Cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) selectivity of COX inhibitors

In vitro evaluations of the selectivity of COX inhibitors are based on a great variety of experimental protocols. As a result, data available on cyclooxygenase (COX)-1/COX-2/5- lipoxygenase (LOX) selectivity of COX inhibitors lack consistency. We, therefore, performed a systematic analysis of the COX-1/COX-2/5-LOX selectivity of 14 compounds with selective COX inhibitory activity (Coxibs). The compounds belonged to different structural classes and were analyzed employing the well-recognized whole-blood assay. 5-LOX activity was also tested on isolated human polymorphonuclear leukocytes. Among COX inhibitors, celecoxib and ML-3000 (licofelone) inhibited 5-LOX in human neutrophils at micromolar ranges. Surprisingly, ML-3000 had no effect on 5-LOX product synthesis in whole-blood assay. In addition, we could show that inhibition of COX pathways did not increase the transformation of arachidonic acid by the 5-LOX pathway.     

Characterization of spirostanol glycosides and furostanol glycosides from anemarrhenae rhizoma as dual targeted inhibitors of 5-lipoxygenase and Cyclooxygenase-2 by employing a combination of affinity ultrafiltration and HPLC/MS

Background : Modulation of the arachidonic acid (AA) cascade via 5-lipoxygenase (5-LOX) and cyclooxygenase-2 (COX-2) represent the two major pathways for treatments of inflammation and pain. The design and development of inhibitors targeting both 5-LOX and COX-2 has gained increasing popularity. As evidenced, 5-LOX and COX-2 dual targeted inhibitors have recently emerged as the front runners of anti-inflammatory drugs with improved efficacy and reduced side effects. Natural products represent a rich resource for the discovery of dual targeted 5-LOX and COX-2 inhibitors. By combining affinity ultrafiltration and high-performance liquid chromatography-mass spectrometry (AUF-LC-MS), an efficient method was developed to identify spirostanol glycosides and furostanol glycosides as the 5-LOX/COX-2 dual inhibitors from saponins extract of Anemarrhenae Rhizoma (SEAR).Methods: A highly efficient method by combining affinity ultrafiltration and high-performance liquid chromatography-mass spectrometry (AUF-LC-MS) was first developed to screen and characterize the 5-LOX/COX-2 dual targeted inhibitors from SEAR. The structures of compounds in the ultrafiltrate were characterized by high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). In addition, in vitro 5-LOX/COX-2 inhibition assays and their dual expression in vivo were performed to confirm the inhibitory activities of the compounds screened by AUF-LC-MS. Molecular docking studies with the corresponding binding energy were obtained which fit nicely to both 5-LOX and COX-2 protein cavities and in agreement with our affinity studies.Results: A total of 5 compounds, timosaponin A-II, timosaponin A-III, timosaponin B-II, timosaponin B-III and anemarrhenasaponin I, were identified as potential 5-LOX/COX-2 dual targeted inhibitors with specific binding values > 1.5 and IC₅₀ ≤ 6.07 μM.Conclusion: The present work demonstrated that spirostanol glycoside and furostanol glycoside were identified as two novel classes of dual inhibitors of 5-LOX/COX-2 enzymes by employing a highly efficient screening method of AUF-LC-MS. These natural products represent a novel class of anti-inflammatory agents with the potential of improved efficacy and reduced side effects.     

Development of 3,5-dinitrobenzoate-based 5-lipoxygenase inhibitors

Human 5-lipoxygenase (5-LOX) is a well-validated target for anti-inflammatory therapy. Development of novel 5-LOX inhibitors with higher activities is highly demanded. In previous study, we have built a model for the active conformation of human 5-LOX, and identified naphthalen-1-yl 3,5-dinitrobenzoate (JMC-4) as a 5-LOX inhibitor by virtual screening. In the present work, 3,5-dinitrobenzoate-based 5-lipoxygenase inhibitors were developed. Twenty aryl 3,5-dinitrobenzoates, N-aryl 3,5-dinitrobenzamides and analogues were designed and synthesized. Several of them were found with significantly increased activities according to cell-free assay and human whole blood assay. The structure–activity relationship study may provide useful insights for designing effective 5-LOX inhibitors.     

