Cinnamaldehyde Analogs: Docking Based Optimization, COX-2 Inhibitory In Vivo and In Vitro Studies.

BACKGROUND: In the past decade CADD has emerged as a rational approach in drug development so with the help molecular docking approach we planned to perform virtual screening of the designed data set of Schiff bases of cinnamaldehyde. The research work will be helpful to put some light on the drug receptor interactions required for anti-inflammatory activity. METHODS: For carrying out virtual screening of the developed cinnamaldehyde Schiff base data set, AutoDock 4.0 was used. The active hits identified through in silico screening were synthesized. Anti-inflammatory evaluation was carried out using Carrageenan-induced paw oedema method. RESULTS: Compounds V2A44, V2A55, V2A76, V2A82, V2A119, V2A141 and V2A142 has shown highest binding energy (-4.84, -4.76, -4.59, -4.78, -4.74, -4.85 and -4.72 kcal/mol, respectively) and the binding interactions with amino acids namely, Phe478, Glu479, Lys492, Ala493, Asp497 and Ile498. Some of the analogs have shown significant activity and were comparable to Indomethacin (standard drug). CONCLUSION: Five new compounds have shown significant activity and the results obtained from in silico studies are parallel to those of in vivo studies.

Arg-513 and Leu-531 Are Key Residues Governing Time-Dependent Inhibition of Cyclooxygenase-2 by Aspirin and Celebrex.

Aspirin and Celebrex are well-known time-dependent inhibitors of the cyclooxygenases (COX). Molecular dynamics simulations suggest that Arg-513 and Leu-531 contribute to the structural mechanisms of COX inhibition. We used mutagenesis and functional analyses to characterize how substitutions at these positions influence time-dependent inhibition by aspirin and Celebrex. We show that substitutions of Leu-531 with asparagine and phenylalanine significantly attenuate time-dependent inhibition of COX-2 by these drugs. The introduction of side chain bulk, rigidity, and charge would disrupt the formation of the initial noncovalent complex, in the case of aspirin, and the "high-affinity" binding state, in the case of Celebrex. Substitution of Arg-513 with histidine (the equivalent residue in COX-1) resulted in a 2-fold potentiation of aspirin inhibition, in support of the hypothesis that the presence of histidine in COX-1 lowers the activation barrier associated with the formation of the initial noncovalent enzyme-inhibitor complex. As a corollary, we previously hypothesized that the flexibility associated with Leu-531 contributes to the binding of arachidonic acid (AA) to acetylated COX-2 to generate 15R-hydroxyeicosatetraenoic acid (15R-HETE). We determined the X-ray crystal structure of AA bound to Co3+-protoporphyrin IX-reconstituted V349I murine COX-2 (muCOX-2). V349I muCOX-2 was utilized as a surrogate to trap AA in a conformation leading to 15R-HETE. AA binds in a C-shaped pose, facilitated by the rotation of the Leu-531 side chain. Ile-349 is positioned to sterically shield antarafacial oxygen addition at carbon-15 in a manner similar to that proposed for the acetylated Ser-530 side chain.

Prokaryotic expression, purification and characterization of human cyclooxygenase-2.

Cyclooxygenase-2 (COX-2) is a key enzyme which catalyzes the conversion of arachidonic acid (AA) into prostaglandins (PGs). It plays an important role in pathophysiological processes, such as tumorigenesis, angiogenesis, inflammation and tumor cell drug resistance. Therefore, COX-2 has been viewed as an important target for cancer therapy. The preparation of COX-2 protein is an important initial step for the subsequent development of COX-2 inhibitors. In this study, we report a strategy to heterologously express truncated human COX-2 (trCOX-2) in Escherichia coli (E. coli) BL21(DE3) host cells. Following denaturation, purification and renaturation, we successfully obtained enzymatically active trCOX-2 containing 257 residues of the C-terminus. Homology modeling and molecular docking analyses revealed that trCOX-2 retained the predicted 3D catalytic domain structure and AA could still bind to its hydrophobic groove. Western blot analysis and ELISA indicated that the trCOX-2 still retained its characteristic antigenicity and binding activity, while COX assays revealed that trCOX-2 maintained its enzyme activity. On the whole, in this study, we provided a novel method to isolate trCOX-2 possessing AA binding and catalytic activities. This study thus lays a foundation to facilitate further investigations of COX-2 and offers a valuable method with which to achieve the prokaryotic expression of a eukaryotic membrane protein.

