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Drug design is the approach of finding new drugs, based on the biological targets(the protein). Typically a drug target is a key molecule involved in a particular metabolic or signalling pathway that is specific to a disease condition or pathology, or to the infectivity or survival of a microbial pathogen. Rational Drug Design (RDD) is a process used in the biopharmaceutical industry to discover and develop new drug compounds. RDD uses a variety of computational methods to identify novel compounds, design compounds for selectivity, efficacy and safety, and develop compounds into clinical trial candidates. These methods fall into several natural categories – structure-based drug design, ligand-based drug design, de novo design and homology modeling – depending on how much information is available about drug targets and potential drug compounds. In this project main emphasis is on Cyclooxygenase 2 which is an enzyme which is present in human body and is responsible for inflamatory response of the immune system of human body.The cyclooxygenase is naturally occuring enymes and produces hormones called protaglandins.E.C number of cyclooxygenase is 22.214.171.124 .There are two isoforms of cyclooxygenase .Cyclooxygenase-1(Cox-1) & cyclooxygenase-2(Cox2). The genes for cox-1 and cox-2 are located on chromosomes 9 and 1. The human cox2 gene is 8.3 kilobases (kb) whereas the cox-1 gene is much larger 22 kb . In terms of their molecular biology, COX-1 and COX-2 are of similar molecular weight (67 and 72 kDa respectively), and having 65% amino acid sequence homology and near-identical catalytic sites. Cox-1 Is a “House Keeping Enzyme” and Is Normally Present in a Variety of Area of the Human Body Including Stomach.The cox-1 enzyme of the stomach produces certain chemical messanger called prostaglandins that ensure the natural mucus lining which protects the inner stomatch, mediate normal platelet function and regulate renal blood flow.
Cox 2 is 74 kD protein,Cox 2 contains 604 amino acids, it is located at chromosome number 1,TATA box is present upstream of the start codon,it is usually absent in a cell under normal condition and is expressed only in response to inflammatio,its over expression in our system is a matter of concern as it may cause tumor formation.The tumor formed may,under the influence of mitogens and other carcinogens, lead to cancer formation. There are many differences between cox 1 and cox 2.COX 1 is detectable under normal conditions while COX 2 is not.COX 1 is located on chromosome number 9 while COX 2 is located on chromosome number 1 in human DNA.In COX 1 TATA Box is is ABSENT while in COX 2 it is present.The lumen of COX 1 is thinner while that of COX 2 is wider,so sometimes antibodies for COX 2 tend to affect COX 1 as well.The gene for COX 1 is longer as compare to COX 2. COX 1 is found in most mammalian cells while COX 2 is abundant only in macrophages and other sites of inflammation.At position 523,in the gene,COX 1 has isoleucine while COX 2 has valine.
1.2 Pathways Of Formation And Action Of Cox-1 And Cox-2
1.3 Production And Action Of Prostagladin Arachidonic acid(a 20 carbon FA containing 4 double bonds) is liberated from the membrane phospholipids by phospholipase A2, Which is activated by diverse stimuli. Arachidonic acid is converted by cyclooxygenase to the unstable intermediate prostaglandinH2 . Prostaglandin H2 is converted by tissue specific isomerases to multiple prostaglandin such as prostacyclin, thromboxane,prostaglandinD,E,F.
FIG. . The arachidonic acid cascade
While initial studies upheld the concept that COX-2 is mainly an inflammatory, inducible enzyme, more recent studies are beginning to reveal additional functions. We will now turn our attention to the various organ systems and disease states where COX-2 appears to have functional significance. Prostaglandins are known to serve as important physiologic modulators of vascular tone and sodium and water homeostasis in the mammalian kidney, including modulation of glomerular hemodynamics, tubular reabsorption of sodium and water, and regulation of renin secretion. COX-2 seems to have some role in regulating brain function. PGs have long been known as mediators of fever, of inflammatory reactions in neural tissue, and, more recently, of brain function. The recognition that each of these processes involves induction of PG synthesis has led to an appreciation of the role COX-2 plays in the PG-mediated functions. In turn, COX-2 inhibition by an isoform-specific NSAID can effectively block fever . Communication between local inflammatory sites and the brain endothelium is mediated by cytokines such as IL-1, which can directly induce COX-2 expression in these cells . The use of NSAIDs causes a variety of problems in the gastrointestinal tract including irritation and ulceration of the stomach lining . In animal studies, COX-2 is not induced after exposure to radiation, and its presence is not essential for crypt cell survival . Under these circumstances, COX-1 appears to play a major role, as it does in the stomach, in maintaining proper glandular architectureIn addition, COX-2 is expressed during inflammation and wound healing, and in animal models, treatment with COX-2 inhibitors can exacerbate inflammation and inhibit healing. Nevertheless, COX-2 selective inhibitors appear to be associated with less gastrointestinal damage than conventional NSAIDs .
