Asymmetric Synthesis of a Chiral Quaternary FLAP Inhibitor,J. Org. Chem. 80 (2015) 1651-1660. [49] N.S. Prasad, R. Raghavendra, B.R. Lokesh, K.A. Naidu, Spice phenolics inhibit human PMNL 5lipoxygenase,Prostaglandins Leukotrienes Essent Fatty Acids. 70 (2004) 521-528. [50] H. Raghavenra, B.T. Diwakr, B.R.Lokesh, K.A. Naidu, Eugenol-the active principle from cloves inhibits 5lipoxygenase activity and leukotriene C4 in human PMNL cells, Prostaglandins Leukotrienes Essent Fatty Acids.74(2006) 23-27. [51] D. Aharony, R.L. Stein,Kinetic mechanism of guinea pig neutrophil 5-lipoxygenase,J Biol. Chem. 261(25)(1986) 11512-11519. [52] J. Zhou, D.G. Joplin, J.V. Cross, D.J. Templeton, Sulforaphane Inhibits Prostaglandin E2 Synthesis by Suppressing Microsomal Prostaglandin E Synthase 1, PLOS One. 7(11) (2012) [53] C.M. Recio, I. Andujar, L.J. Rios,Anti-Inflammatory Agents from Plants: Progress and Potential, Curr. Med. Chem. 19(14) (2012) 2088-2103. [54] V. Madka, V.C. Rao, Anti-inflammatory Phytochemicals for Chemoprevention of Colon Cancer,Curr. Cancer Drug Targets. 13(5) (2013) 542-557. [55] M.A. Hull, S.C. Ko, G. Hawcroft, Prostaglandin EP receptors: targets for treatment and prevention of colorectal cancer?, Mol.Cancer Ther. 3 (2004) 1031-1039. [56] M. Murakami, H. Naraba, T. Tanioka, N. Semmyo, Y. Nakatani, F. Kojima, T. Ikeda, M. Fueki, A. Ueno, A. Oh-ishi,Regulation of prostaglandin E2 biosynthesis by inducible membrane-associated prostaglandin E2 synthase that acts in concert with cyclooxygenase-2,J. Biol. Chem. 275(2000) 32783-32792. [57] Y. Li, S. Yin, D. Nie, S. Xie, L. Ma, X. Wang, Y. Wu, J. Xiao, MK886 inhibits the proliferation of HL-60 leukemia cells by suppressing the expression of mPGES-1 and reducing prostaglandin E2 synthesis, Int. J.Hematol. 94 (2011) 472-478.
ACCEPTED MANUSCRIPT
M AN U
SC
RI PT
1
AC C
EP
TE D
Figure 1: Identification of multi-targeted anti-inflammatory molecules; Known anti-inflammatory compounds were used to derive three pharmacophores, viz, Ph-dual, Ph-alk and Ph-FLAP. The query molecules were searched for these pharmacophores and the hits documented. * represents hits molecules present in all the three pharmacophores; # represents those that are mapped onto Ph-dual and Ph-FLAP; ^ shows the compound present in both Ph-dual and Ph-alk; $ represents the molecules mapped onto Ph-alk and Ph-FLAP
ACCEPTED MANUSCRIPT
TE D
M AN U
SC
RI PT
2
AC C
EP
Figure 2: Positioning of the pharmacophoric features of A) Ph-dual B) Ph-alk C) Ph-FLAP as deduced by the pharmacophore elucidation module of MOE2015.1001; Hyd- Hydrophobic centroids & Acc2- Projected acceptor; The distances between the pharmacophores are given in A⁰.
ACCEPTED MANUSCRIPT
SC
RI PT
3
M AN U
Figure 3: Structure of 2,4,5 trimethoxycinnamic acid mapped onto Ph-dual (A), Ph-alk (B) and Ph-FLAP (C); The hydrophobic centroids are depicted in blue spheres and the projected acceptor is shown as red sphere; denotes the element that has hydrophobic property and denotes the atom that possess acceptor property.
Eugenol
100
50
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
TA
0
EP
150
3,5 dimethoxycinnamicacid
2,4,5 trimethoxycinnamicacid
100
AC C
% Inhibition of PGE2
Berberine
Ferulic acid
TE D
% Inhibition of 5-LOX
150
Berberine
50
19
16 17 18
13 14 15
9 10 11 12
7 8
5 6
4
2 3
1
TA
0
Figure 4: Percentage inhibition of (a) 5-LOX extracted from PMNL by natural products at 100 µM concentration and (b) mPGES1 in HeLa cells by natural products at 50 µM concentration; Zileuton(2 µM- 90% inhibition for 5LOX) and Licofelone (6 µM- 55% inhibition for PGE2) are included as reference molecules; TA-Total activity of the enzyme in the absence of test compounds ( Highlights the three best compounds); Compounds 7&8 appear in all the three pharmacophores.
ACCEPTED MANUSCRIPT
EP
TE D
M AN U
SC
RI PT
Pharmacophores were elucidated using three sets of anti-inflammatory molecules. All pharmacophores had three hydrophobic centroids and a projected acceptor feature. Two methoxy derivatives of cinnamic acid mapped onto all the three pharmacophores. Berberine showed good inhibition of 5-LOX activity and PGE2 production.
AC C
1) 2) 3) 4)