Enhanced expression of human prostaglandin H synthase-2 in the yeast Pichia pastoris and removal of the C-terminal tag with bovine carboxypeptidase A.

Vertebrate prostaglandin H synthases (PGHSs) are membrane-bound disulphide-containing hemoglycoproteins. Therefore, eukaryotic expression systems are required for the production of recombinant PGHSs. Recently we announced the expression of human PGHS-2 (hPGHS-2) in the yeast Pichia pastoris. Here we report improved production of hPGHS-2 in P. pastoris and a convenient method for the purification and de-tagging of the protein. An affinity tag comprised of a proline, a glycine and eight histidines was introduced into the C-terminal end of hPGHS-2. The tagged hPGHS-2 was expressed intracellularly in P. pastoris under the control of a constitutive or methanol-inducible promoter. Compared to constitutive expression, methanol-induced expression yielded approximately four times more protein. The analysis of high and low gene copy number recombinants revealed a positive correlation between the gene copy number and the expression level of hPGHS-2. The recombinant hPGHS-2 was purified using immobilised metal ion affinity chromatography. A novel elution method, treatment of the affinity resin with bovine carboxypeptidase A, was employed. The yield of pure de-tagged hPGHS-2 from 1l of yeast culture was approximately 3mg. The protein purification process with simultaneous removal of the C-terminal polyhistidine tag could be easily applied for the affinity purification of other proteins.

Purification and characterization of the recombinant human prostaglandin H synthase-2 expressed in Pichia pastoris.

Prostaglandin H synthase-1 and -2 (PGHS-1 and PGHS-2, EC 1.14.99.1) are membrane associated glycoproteins that catalyze the first two steps in prostaglandin synthesis. As the enzymes play an important regulatory role in several physiological and pathophysiological processes, recombinant PGHS isoforms are widely used in biomedical research. In the present study, we expressed human PGHS-2 (hPGHS-2) with and without a six histidine sequence tag (His(6) tag) near the amino- or carboxy-terminus of the protein in the Pichia pastoris (P. pastoris) expression system using native or yeast signal sequences. The recombinant His(6) tagged hPGHS-2 was purified using Ni-affinity and anion exchange chromatography, whereas the purification of the C-terminally His(6) tagged hPGHS-2 was more efficient. K(m), k(cat) and IC(50) values were determined to characterize the protein. The data obtained indicate that both the N- and C-terminally His(6) tagged hPGHS-2 are functional and the catalytic properties of the recombinant protein and the enzyme produced in other expression systems are comparable. As the yeast culture is easy to handle, the P. pastoris system could serve as an alternative to the most commonly used baculovirus-insect cell expression system for the production of the recombinant PGHS-2.

Purification of recombinant human COX-1 and COX-2.

Human recombinant COX-1 and COX-2 was prepared in a purified form. The genes were cloned and expressed in insect cells, extracted by detergents, and purified by ion-exchange followed by size exclusion chromatography. Insect cells from 10 L fermentation yielded 2.3 mg of COX-1 with an overall yield of 75%, and 29 mg of COX-2 with an overall yield of 45%. Enzyme prepared in this manner was fully active and proved to be useful in biophysical studies including direct binding studies. The COX-2 provided material that was subsequently used in X-ray crystallography studies.

Cloning and expression of cyclooxygenase-1 and cyclooxygenase-2.

Cyclooxygenase (COX) enzymes play important roles in normal physiology and during inflammation of various cells and tissues. In order to help understand the functions of these enzymes, their genes can be cloned to facilitate the production of the proteins in recombinant form. We outline a method to clone the genes from a human macrophage cell line for expression in an insect cell line infected with recombinant baculovirus encoding these enzymes.