Synthesis of celecoxib analogs that possess a N-hydroxypyrid-2(1H)one 5-lipoxygenase pharmacophore: biological evaluation as dual inhibitors of cyclooxygenases and 5-lipoxygenase with anti-inflammatory activity

A hitherto unknown class of celecoxib analogs was designed for evaluation as dual inhibitors of the 5-lipoxygenase/cyclooxygenase-2 (5-LOX/COX-2) enzymes. These compounds possess a SO(2)Me (11a), or SO(2)NH(2) (11b) COX-2 pharmacophore at the para-position of the N(1)-phenyl ring in conjunction with a 5-LOX N-hydroxypyrid-2(1H)one iron-chelating moiety in place of the celecoxib C-5 tolyl group. The title compounds 11a-b are weak inhibitors of the COX-1 and COX-2 isozymes (IC(50)=7.5-13.2 microM range). In contrast, the SO(2)Me (11a, IC(50)=0.35 microM), and SO(2)NH(2) (11b, IC(50)=4.9 microM), compounds are potent inhibitors of the 5-LOX enzyme comparing favorably with the reference drug caffeic acid (5-LOX IC(50)=3.47 microM). The SO(2)Me (11a, ED(50)=66.9 mg/kg po), and SO(2)NH(2) (11b, ED(50)=99.8 mg/kg po) compounds exhibited excellent oral anti-inflammatory (AI) activities being more potent than the non-selective COX-1/COX-2 inhibitor drug aspirin (ED(50)=128.9 mg/kg po) and less potent than the selective COX-2 inhibitor celecoxib (ED(50)=10.8 mg/kg po). The N-hydroxypyridin-2(1H)one moiety constitutes a novel pharmacophore for the design of cyclic hydroxamic mimetics capable of chelating 5-LOX iron for exploitation in the design of 5-LOX inhibitory AI drugs.     

Novel dual cyclooxygenase and lipoxygenase inhibitors targeting hyaluronan–CD44v6 pathway and inducing cytotoxicity in colon cancer cells

Cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) enzyme have been found to play a role in promoting growth in colon cancer cell lines. The di-tert-butyl phenol class of compounds has been found to inhibit both COX-2 and 5-LOX enzymes with proven effectiveness in arresting tumor growth. In the present study, the structural analogs of 2,6 di-tert-butyl-p-benzoquinone (BQ) appended with hydrazide side chain were found to inhibit COX-2 and 5-LOX enzymes at micromolar concentrations. Molecular docking of the compounds into COX-2 and 5-LOX protein cavities indicated strong binding interactions supporting the observed cytototoxicities. The signaling interaction between endogenous hyaluronan and CD44 has been shown to regulate COX-2 activities through ErbB2 receptor tyrosine kinase (RTK) activation. In the present studies it has been observed for the first time, that three of our COX/5-LOX dual inhibitors inhibit proliferation upon hydrazide substitution and prevent the activity of pro-angiogenic factors in HCA-7, HT-29, Apc10.1 cells as well as the hyaluronan synthase-2 (Has2) enzyme over-expressed in colon cancer cells, through inhibition of the hyaluronan/CD44v6 cell survival pathway. Since there is a substantial enhancement in the antiproliferative activities of these compounds upon hydrazide substitution, the present work opens up new opportunities for evolving novel active compounds of BQ series for inhibiting colon cancer.     