Evidence provided by animal models of inflammatory arthritis strongly suggests that increased expression of COX-2 is responsible for increased PG production seen in inflamed joint tissues . COX-2 induction has been observed in both human osteoarthritisaffected cartilage as well as in synovial tissue taken from patients afflicted with The pro-inflammatory agents IL-1, TNF-, and LPS, as well as the growth factors TGF-ß, EGF, PDGF, and FGF, have all been shown to induce COX-2 expression in this system. On the other hand, the antiinflammatory cytokines IL-4 and IL-13, as well as the immunosuppresive glucocorticoids, were shown to decrease COX-2 levels . Although the synovial tissues of patients with osteoarthritis express lesser amounts of COX-2, primary explant cultures of human osteoarthritis-affected cartilage spontaneously express large amounts of COX-2 and PGs .The rapid expansion of knowledge about the role of COX-2 in inflammation led to drug screens attempting to identify antiinflammatory agents selective for COX-2 as well as to the rational design of highly selective COX-2 inhibitors. COX-2 is induced in both local and central sites , and the question of whether COX-2 mediates pain reception or transmission is being investigated, primarily through the use of COX-2 specific NSAIDs.. In fact, the COX-2 specific inhibitor Celecoxib was shown in short-term human studies to effectively suppress the pain associated with dental work, osteoarthritis, or rheumatoid arthritis without causing any significant gastroduodenal lesions . NSAID use reduces risk for Alzheimer's Disease (AD), with users of these agents having as little as one half the risk of acquiring AD as those not taking NSAIDs. Several population-based studies have detected a 40–50% decrease in relative risk for colorectal cancer in persons who regularly use aspirin and other NSAIDs .
The stimulus that induces COX-2 and cell cycle protein expression in AD is still elusive.02 and 0. we investigated whether the COX-2 expression level might be a useful immunohistochemical marker for distinguishing cutaneous melanomas from benign melanocytic lesions.02. Areas under the receiver operating characteristic curves were.5 and 51.98+/-0. respectively.the respective areas under the ROC curve values were 0. Braak A). The expression of COX-2 was determined immunohistochemically in formalin-fixed. paraffin-embedded specimens of 33 early Clark I/II melanomas and 58 naevi. Using post mortem brain tissue we have investigated whether activation of microglia and astrocytes in AD brain can be correlated with the expression of COX-2 and phosphorylated retinoblastoma protein (ppRb). 0.7%). For all the melanomas (not only the early ones). In this study. 2005 worked on neuronal expression of cyclooxygenase-2 (COX-2) and cell cycle proteins is suggested to contribute to neurodegeneration during Alzheimer's disease (AD). Up to now.97+/-0. COX-2 is the first immunohistochemical marker that allows the distinguishing of early melanomas from benign melanocytic lesions with both high sensitivity and specificity Jeroen. immunohistochemical markers have not ensured satisfactory sensitivity and specificity of differential pathologic diagnosis of melanoma.04 for the COX-2 expression in central and border regions of the lesions. No significant difference in COX-2 or ppRb neuronal immunoreactivity is observed between Braak stage 0 and later 6 6 . A simple diagnostic algorithm using threshold values of the COX-2 expression level allows for differentiation between early melanomas and naevi with high sensitivity (Se) and specificity (Sp) (for Se between 91 and 100%.REVIEW OF LITERATURE Barbara et-al. Mean COX-2 expression in melanomas was significantly stronger than in naevi (P[almost equal to]10-13).86+/-0. The highest levels of neuronal COX-2 and ppRb immunoreactivity are observed in the first stages of AD pathology (Braak 0–II. Sp values change between 96. In conclusion. Activated glia cells are shown to secrete substances that can induce expression of COX-2 and cell cycle proteins in vitro. et-al.97+/-0.01 and 0. 2007 reported recently that changes in expression level of COX-2 are correlated with development and progression of human melanoma.