Liposomal reconstitution of monotopic integral membrane proteins.

In spite of considerable progress in the methodology for reconstitution of membrane proteins into the liposomes, a successful reconstitution still appears to be more an art than a science. Reconstitution of membrane proteins into bilayers is required for establishing several aspects of the functions of membrane proteins and lipids and for elaborating models of naturally occurring membranes.Cyclooxygenase (COX)-1 and -2 (also prostaglandin endoperoxide H(2) synthase, PGHS-1 and -2) belong to the class of monotopic membrane proteins. Membrane-binding domains of both COX-1 and -2 contain four short, consecutive, amphipathic alpha-helices (A, B, C, and D). Crystal structures of the COXs indicate that basic, hydrophobic, and aromatic residues in the membrane-binding domain are oriented away from the protein core and form a surface on the enzyme, which has been proposed to interact with the lipid bilayer (1).In this chapter, we describe a fast and efficient method for direct incorporation of COX-1 and -2 isozymes - as models for monotopic integral membrane proteins - into preformed liposomes containing fatty acids without loss of activity.

MK-886, an inhibitor of the 5-lipoxygenase-activating protein, inhibits cyclooxygenase-1 activity and suppresses platelet aggregation.

MK-886, an inhibitor of the 5-lipoxygenase-activating protein (FLAP), potently suppresses leukotriene biosynthesis in intact cells and is frequently used to define a role of the 5-lipoxygenase (EC 1.13.11.34) pathway in cellular or animal models of inflammation, allergy, cancer, and cardiovascular disease. Here we show that MK-886 also interferes with the activities of cyclooxygenases (COX, EC 1.14.99.1). MK-886 inhibited isolated COX-1 (IC(50)=8 microM) and blocked the formation of the COX-1-derived products 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid (12-HHT) and thromboxane B(2) in washed human platelets in response to collagen as well as from exogenous arachidonic acid (IC(50)=13-15 microM). Isolated COX-2 was less affected (IC(50)=58 microM), and in A549 cells, MK-886 (33 microM) failed to suppress COX-2-dependent 6-keto-prostaglandin (PG)F(1alpha) formation. The distinct susceptibility of MK-886 towards COX-1 and -2 is apparent in automated molecular docking studies that indicate a preferred binding of MK-886 to COX-1 into the active site. MK-886 (10 microM) inhibited COX-1-mediated platelet aggregation induced by collagen or arachidonic acid whereas thrombin- or U-46619-induced (COX-independent) aggregation was not affected. Since leukotrienes and prostaglandins share (patho)physiological properties in the development and regulation of carcinogenesis, inflammation, and vascular functions, caution should be used when interpreting data where MK-886 is used as tool to determine the involvement of FLAP and/or the 5-lipoxygenase pathway in respective experimental models.

Large-scale expression, purification, and characterization of an engineered prostacyclin-synthesizing enzyme with therapeutic potential.

Recently, we reported that a novel hybrid enzyme (TriCat enzyme), engineered by linking human cyclooxygenase-2 (COX-2) with prostacyclin (PGI(2)) synthase (PGIS) together through a transmembrane domain, was able to directly integrate the triple catalytic (TripCat) functions of COX-2 and PGIS and effectively convert arachidonic acid (AA) into the vascular protector, PGI(2) [K.H. Ruan, H. Deng, S.P. So, Biochemistry 45 (2006) 14003-14011]. In order to confirm the important biological activity and evaluate its therapeutic potential, it is critical to characterize the properties of the enzyme using the purified protein. The TriCat enzyme cDNA was subcloned into a baculovirus vector and its protein was expressed in Sf-9 cells in large-scale with a high-yield ( approximately 4% of the total membrane protein), as confirmed by Western blot and protein staining. The Sf-9 cells' membrane fraction, rich in TriCat enzyme, exhibited strong TriCat functions (K(m)=3 microM and K(cat)=100 molecules/min) for the TriCat enzyme and was 3-folds faster in converting AA to PGI(2) than the combination of the individual COX-2 and PGIS. Another superiority of the TriCat enzyme is its dual effect on platelet aggregation: it completely inhibited platelet aggregation at the low concentration of 2 microg/ml and then displayed the ability to reverse the initially aggregated platelets to their non-aggregated state. Furthermore, multiple substrate-binding sites were confirmed in the single protein by high-resolution NMR spectroscopy, using partially purified TriCat enzyme. These studies have clearly demonstrated that the isolated TriCat enzyme protein functions in the selective biosynthesis of the vascular protector, PGI(2), and revealed its potential for anti-thrombosis therapeutics.