Pharmacophore modeling and virtual screening for designing potential 5-Lipoxygenase inhibitors

Inhibitors of the 5-Lipoxygenase (5-LOX) pathway have a therapeutic potential in a variety of inflammatory disorders such as asthma. In this study, chemical feature based pharmacophore models of inhibitors of 5-LOX have been developed with the aid of HipHop and HypoGen modules within Catalyst program package. The best quantitative pharmacophore model, Hypo1, which has the highest correlation coefficient (0.97), consists of two hydrogen-bond acceptors, one hydrophobic feature and one ring aromatic feature. Hypo1 was further validated by test set and cross validation method. The application of the model shows great success in predicting the activities of 65 known 5-LOX inhibitors in our test set with a correlation coefficient of 0.85 with a cross validation of 95% confidence level, proving that the model is reliable in identifying structurally diverse compounds for inhibitory activity against 5-LOX. Furthermore, Hypo1 was used as a 3D query for screening Maybridge and NCI databases within catalyst and also drug like compounds obtained from Enamine Ltd, which follow Lipinski’s rule of five. The hit compounds were subsequently subjected to filtering by docking and visualization, to identify the potential lead molecules. Finally 5 potential lead compounds, identified in the above process, were evaluated for their inhibitory activities. These studies resulted in the identification of two compounds with potent inhibition of 5-LOX activity with IC₅₀ of 14 μM and 35 μM, respectively. These studies thus validate the pharmacophore model generated and suggest the usefulness of the model in screening of various small molecule libraries and identification of potential lead compounds for 5-LOX inhibition.     

Synthesis and biological evaluation of purine-pyrazole hybrids incorporating thiazole,thiazolidinone or rhodanine moiety as 15-LOX inhibitors endowed with anticancer and antioxidant potential

Novel purine-pyrazole hybrids combining thiazoles, thiazolidinones and rhodanines, were designed and tested as 15-LOX inhibitors, potential anticancer and antioxidant agents. All tested compounds were found to be potent 15-LOX inhibitors with IC₅₀ ranging from 1.76 to 6.12 µM. The prepared compounds were evaluated in vitro against five cancer cell lines: A549 (lung), Caco-2 (colon), PC3 (prostate), MCF-7 (breast) and HepG-2 (liver). Compounds 7b and 8b displayed broad spectrum anticancer activity against the five tested cell lines (IC₅₀ = 18.5–95.39 µM). While, compound 7h demonstrated moderate anticancer activity against lung A549 and colon Caco-2 cell lines. Antioxidant screening revealed that six compounds (5a, 5b, 6b, 7b, 7h and 8b) with IC₅₀ ranging from 0.93 to 14.43 µg/ml were found to be more potent scavengers of 2,2- diphenyl-1-picrylhydrazyl (DPPH) than the reference ascorbic acid with IC₅₀ value of 15.34 µg/ml. Compounds 7b, 7h and 8b, when evaluated for their antioxidant activity, where found to be potent DPPH scavengers. Moreover, compound 7b displayed twice the potency of ascorbic acid as NO scavenger. Docking study was performed to elucidate the possible binding mode of the most active compounds with the active site of 15-LOX enzyme. Collectively, the purine-pyrazole hybrids having thiazoline or thizolidinone moieties (7b, 7h and 8b) constitute a promising scaffold in designing more potent 15-LOX inhibitors with anticancer and antioxidant potential.     

Molecular modelling insights into a physiologically favourable approach to eicosanoid biosynthesis inhibition through novel thieno[2,3-b]pyridine derivatives

In this research, we exploited derivatives of thieno[2,3-b]pyridine as dual inhibitors of the key enzymes in eicosanoid biosynthesis, cyclooxygenase (COX, subtypes 1 and 2) and 5-lipoxygensase (5-LOX). Testing these compounds in a rat paw oedema model revealed potency higher than ibuprofen. The most active compounds 7a, 7b, 8b, and 8c were screened against COX-1/2 and 5-LOX enzymes. Compound 7a was the most powerful inhibitor of 5-LOX with IC₅₀?=?0.15?µM, while its p-chloro analogue 7b was more active against COX-2 (IC₅₀?=?7.5?µM). The less desirable target COX-1 was inhibited more potently by 8c with IC₅₀?=?7.7?µM. Surflex docking programme predicted that the more stable anti- conformer of compound (7a) formed a favourable complex with the active site of 5-LOX but not COX-1. This is in contrast to the binding mode of 8c, which resembles the syn-conformer of series 7 and binds favourably to COX-1.     