427 and P=0. At RT-PCR 9 meningiomas. and hormonal therapy. the common COX-2 overexpression in meningiomas may suggest considering the COX-2 inhibitors.251. NSAIDs can also improve the efficacy of radiotherapy. In conclusion. even if further studies on larger series are necessary. Moreover. Experimental data suggest a possible therapeutic use of the COX-inhibitors nonsteroidal antiinflammatory drugs (NSAIDs). This study reviews the COX-2 expression as evaluated through immunohistochemistry and real time polymerase chain reaction (RT-PCR) in 23 meningiomas [14 World Health Organization (WHO) grade I. showed a COX-2 expression greater than the reference value (average expression of all meningiomas that we studied). and angiogenesis. 8 WHO grade I and 1 WHO grade II. especially through antiangiogenic and proapoptotic effects.Braak stages for neurofibrillary changes or amyloid plaques. NSAIDs can block tumor growth through many mechanisms. 2007 work showed that Cyclooxygenase-2 (COX-2) is the inducible form of the enzyme involved in the first steps of the prostaglandins and thromboxane synthesis. These data show that maximal COX-2 and ppRb immunoreactivity in neurons occurs during early Braak stages prior to the maximal activation of astrocytes and microglia. COX-2 up-regulation is demonstrated in tumors where it can modulate tumoral progression. alone or in combination 7 7 . At immunohistochemistry all the lesions but 4 (83%) were COX-2 positive. 3 WHO grade III. chemotherapy.et-al. In addition. Immunoreactivity for glial markers KP1. In contrast to in vitro studies. CR3/43 and GFAP immunoreactivity and the presence of neuronal immunoreactivity for COX-2 and ppRb. a significant negative correlation is observed between the presence of KP1. 1 oncocytic meningioma]. The mean number of COX2 or ppRb immunoreactive neurons is significantly decreased in Braak stage C compared to Braak stage A for amyloid deposits. respectively). multidrug resistance. The association between tumor grade and immunohistochemical or RT-PCR COX-2 expression was not significant (P=0. CR3/43 and GFAP appears in the later Braak stages and is significantly increased in Braak stage V-VI compared to Braak stage 0 for neurofibrillary changes. 5 WHO grade II. metastasis. post mortem data do not support a causal relation between the activation of microglia and astrocytes and the expression of neuronal COX-2 and ppRb in the pathological cascade of AD Anna .
inhibited glucose-induced insulin secretion from islets. et-al. to identify which EP receptor type mediates PGE2 inhibition of insulin secretion in pancreatic islets. Multiple myeloma (MM) is known to involve a deregulated cytokine network with secretion of inflammatory mediators. Real-time PCR showed COX-2 mRNA overexpression. including MM and monoclonal gammopathy of undetermined significance (MGUS). COX-2 was expressed in 11% (2 of 18) of MGUS specimens. Electromobility shift assays demonstrated that sodium 8 8 . but little is known about its presence and role in hematologic neoplasms. and epididymal fat. abundant type in skeletal muscle. liver. or major degradative sites. kidney. COX-2 positivity was associated with a poor outcome in terms of progression-free (18 vs 36 months.et-al. Real-time fluorescencebased RT-PCR indicated that EP3 is the most abundant EP receptor type in islets. 31% (29 of 94) of MM at diagnosis. Misoprostol. Selected cases underwent further evaluation by WB on purified CD138+ cells. and real-time polymerase chain reaction (PCR) for mRNA expression. IC and cell separation studies demonstrated COX-2 expression to be restricted to malignant plasma cells. 2005 work showed that Cyclooxygenase 2 (COX-2) is an inflammationassociated enzyme involved in the pathogenesis of many solid tumors. a potential area of therapeutic intervention in some selected meningiomas.with radiotherapy. EP3 mRNA is the least. whereas EP2 mRNA is the most. P < .05). and to examine possible sites of action through which sodium salicylate might affect IL-1ß/PGE2 interactions. Phuong. Future studies will assess COX-2 involvement in other hematologic tumors and its potential use as a therapeutic or chemo-preventive target in onco-hematology. of insulin.001) and overall survival (28 vs 52 months. Marco . We thus decided to investigate the involvement of COX-2 in this neoplasm. P < . and 47% (14 of 30) of MM with relapsed/refractory disease. Western blotting (WB) was used to evaluate 142 bone marrow (BM) specimens. by decreasing cAMP. an EP3 agonist. 2002 Their studies were performed to ascertain the relative abundance of E prostaglandin (EP) receptor mRNAs in tissues that are major targets. an event that was prevented by preincubation with pertussis toxin. This is the first report of the presence and prognostic role of COX-2 expression in MM. immunohistochemistry (IC).