5-aminosalicylic acid inhibits colitis-associated colorectal dysplasias in the mouse model of azoxymethane/dextran sulfate sodium-induced colitis.

BACKGROUND: The impact of the antiinflammatory agent 5-aminosalicylic acid (5-ASA) on the risk for colitis-associated colorectal cancer remains controversial. The chemopreventive activity of 5-ASA was evaluated in the Swiss Webster model of azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced colitis-associated neoplasia. METHODS: Mice were injected with AOM (7.4 mg/kg i.p.) and randomized to receive either vehicle or 5-ASA (75, 150, and 225 mg/kg) for the remainder of the study. DSS treatment began at 9 weeks of age and continued for 3 cycles. At the time of sacrifice (18 weeks of age), the entire colon and rectum were processed for histopathologic examination. RESULTS: An inverse trend was observed between dose and multiplicity of colonic dysplasias in all drug-treated groups (P = 0.03), with animals receiving 75 mg/kg 5-ASA exhibiting 56% of the number of dysplasias of the AOM/DSS controls (mean +/- SEM: 7.6 +/- 1.4 and 13.6 +/- 2.7, respectively). Administration of 75 mg/kg 5-ASA decreased both the mean multiplicity of flat dysplasias (1.8 +/- 0.4 for drug-treated versus 5.6 +/- 1.2 for AOM/DSS control) and the burden of polypoid dysplasias (tumor burden: 6.7 +/- 2.7 for drug-treated versus 14.9 +/- 3.9 units for AOM/DSS controls) significantly (P = 0.002 and 0.04, respectively). Inflammation was least severe in the 75 mg/kg group, which exhibited the fewest number of colorectal tumors. CONCLUSIONS: These data suggest that low-dose 5-ASA may be efficacious in preventing colitis-associated dysplasias and provide strong support for optimizing this therapy for the prevention of colonic neoplasms in patients with ulcerative colitis.

Quantitative promoter methylation analysis of multiple cancer-related genes in renal cell tumors.

BACKGROUND: Aberrant promoter hypermethylation of cancer-associated genes occurs frequently during carcinogenesis and may serve as a cancer biomarker. In this study we aimed at defining a quantitative gene promoter methylation panel that might identify the most prevalent types of renal cell tumors. METHODS: A panel of 18 gene promoters was assessed by quantitative methylation-specific PCR (QMSP) in 85 primarily resected renal tumors representing the four major histologic subtypes (52 clear cell (ccRCC), 13 papillary (pRCC), 10 chromophobe (chRCC), and 10 oncocytomas) and 62 paired normal tissue samples. After genomic DNA isolation and sodium bisulfite modification, methylation levels were determined and correlated with standard clinicopathological parameters. RESULTS: Significant differences in methylation levels among the four subtypes of renal tumors were found for CDH1 (p = 0.0007), PTGS2 (p = 0.002), and RASSF1A (p = 0.0001). CDH1 hypermethylation levels were significantly higher in ccRCC compared to chRCC and oncocytoma (p = 0.00016 and p = 0.0034, respectively), whereas PTGS2 methylation levels were significantly higher in ccRCC compared to pRCC (p = 0.004). RASSF1A methylation levels were significantly higher in pRCC than in normal tissue (p = 0.035). In pRCC, CDH1 and RASSF1A methylation levels were inversely correlated with tumor stage (p = 0.031) and nuclear grade (p = 0.022), respectively. CONCLUSION: The major subtypes of renal epithelial neoplasms display differential aberrant CDH1, PTGS2, and RASSF1A promoter methylation levels. This gene panel might contribute to a more accurate discrimination among common renal tumors, improving preoperative assessment and therapeutic decision-making in patients harboring suspicious renal masses.