靶向5-LOX中药黄酮类化合物的活性筛选及其作用方式和共性规律研究

筛选靶向结合炎症相关蛋白5-LOX(5-lipoxygenase,5-脂氧合酶)的中药黄酮类天然产物,分析与5-LOX结合的黄酮类成分及其来源中药的共性规律。本研究借助Discovery Studio 2017 R2分子对接和药效团构建模块,结合SPR分子筛选实验,以及关联网络构建的方法进行研究。研究结果显示,来源于17种中药的18个黄酮类小分子中有11个能够与5-LOX结合,并从分子对接以及药效团构建研究中发现其作用的3种方式和共性特征:(1)部分中药黄酮成分(如木犀草素等)通过结构中的B环与5-LOX在活性位点ASP243形成静电中心相结合;(2)部分中药黄酮成分(如芹菜素等)是通过结构中的A环与活性位点VAL520形成疏水键、与活性位点ASP243形成氢键与5-LOX结合;(3)杨梅苷等黄酮类成分由于极性较强,在没有形成疏水键的情况下,也是通过形成静电中心与5-LOX在活性位点ASP243产生相互作用。此外还发现靶向5-LOX的活性中药黄酮类化合物,大多来源于具有利湿、退黄等功效,性味甘苦寒的景天科中药中。本研究发现了部分靶向5-LOX的中药黄酮类成分及其作用方式和共性规律,为开发靶向5-LOX抗肿瘤新药提供思路和方法。     

Synthesis and biological evaluation of a novel class of rofecoxib analogues as dual inhibitors of cyclooxygenases (COXs) and lipoxygenases (LOXs)

A group of 4-(4-methanesulfonylphenyl)-3-phenyl-2(5H)furanones possessing an acetyl, 3-oxobut-1-ynyl, [hydroxyl(or alkoxy)imino]alkyl, [hydroxyl(or alkoxy)imino]alkynyl, and N-alkoxy(or N-phenoxy)carbonyl-N-hydroxy-N-ethylamino substituents at the para-position of the C-3 phenyl ring of rofecoxib were synthesized. This group of compounds was designed for evaluation as dual inhibitors of cyclooxygenases (COXs) and lipoxygenases (LOXs) that exhibit in vivo anti-inflammatory and analgesic activities. In vitro COX-1/COX-2, and 5-LOX/15-LOX, isozyme inhibition structure-activity relationships identified 3-[4-(1-hydroxyimino)ethylphenyl]-4-(4-methanesulfonylphenyl)-2(5H)furanone (17a) having an optimal combination of COX-2 (COX-2 IC50 = 1.4 microM; COX-2 SI > 71), and 5-LOX and 15 LOX (5-LOX IC50 = 0.28 microM; 15-LOX IC50 = 0.32 microM), inhibitory effects. It was also discovered that 3-[4-(3-hydroxyiminobut-1-ynyl)phenyl]-4-(4-methanesulfonylphenyl)-2(5H)furanone (18a) possesses dual COX-2 (IC50 = 2.7 microM) and 5-LOX (IC50 = 0.30 microM) inhibitor actions. Further in vivo studies employing a rat carrageenan-induced paw edema model showed that the oxime compounds (17a, 18a) were more potent anti-inflammatory agents than the 5-LOX inhibitor caffeic acid, and 15-LOX inhibitor nordihydroguaiaretic acid (NDGA), but less potent than the selective COX-2 inhibitor celecoxib. The results of this investigation showed that incorporation of a para-oxime moiety on the C-3 phenyl ring of rofecoxib provides a suitable template for the design of dual inhibitors of the COX and LOX enzymes.     