431 person-years of follow-up of 82.78). These findings indicate that the sites of action through which sodium salicylate inhibits these negative effects of IL-1ß on ß-cell function include activation of NF.000 person-years among regular aspirin users. 95% CI. 2005 sujjested that selective inhibition of cyclooxygenase-2 (COX-2) may be associated with an increased risk of thrombotic events. 0.911 women and 47.000 person-years among those who did not use aspirin regularly. placebo-controlled. 0. the rate for cancers with weak or absent COX-2 expression was 27 per 100.52 to 0.446.96.64. The age-standardized incidence rate for cancers that overexpressed COX-2 was 37 per 100.02).B (NF. as compared with 56 per 100. whereas regular aspirin use had no influence on tumors with weak or absent expression of COX-2 (multivariate relative risk. multicenter.B) activation.363 men. Sodium salicylate also prevented IL-1ß from inducing EP3 and cyclooxygenase (COX)-2 gene expression in islets and thereby prevented IL-1ß from inhibiting glucose-induced insulin secretion. 0.000 person-years among nonregular aspirin users Robert. They applied Cox regression to a competing-risks analysis to compare the effects of aspirin use on the relative risk of colorectal cancer in relation to the expression of COX-2 in the tumor. 2005 They estimated cyclooxygenase-2 (COX-2) expression by immunohistochemical assay of sections from paraffin-embedded colorectal-cancer specimens from two large cohorts of participants who provided data on aspirin use from a questionnaire every 2 years.000 person-years among regular aspirin users. 423 (67%) had moderate or strong COX-2 expression. randomized. et-al. The effect of aspirin use differed significantly in relation to COX-2 expression (P for heterogeneity=0.salicylate inhibits IL-1ß-induced nuclear factor. Regular aspirin use conferred a significant reduction in the risk of colorectal cancers that overexpressed COX-2 (multivariate relative risk. During 2. et-al. They worked on the cardiovascular outcomes associated with the use of the selective COX-2 inhibitor rofecoxib in a long-term. double-blind trial designed to determine the effect of three years of treatment with rofecoxib on the risk of 9 9 . as compared with 28 per 100.B as well as generation of PGE2 by COX-2. we found 636 incident colorectal cancers that were accessible for determination of COX-2 expression.26). Andrew. in contrast. Of the tumors.73 to 1. 95% confidence interval [CI]. 0.
Twenty-nine adult male Fischer 344 rats were implanted with intracerebral 9L gliosarcomas and divided into 3 treatment groups.19 to 3. 1. the corresponding relative risk was 1.83).61. the event rates were similar in the two groups. CONCLUSIONS: Among patients with a history of colorectal adenomas. pulmonary edema. Overall and cardiovascular mortality was similar in the two groups. during the first 18 months. and 1299 to receive placebo.o. The results primarily reflect a greater number of myocardial infarctions and ischemic cerebrovascular events in the rofecoxib group.4. the use of rofecoxib was associated with an increased cardiovascular risk Jana.50 events per 100 patientyears). The purpose of this study was to determine in a rat brain tumor model whether SC-236.A total of 2586 patients with a history of colorectal adenomas underwent randomization: 1287 were assigned to receive 25 mg of rofecoxib daily. another (n = 9) was treated with dexamethasone (3 mg/kg p. A total of 46 patients in the rofecoxib group had a confirmed thrombotic event during 3059 patient-years of follow-up (1. One group (n = 9) served as controls.recurrent neoplastic polyps of the large bowel in patients with a history of colorectal adenomas. 1.o. A survival study was 1 10 . There was earlier separation (at approximately five months) between groups in the incidence of nonadjudicated investigator-reported congestive heart failure. P=0. a selective COX-2 inhibitor. it is associated with distressing side effects that decrease the quality of life for many patients. daily). The increased relative risk became apparent after 18 months of treatment. and a third group (n = 11) received SC-236 (3 mg/kg p. All investigator-reported serious adverse events that represented potential thrombotic cardiovascular events were adjudicated in a blinded fashion by an external committee.50 to 18.78 event per 100 patient-years).95percent confidence interval. One potential mechanism to explain the ability of dexamethasone to repair blood-brain barrier dysfunction is through the inhibition of cyclooxygenase-2 (COX-2). or cardiac failure (hazard ratio for the comparison of the rofecoxib group with the placebo group. et-al. as compared with 26 patients in the placebo group during 3327 patient-years of follow-up (0.11.008). 2002 worked on Dexamethasone and concluded that it is very effective for controlling peritumoral cerebral edema.92 (95 percent confidence interval. is as effective as dexamethasone. daily).