Yin Yang 1 enhances cyclooxygenase-2 gene expression in macrophages.

Expression of cyclooxygenase-2 (COX-2) is associated with the pathogenesis of inflammation and various cancers, including lung cancer. Yin Yang 1 (YY1) is a zinc-finger transcription factor that interacts with histone acetyltransferases and deacetylases for its transcriptional activity and also is involved in inflammation and tumorigenesis. We investigated whether YY1 regulates COX-2 expression. We located a possible YY1 binding site proximal to the transcription initiation site of the COX-2 promoter. Electrophoretic mobility shift assays show that YY1 bound to the putative YY1 site in vitro. To show biological relevance, we performed chromatin immunoprecipitation assays showing that lipopolysaccharide (LPS) treatment induced YY1 binding to the cognate site in the endogenous COX-2 promoter. Overexpression of YY1 in macrophages treated with either LPS or live Pseudomonas aeruginosa increased COX-2 transcriptional activity. Furthermore, YY1 enhanced COX-2 protein expression and prostaglandin D(2) production elicited by LPS treatment. Mechanistically, we observed that LPS treatment resulted in disruption of an interaction between YY1 and p300, a histone acetyltransferase, but did not affect the interaction between YY1 and histone deacetylase 1/2. These data suggest that in response to LPS, YY1 dissociates from p300 and binds to the COX-2 promoter, contributing to COX-2 expression in an inflammatory milieu.

An ELISA method to measure inhibition of the COX enzymes.

The cyclooxygenase (COX) reaction can be monitored by measurement of oxygen consumption, peroxidase co-substrate oxidation or prostaglandin (PG) detection. This protocol describes a procedure measuring cyclooxygenase activity by quantifying PGE2 produced by enzymatic conversion of arachidonic acid, in the presence or absence of potential inhibitors. This high-throughput method has the advantage that it directly measures cyclooxygenase activity and requires little enzyme. The first part of the assay consists of incubating arachidonic acid, cyclooxygenase and the test samples to generate prostaglandins. The second part uses an ELISA method to quantify the amount of PGE2 produced by the enzymatic reaction. The isolation of COX-1 and COX-2 enzymes is also described. This protocol can be completed in approximately 23 h, including 16-h and 4-h incubation phases. This does not include enzyme preparation (3 h for COX-1 and 24 h for COX-2) or preparation of ELISA plates (23 h, including incubation).

Fast, efficient reconstitution of the cyclooxygenases into proteoliposomes.

To study the physical and catalytic properties of purified membrane proteins, it is often necessary to reconstitute them into lipid bilayers. Here, we describe a fast efficient method for the direct incorporation of cyclooxygenase-1 and -2 (COX-1 and -2) isozymes into liposomes without loss of activity. Purified COX-1 and -2 spontaneously incorporate into large unilamellar vesicles produced from a mixture of DOPC:DOPS (7:3) that has been doped with oleic acid. When incorporation was measured by comparing cyclooxygenase activity to total phospholipid in the proteoliposomes, molar reconstitution ratios of 1000:1 (phospholipid:COX) were obtained. Electron paramagnetic resonance spectroscopic spin counting analysis of proteoliposomes formed with nitroxide spin-labeled COX-2 gave a nearly identical phospholipid:COX ratio, confirming that incorporation had no effect on enzyme activity, and demonstrating that the efficiency of protein incorporation is sufficient for EPR spectroscopic analysis. The spontaneous incorporation of cyclooxygenase into intact liposomes allows only insertion into the outer leaflet for this monotopic enzyme, an orientation confirmed by immunogold staining of the proteoliposomes. This method of reconstitution into liposomes may be generally applicable to the class of monotopic integral membrane proteins typified by the cyclooxygenase isozymes.