N-1, C-3 substituted indoles as 5-LOX inhibitors—In vitro enzyme immunoaasay,mass spectral and molecular docking investigations

Based upon the structures of some known 5-LOX inhibitors, a set of five compounds carrying appropriate substituents at N-1 and C-3 of indole were synthesized and investigated for 5-LOX inhibitory activities. Fifty percent inhibitory concn (IC₅₀) of these compounds ranges from 0.6 to 5 μM and found to be comparable to that of clinically used 5-LOX inhibitor, zileuton. The compounds under present investigations exhibited appreciable interactions with 5-LOX as apparent from their association constants calculated from the mass spectral data. Compound 5a with a tosyl group at N-1 and pyrolidinyl-1,2-dione substituent at C-3 of indole, exhibiting IC₅₀ 0.6 μM and stoichiometry of 1:7 in the enzyme–compound complex was identified as highly potent 5-LOX inhibitor and seems to be suitable for further investigations.     

Synthesis and biological evaluation of isoxazolo[4,5-d]pyridazin-4-(5H)-one analogues as potent anti-inflammatory agents

In this study, eighteen new isoxazolo[4,5-d]pyridazin-4(5H)-one derivatives possessing either a 1,3,4-thiadiazole or a 1,2,4-triazole-5-thione moiety were synthesized and tested for anti-inflammatory activity in vitro (COX-1/COX-2, 5-LOX) and in vivo (rat paw edema assay). Compounds 15, 16, 25, 26 and 28-30 showed dual COX-2 (IC(50)'s in the 2.1-10.9 μM range), and 5-LOX (IC(50)'s in the 6.3-63.5 μM range) inhibitory activity. When administered orally to rats, dual COX-2/5-LOX inhibitors showed higher anti-inflammatory activity in vivo (30-45% reduction of the inflammatory response) than the reference drug ibuprofen (18%). Among dual COX-2/5-LOX inhibitors, the most potent compound (28) exhibited the best anti-inflammatory profile by inhibiting both COX-2 (IC(50)=2.1 μM) and 5-LOX (IC(50)=6.3 μM) enzymes. We investigated the binding interactions of compound 28 by an enzyme-ligand molecular modeling (docking) studies, which showed favorable binding interactions in both COX-2 and 5-LOX active sites. Furthermore, the dual acting COX-2/5-LOX compound 28 exhibited a superior gastrointestinal safety profile (ulcer index=0.25) compared to the reference drug ibuprofen (UI=7.0) when administered orally at the same molar dose. These observations suggest that isoxazolo[4,5-d]pyridazin-4(5H)-one analogs represent a new scaffold to design potent, effective, and safe anti-inflammatory agents possessing dual COX-2/5-LOX inhibitory activity.     

Synthesis and evaluation of benzoxazole derivatives as 5-lipoxygenase inhibitors

5-Lipoxygenase (5-LOX) is important enzyme in the biosynthesis of leukotrienes, and is a potential target in the treatment of asthma and allergy. We designed and synthesized a series of benzoxazoles and benzothiazoles as 5-LOX inhibitors. Fourteen compounds prepared showed the inhibition of LTC4 formation with IC₅₀ value of 0.12–23.88 μM. Also two compounds 2d and 2g showed improved airway hypersensitiveness.     

Synthesis and biological evaluation of acyclic triaryl (Z)-olefins possessing a 3,5-di-tert-butyl-4-hydroxyphenyl pharmacophore: dual inhibitors of cyclooxygenases and lipoxygenases