selective inhibition of COX-1 via post-training piroxicam administration had no effect on retention of either task. Injections of indomethacin or NS-398 that were delayed 2 h post-training had no effect on retention.-cyclodextrin in distilled water). Kaplan-Meier analysis on pairwise group comparisons showed improved survival that was statistically significant for each treatment group compared with the control group (log-rank test P = 0.2). These results suggest that a selective COX-2 inhibitor appears to be as effective as dexamethasone in prolonging survival in a rat brain tumor model Lisa. The median survival in the control group was 16 days. and no significant difference in survival for the COX-2 compared with dexamethasone (log-rank test P = 0. the significance of the COX isoforms cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) in post-training memory processes was assessed. 2002 indicated that prostanoids.005 for COX-2 to control). indicating an impairment in retention. rats received an intraperitoneal injection of the nonselective COX inhibitor indomethacin. such as prostaglandins. Furthermore. a cellular model for certain forms of learning and memory. or saline. These findings indicate that COX2 is a required biochemical component mediating the consolidation of hippocampaldependent memory 1 11 . In these experiments. the COX-2-specific inhibitor N-[2-cyclohexyloxy-4-nitrophenyl]-methanesulfonamide (NS-398). Post-training indomethacin or NS-398 had no influence on retention of the visible platform version of the water maze at any of the doses administered. the retention test escape latencies of rats administered indomethacin (5 and 10 mg/kg) or NS-398 (2 and 5 mg/kg) were significantly higher than those of vehicle-treated rats.009 for dexamethasone to control and P = 0. play a regulatory role in several forms of neural plasticity.performed. latency to mount the escape platform was used as a measure of memory. compared with 23 days for the dexamethasone group and 23 days for the COX-2 inhibitor group. et-al. including long-term potentiation. On a two-trial retention test session 24 h later. vehicle (45% 2-hydroxypropyl. After the completionoftraining. Adult male Long-Evans rats underwent an eight-trial (30-sec intertrial interval) training session on a hippocampus-dependent (hidden platform) or dorsal striatal-dependent (visible platform) tasks in a water maze. In the hidden platform task. the COX-1-specific inhibitor piroxicam.
The sequence alignment and template stucture are used to produce a structural model of the target.1. Different softwares are used for Homology Modelling such as SWISS MODEL SERVER.it does not show any results.ncbi.. The sequence of protein was in fasta formet.nih. 3.2.. In this project Swiss Model server is used for Homology Modelling.Here I carried out protein-protein 1 12 .ogy modeling techniques depend on identificatiction of one or more stuctures known as ‘template’.CPH MODEL SERVER.MATERIALS AND METHODS 3.nlm.The structure of cyclooxygenase 2 was also unavailable.nlm.(www. Retrieval of Protein Sequence of Cox 2 in Homo sapiens: Protein sequence of cox 2 in Homo sapiens was done from National Center Of Biotechnology information(www.2 Homology Modelling : Homology modelling is required when the exact structure of the protein is not available.Here we model the molecule(protein) from amino acid sequence by following a protocol to model.which resembles the sructure of query sequence.against the database of BLAST.gov/BLAST)is a tool by which we can find alignment between our query in form of nucleotide or protein sequence.nih. BLAST BLAST (Basic Local Alignment Search Tool).ncbi.The methodology for homology modeling with swiss Model Server is: 3.In case our sequence is a novel entry.The results show us the extent to which our query sequence matches the sequences stored in the BLAST database.1.It is also known as ‘comperative modelling’.MODELLER etc.The amino acid sequence is ‘query’ or ‘target’ sequence.Usually sequence similarity corresponds to high structural similarity.gov/).so homology modeling was required.Homo.
2.swissmodel.or opload co-ordinate files.It gives the match between the query sequences and allows us to have the idea of best match between our target sequence and template.uk/clustalw/) is a multiple sequence alignment programme for DNA or proteins.which are: 1.2. 2.(www. 1 13 .BLAST of my query sequence.html).Basically there are three moes os SWISS MODEL. 3.The reliability of model decreases as sequence identity decreases.which consists of amino acid sequences of the proteins submitted in pdb . First approach mode:it only requires a single amino acid sequence information as input data.against pdb(protein data bank).It provides multiple sequence alignment forgiven sequence. CLUSTAL W CLUSTAL-W (www.ebi.The server builds yhe model according to given alignment.The ser predicts the target sequence and the one .The process starts if atleast one template sequence has a identity of more than 25% with submitted target sequence. Swiss Model: It is totally automated protein structure homology modeling server . 3. After we get BLAST results we carry out CLUSTAL W.This informationis furthur used in swiss model.org//SWISSMODEL.expasy.accessible via ExPASy web server or from swiss pdb viewer.However the user may specify upto five template structures either from ExPDB library.3.which is structurally known protein FROM ExPDB library.The server automatically selects suitable template.The results generated by BLAT were furthur used for the modeling of the protein. Alignment Mode:it is done by submitting a sequence alignment.2.Evolutionary relationships can be viewed by cladograms or phylograms. SWISS MODEL SERVER: It is used for final modeling of protein.ac.using results of CLUSTAL-W.