A new class of regioisomeric acyclic triaryl (Z)-olefins possessing a 3,5-di-tert-butyl-4-hydroxyphenyl (DTBHP) 5-lipoxygenase (5-LOX) pharmacophore that is vicinal to a para-methanesulfonylphenyl cyclooxygenase-2 (COX-2) pharmacophore were designed for evaluation as selective COX-2 and/or 5-LOX inhibitors. The target compounds were synthesized via a highly stereoselective McMurry olefination cross-coupling reaction. This key synthetic step afforded a (Z):(E) olefinic mixture with a predominance for the (Z)-olefin stereoisomer. Structure-activity studies for the (Z)-1-(3,5-di-tert-butyl-4-hydroxyphenyl)-2-(4-methanesulfonylphenyl)-1-phenylalk-1-ene regioisomers showed that COX-1 inhibition decreased, COX-2 inhibition increased, and the COX-2 selectivity index (SI) increased as the chain length of the alkyl substituent attached to the olefinic double bond was increased (Et-->n-butyl-->n-heptyl). In this group of compounds, inhibition of both 5-LOX and 15-LOX was dependent upon the length of the alkyl substituent with the hex-1-ene compound 9c having a n-butyl substituent exhibiting potent inhibition of both 5-LOX (IC50=0.3 microM) and 15-LOX (IC50=0.8 microM) relative to the inactive (IC50>10 microM) Et and n-heptyl analogs. Compound 9c is of particular interest since it also exhibits a dual inhibitory activity against the COX (COX-1 IC50=3.0 microM, and COX-2 IC50=0.36 microM, COX-2 SI=8.3) isozymes. A comparison of the relative inhibitory activities of the two groups of regioisomers investigated shows that the regioisomers in which the alkyl substituent is attached to the same olefinic carbon atom (C-2) as the para-methanesulfonylphenyl moiety generally exhibit a greater potency with respect to COX-2 inhibition. The 4-hydroxy substituent in the 3,5-di-tert-butyl-4-hydroxyphenyl moiety is essential for COX and LOX inhibition since 3,5-di-tert-butyl-4-acetoxyphenyl derivatives were inactive inhibitors. These structure-activity data indicate acyclic triaryl (Z)-olefins constitute a suitable template for the design of dual COX-2/LOX inhibitors.     

Essential structural profile of a dual functional inhibitor against cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX): molecular docking and 3D-QSAR analyses on DHDMBF analogues

It is recently proposed that compounds with equal capabilities of inhibiting COX and 5-LOX, both are key enzymes involved in the arachidonic acid (AA) cascade, are expected to be safer non-steroidal anti-inflammatory drugs (NSAIDs). To dig out helpful information in designing dual functional inhibitors against the two enzymes, homology modeling, molecular dynamics (MD) simulations, automated docking, and 3D-QSAR analyses were performed in this study on 21 COX-2/5-LOX dual inhibitors, namely, 7-tert-butyl-2,3-dihydro-3,3-dimethylbenzofuran (DHDMBF) analogues. A 3D-model of 5-LOX was built based on the high-resolution X-ray structure of rabbit reticulocyte 15-lipoxygenase. Molecular docking was then applied to locate the binding orientations and conformations of DHDMBF analogues with COX-2 and 5-LOX, respectively, leading to highly predictive CoMFA models constructed on the basis of the binding conformations with q2 values of 0.782 and 0.634 for COX-2 and 5-LOX, respectively. In addition, CoMFA field distributions were found in good agreement with the structural characteristics of the corresponding binding sites. Both the docking simulations and QSAR analyses suggest that new potent dual inhibitors should share a structural feature with a moderately bulky group at R2 position and a rather negatively charged group around the position of the carbonyl group of DHDMBFs. Therefore, the final 3D-QSAR models and the information of the inhibitor-enzyme interaction should be useful in developing new NSAIDs as anti-inflammation drugs with favorable safety profile.     