• Puting the results obtained in last step alongwith target protein sequence of protein in a notepad.to save a pdb file.It can also be used to improve the output of first approach mode. Open the saved file with rasmol viewer to view 3-D image of the modeled protein.fourth match results and obtaining their FASTA format of sequence.third.asve the result file with (.plates. Here ‘alignment mode’ of SWISS MODEL was used to predict structure of cox 2 model. • Protein BLAST of protein sequence obyained in last step against pdb(protein data bank). • Open CLUSTAL W page and paste the sequence obtained in last step in window displayed and submit.3. Project Mode:here user submits a manually optimized modeling request to SWISS MODEL server The starting moe is a ‘Deep View’ project file. There are certain steps to be followed in this process.The sequences obtained in last steo are pot\entail te. • Open SWISS MODEL SERVER page and paste the sequence in window and submit.which are: • Retrieval of protein seuence from NCBI in FASTA format.It contains superposed template structures and alignment between target and template. • • The results are obtained. • Selecting the second. 1 14 .pdb) extension.It allows template selection or gap placement in the alignment.
3 Building of 3d structure (PDB file) of Inhibitors: 2D structure of potent inhibitors are obtained by submitting the CID no to the NCBI’s Pubchem compound and convert it into SDF format then convert it into PDB format to get the 3D-structure. same stereochemistry. Molecular Formula. 1. SMARTS. BRENDA (www.uni-koeln. The compounds are grouped into levels of chemical similarity from most general to most specific: same bonding connectivity and any tautomer. and octanol-water partition coefficients (XlogP).3 Retrieval of inhibitor against Cox-2: Inhibitor against Cox-2 protein retrieved through two major sources. total formal charge. the number of hydrogen bond donors/acceptors. and same stereochemistry and isotopes. same bonding connectivity. PubChem Compound also indexes these chemicals using 34 fields. SMILES. You may also specify the structural query input by PubChem Compound Identifier (CID). NCBI Pubchem Compound: PubChem Structure Search allows PubChem Compound Database to be queried using a chemical structure. or by upload of a supported structure file format. InChI.de) is the main collection of enzyme functional data available to the scientific community. This standardizing allows NCBI to compute chemical parameters and similarity relationships between compounds. 1 15 . These groups are provided as Entrez links that allow similar compounds to be retrieved quickly 3. same isotopes. Chemical structure queries may be sketched using the PubChem Sketcher. molecular formula and weight.3.BRENDA is maintained and developed at the institute of Biochemistry at the University of Cologne 2. many of which represent computed chemical properties such as the number of chiral centers.brenda. Procedure of converting 2D-structure into 3D-structure 1) To select SDF format from NCBI.
Choose pubchem compound from search drop-down menu. Type CID no.Change display format to SDFand save it 1 16 . When answers come .Open google and enter NCBI home page.
Babel is a chemical toolbox designed to allowing anyone. analyze. copy that data and paste in the word pad and save that file with (.4.convert.e in the form of PDB file.2 Procedure to convert the 2-D file in the 3-D file or PDB file First of all open the Babel page.pdb) extension.4. 3.3.4 To retrieve PDB file of Inhibitors: 2-D structure of protein has been obtained by put the specific CID no. or related areas.1 Babel Molecule format Converter: Babel is a cross-platform program designed to interconvert between many file formats used in molecular modeling and computational chemistry and related areas. Paste the data of 2-D file in the input section or upload the SDF file. 3.e. Set the parameter for input and output file i. The result will show in the output section in the form of PDF file. using software Babel. chemistry.in the pubchem compound it retrieved the 2-D or SDF file of inhibitor we save it & will convert this 2-D file in the 3-D file i. biochemistry. SDF for input file & PDF for output file. 1 17 . solid-state materials. Click on the convert file. or store data from molecular modeling.
SGI 9 10 HINT GOLD Commercial Free evaluation Windows 2000.UNIX UNIX UNIX Keyword GA/LGA.LINUX.LINUX UNIX mechanics Hydropathic interaction GA 3.SGI.IRIS Supercomputers.5 Docking Of Flexible Ligands to the Receptors For docking the flexible ligands to the receptors following softwares can be used which are listed below: SN 1 2 3 4 5 6 Name Autodock Affinity Dock Vision DOT(Daughter of Turnip) Flex X Shape License Term Commercial Commercial Commercial Free Commercial E-mail request Platform UNIX.SGI SGI LINUX.3.2 Cygwin: It is a collection of free software tools originally developed from ‘Cygnus solutions’to allow various versions of Microsoft windows to act similarto a Linux operating system.As Autodock is programmed to run on Linux operating system.GA Fragnent Based Structure and chemistry molecular surface ligand design Mixed quantum molecular and of 7 8 LEAPFROG Q site Commercial Commercial SGI UNIX.3.LINUX.MC Monte Carlo method MC.so 1 18 .
Go to ‘Ligand’ again in autodock window and in it ‘Torsion Tree’ and click on’detect route’. AUTOGRID: A. 3. In additions to using them for docking. 1 19 . the atomic affinity grids can be visualized.Go to ‘Ligand’ again and select ‘Torsion Tree’ and select ‘Set number Of Torsions’.3.go to ‘ligand’ and in it click on ‘input’. Auto Grid pre-calculates these grids.pdb) file of the inhibitor.3. such as substrates or drug candidates. It is designed to predict how small molecules. 1.for those systems which run on windows.3.Set number of torsions less than or equal to 6 and click on ‘Dismiss’. iv.Autodock.It can be freely downloaded from the internet.Peparing a Ligand for Autodock: i.The number of torsions vii. for example.In autodock page. 3. AUTODOCK: Autodock is a suite of automated docking tools.In ‘Ligand’ select ‘Torsion Tree’ and click on ‘Choose Torsion’and click on ‘done’.1. iii.In ‘input’ click on ‘open AD3’. AutoDock actually consists of two main programs: AutoDock performs the docking of the ligand to a set of grids describing the target protein. vi. ii. 2.3. to guide organic synthetic chemists design better binders. This can help.Go to your folder and open the (.Autogrid.cygwin is a must. bind to a receptor of known 3D structure. v.
iii.y.Now click on ‘Ligand’ and then click on ‘Accept’.out.Preparing The Grid Parameter File: i.pdbq).viii.Now select the inhibitor file.set x. ii. iv.pdbqs’.Come back to autodock window and press ‘shift key+n’ to visualize the protein on screen.click on ‘File’ and there click on ‘Saving Current Setting’.z co-ordinate axes so that the macromolecule is completely covered. 2 20 .Go to ‘Grid’ again and in it go to ‘Output’.Go to ‘Logand’ and in it to ‘Output’ and then click on ‘Save As PDBQ’.then click on ‘SaveGPF(AG3) viii.Now save this file as ‘protein name.Open the (.Save this file in your folder as ‘protein name.pdb) file from your folder and click on ‘OK’. C.On the window that opens. ii.gpf’. v.Go to ‘Grid’ and select’Grid Box’. iv. Preparing A Macromolecule For Autodock: i.You can rotate the molecule by pressing ‘shift+right click of mouse’. B.On the same window. vi.Go to ‘Grid’ and select ‘Set Map Types’ and in it select ‘Choose Ligand AG3’. vii. iii.Then go to your folder a save your this file as (inhibitor name.Go to ‘Grid’ and in it click on ‘Macromolecule’ and in it click on ‘Open AG3’.
iii.Select your protein in the window that opens and click on ‘OK’.Click on ‘Save’ in this window after you finish. the path) of the ‘Browse’ window and press ‘cntrl+c’.ix.Preparing A docking Parameter For Autodock: i.On the window that opens on the first ‘Browse’ option click and select ‘autogrid.Open ‘Cygwin’ and in it go to ‘Edit’ and ‘Paste’ the path copied and press ‘Enter’.then click on ‘Accept’.e. x.In it select on ‘Macromolecule’ and in it click on ‘Choose AD3’.Go to your folder and copy the path of your folder eg(C:\probir) and open the (. select the entire bottom line(i.Startimg Autodogrid: i.gpf) file in your folder wit ‘wordpad’ and paste this path followed by ’\’ on left of wherever you find protein name in this file. v.It is to be noted that there should not be any gap between path and protein name. 2 21 . B.gpf) file. iii. xi.Go to ‘Grid’ and in it go to ‘Edit Grid’ and clik it. iv. 3.Back to autogrid.3.exe’ file.2. ii.3. ii.Click on ‘Docking’ on the the autodock window.Then in second ‘Browse’ option click and go to your folder and open the (.Go to ‘Run’ and in it click on ‘Run Autogrid’.AUTODOCK: A.
Go to your folder and copy the path of (.click and go to your folder and open the the (. xi.Click on ‘Select Ligand’ and click on ‘Accept’ on the window.e. iii.Click ‘Accept’ on yhe window that opens. C.Then go to your folder and save the file as (inhibitor name. 2 22 in it click on ‘Genetic .Click on ‘Select Macromolecule’ and click on window that opens twice.dpf) file.Go to ‘Docking’ again and in it click on ‘Docking Parameters’.’path+\’. vii.dpf) file. viii.Go to ‘Docking’ and in it go to ‘Ligand’ and in it click on ‘Choose (AD3)’.dpf) file in ‘wordpad’ and paste the copied path everywhere you find inhibitor name. ii.Go to ‘Docking’ again and in it select ‘Output’ and in it click on ‘Lamarkian GA(AD3)’.followed by ‘\’ i.Go to docking again and select’Search Parameter’and Algorithm’.Continue till you reach on yhe line with ‘move’ and here do the same. xii.Back to autodock. iv.On the window that opens on first ‘Browse’ option.Starting Autodock: i.Open this (.exe’ file.Come back to ‘Browse’ window and copy the path at the bottom by selecting it and then pressing ‘cntrl+c’.click on ‘Run’ and in it click on ‘Run Autodock’.Save the page. ix.click it and open the ‘autodock. xi. v. x.dpf). vi.on left of the inhibitor name.On the second ‘Browse’ option.iv.On the window that opens click and select the ligand.Click on ‘Accept’ on the window that opens.