Novel di-tertiary-butyl phenylhydrazones as dual cyclooxygenase-2/5-lipoxygenase inhibitors: Synthesis,COX/LOX inhibition,molecular modeling,and insights into their cytotoxicities

Although dual inhibition of Cyclooxygenase-2 (COX-2) and 5-Lipoxygenase (5-LOX) enzymes is highly effective than targeting COX or LOX alone, there are only a few reports of examining such compounds in case of colorectal cancers (CRC). In the present work we report that the novel di-tert-butyl phenol-based dual inhibitors DTPSAL, DTPBHZ, DTPINH, and DTPNHZ exhibit significant cytotoxicity against human CRC cell lines. Molecular docking studies revealed a good fit of these compounds in the COX-2 and 5-LOX protein cavities. The inhibitors show significant inhibition of COX-2 and 5-LOX activities and are effective against a panel of human colon cancer cell lines including HCA-7, HT-29, SW480 and intestinal Apc10.1 cells as well as the hyaluronan synthase-2 (Has2) enzyme over-expressing colon cancer cells, through inhibition of the Hyaluronan/CD44v6 cell survival pathway. Western blot analysis and qRT-PCR analyses indicated that the di-tert-butyl phenol-based dual inhibitors reduce the expression of COX-2, 5-LOX, and CD44v6 in human colon cancer HCA-7 cells, while the combination of CD44v6shRNA and DTPSAL has an additional inhibitory effect on CD44v6 mRNA expression. The synergistic inhibitory effect of Celecoxib and Licofelone on CD44v6 mRNA expression suggests that the present dual inhibitors down-regulate cyclooxygenase and lipoxygenase enzymes through CD44v6. The compounds also exhibited enhanced antiproliferative potency compared to standard dual COX/LOX inhibitor, viz. Licofelone. Importantly, the HA/CD44v6 antagonist CD44v6shRNA in combination with synthetic compounds had a sensitizing effect on the cancer cells which enhanced their antiproliferative potency, a finding which is crucial for the anti-proliferative potency of the novel synthetic di-tert-butyl phenol based dual COX–LOX inhibitors in colon cancer cells.     

Design,synthesis and identification of novel coumaperine derivatives for inhibition of human 5-LOX: Antioxidant,pseudoperoxidase and docking studies

5-Lipoxygenase (5-LOX) is a key enzyme involved in the biosynthesis of pro-inflammatory leukotrienes, leading to asthma. Developing potent 5-LOX inhibitors especially, natural product based ones, are highly attractive. Coumaperine, a natural product found in white pepper and its derivatives were herein developed as 5-LOX inhibitors. We have synthesized twenty four derivatives, characterized and evaluated their 5-LOX inhibition potential. Coumaperine derivatives substituted with multiple hydroxy and multiple methoxy groups exhibited best 5-LOX inhibition. CP-209, a catechol type dihydroxyl derivative and CP-262-F2, a vicinal trihydroxyl derivative exhibited, 82.7% and 82.5% inhibition of 5-LOX respectively at 20?µM. Their IC₅₀ values are 2.1?±?0.2?µM and 2.3?±?0.2?µM respectively, and are comparable to zileuton, IC₅₀?=?1.4?±?0.2?µM. CP-155, a methylenedioxy derivative (a natural product) and CP-194, a 2,4,6-trimethoxy derivative showed 76.0% and 77.1% inhibition of 5-LOX respectively at 20?µM. Antioxidant study revealed that CP-209 and 262-F2 (at 20?µM) scavenged DPPH radical by 76.8% and 71.3% respectively. On the other hand, CP-155 and 194 showed very poor DPPH radical scavenging activity. Pseudo peroxidase assay confirmed that the mode of action of CP-209 and 262-F2 were by redox process, similar to zileuton, affecting the oxidation state of the metal ion in the enzyme. On the contrary, CP-155 and 194 probably act through some other mechanism which does not involve the disruption of the oxidation state of the metal in the enzyme. Molecular docking of CP-155 and 194 to the active site of 5-LOX and binding energy calculation suggested that they are non-competitive inhibitors. The In-Silico ADME/TOX analysis shows the active compounds (CP-155, 194, 209 and 262-F2) are with good drug likeliness and reduced toxicity compared to existing drug. These studies indicate that there is a great potential for coumaperine derivatives to be developed as anti-inflammatory drug.     

Docking,steered molecular dynamics,and QSAR studies as strategies for studying isoflavonoids as 5-, 12-, and 15-lipoxygenase inhibitors

Lipoxygenases (LOX) are enzymes that catalyze polyunsaturated fatty acid peroxidation and have a non-heme iron atom located in their active site. They are implicated in the arachidonic acid pathway and involved in inflammation, fever, pain production, and in the origins of several diseases such as cancer, asthma, and psoriasis. The search for inhibitors of these enzymes has emerged in the last years, and isoflavonoids have a broad spectrum of biological activity with low cytotoxicity. Our previous results have shown that isoflavonoids inhibited different LOX isoforms in vitro. For this reason, we studied the most important interactions that govern the potency and selectivity of some isoflavones and isoflavans toward different LOX isoforms using computational methods. The docking results have shown that all the molecules can be located in different zones in the LOX active site. Steered molecular dynamics indicated that selectivity was present at the cavity entry, but not at its exit. We also observed the correlation between the potential mean force and the best (HIR-303) and worst inhibitors (IR-213) in 5-LOX. Finally, structure–activity relationship (QSAR) studies showed a good correlation between theoretical IC₅₀ values and experimental data for 5-LOX and 12-LOX with 96 and 95%, respectively, and a lower correlation for 15-LOX (79%). Conclusively, pharmacophore analysis showed that our proposed molecules should possess a donor–acceptor and aromatic centers to encourage interactions in the active site.     

Binding investigation of human 5-lipoxygenase with its inhibitors by SPR technology correlating with molecular docking simulation

The binding features of a series of 5-lipoxygenase (5-LOX) inhibitors (caffeic acid, NDGA, AA-861, CDC, esculetin, gossypol and phenidone) to human 5-LOX have been studied by using surface plasmon resonance biosensor (SPR) technology based Biacore 3000 and molecular docking simulation analyses. The SPR results showed that the equilibrium dissociation constant (KD) values evaluated by Biacore 3000 for the inhibitors showed a good correlation with its reported IC50, suggesting that SPR technology might be applicable as a direct assay method in screening new 5-LOX inhibitors at an early stage. In addition, the 3D structural model of 5-LOX was generated according to the crystal structure of rabbit reticulocyte 15-lipoxygenase, and the molecular docking simulation analyses revealed that the predicted binding free energies for the inhibitors correlated well with the KD values measured by SPR assay, which implies the correctness of the constructed 3D structural model of 5-LOX. This current work has potential for application in structure-based 5-LOX inhibitor discovery.     

Design, synthesis and biological evaluation of benzo[1.3.2]dithiazolium ylide 1,1-dioxide derivatives as potential dual cyclooxygenase-2/5-lipoxygenase inhibitors

3-(4-Bromophenyl)-6-nitrobenzo[1.3.2]dithiazolium ylide 1,1-dioxide (5) was discovered as a new prototype for dual inhibitors of cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX). Thus, the structure-activity relationships of benzo[1.3.2]dithiazolium ylide 1,1-dioxide skeleton were carried out. The 6-NO(2) group played an essential role in the inhibitory activity. In addition, moderate-sized lipophilic substituents at the para-position of the 3-aryl moiety were required for dual COX-2/5-LOX inhibitory activity. Among the identified potent dual inhibitors, 3-(4-tbutylphenyl) derivative 30c (IC(50) values of 0.27 μM and 0.30 μM against COX-2 and 5-LOX, respectively) and 3-(4-biphenyl) derivative 30f (IC(50) values of 0.50 μM and 0.15μM against COX-2 and 5-LOX, respectively) were the most potent dual COX-2/5-LOX inhibitors. Intraperitoneal administration of 30c at 100mg/kg demonstrated potent acute anti-inflammatory activity. As a result, benzo[1.3.2]dithiazolium ylide 1,1-dioxide represented a novel scaffold for the exploitation in developing dual COX-2/5-LOX inhibitors.