iv.Go to ‘Conformation’ in ‘Analyse’ and in it click on ‘Load’.another window opens.Open ‘Cygwin’ and go to ‘Edit’ and click on ‘Paste’ .Analysing Autodock Results: i.Now go to the window which came in ‘Play’. 2 23 .It shows the docking energy and various other docking parameters.a new window opens.Go to your folder and open the (.dlg) file.the better inhibitor is.Click on ‘Analyse’ on the autodock window and in it click on ‘Docking’.click on (&) sign . D.Now on the first window that came on pressing ‘Load’ go to its second line and click it. vi.pdbqs) file of the target.Press ‘Enter’ to run Autodock. ii.Press ‘Shift+n’ to visualize the macromolecule on the screen. vii.v.Note it.Here we also search for ‘hydrogen bonds’ which are shown on the bottom of that window and we can also find the amino acid to which the ligand binds. vi.The more negative dock energy. ix. v.in it click on the direction buttons to analyse each of the ten active sites. iii. And click ‘ok’.It gives information about various parameters on a particular active site.a box appears.Go to ‘Conformation’ again and click on ‘Play’.Now go to ‘Macromolecule’ in ‘Analyse’ and go to your folder and open the (. viii.In the ‘Play’ window.positive dock energies(if found) are neglected as it is not a proper result.
xi. vii. PMV (Python Molecular Viewer): Python Molecular Viewer is a tool to view the binding of hydrogen bonds in the target molecule.Record the results.pdbq). vi.Again in the adjacent window of ‘pmv’ click on ‘hbond command’ and load this too.Click on ‘Write Current Coords’ and go to your folder and save this file as (inhibitor. v. 2 24 .docked.Go to ‘File’ and click on ‘Browse Command’.then click on ‘Load’.x.Open PMV.Go to ‘Compute’ and in it go to ‘Trace’. iv. xii. ii.4.Expand the bottom of the box which we got on clicking ‘Load’.It helps to visualise and analyse the hudrogen bonds.The process of operation of PMV is enlisted below: Procedure For Operation Of PMV: i.In the adjacent window click on ‘trace command’.Go to file in PMV and click on ‘Read Molecule’ nad go to your folder and open (protein name.click on ‘pmv’ . 3.pdbqs) file.In the window that opens. iii.Here click on ‘Compute Extrude Trace’.
choose any color of your choice and click on ‘Dismiss’. xix. x. ix.In the window that opens adjust the ‘Scale Factor’ to 0. xiv.Click on ‘Dismiss’. xvii. xv.by moving the mouse across the wheels.Go to ‘Display’ of PMV and click on ‘Display’. xxii.viii.Go to ‘Select’ and I it click on ‘Select From String’.Go to ‘Display’ and in it click on ‘cpk’.Click ‘cpk’ in the window that opens. xvi. xviii.In the place where ‘atom type’ is given type ‘*’.In the next window which opens.Click ‘ok’.Go to ‘color’ and click on ‘choose color’.In the that opens click on ‘CAT race’ and click ‘ok’. xiii.Click on ‘Add’.In the window that opens in the box where ‘Residue Number’ is written enter the residue with its number which was noted from ‘Analyse’ of autodock. xii.Go to ‘Color’ and click on ‘By Atom Type’. xxi.7 and ‘Sphere Quality’ to 15.In the next window click on ‘undisplay’ and click ‘ok’. 2 25 . xi.
By last step go to your folder and open the (inhibitor.Go to ‘Display’. xxviii. xxxi.Go to ‘Select’ and in it click on ‘Direct Select’.Click ‘ok’. xxx. xxxvii.pdbq) file and open it.In the next window click on ‘Stick’ and ‘Balls’. xxxiii. xxv.Click ‘ok’. xxxii. xxxv.Go to ‘Color’ and click on ‘By Atom Type’.In it click on ‘Stick and Balls’. xxxvi.Click ‘ok’. xxvi.Click on ‘Molecule’ again and now click on the ligand or the inhibitor.xxiii. xxix. xxiv.docked. xxvii.Click on ‘Dismiss’.Go to ‘File’ and in it go to ‘Read Molecule’.Go to ‘Hydrogen Bond’.In the window that opens click on ‘Molecule’ and in it click on your macromolecule. 2 26 .In the window that opens set ‘Stick Quality’ to 15 and set ‘Ball Quality’ to 15. xxxiv.
2 27 . xxxxv. xxxxiii.to a suitable size.In the next window click on.Click ‘ok’. xxxxx.Go to ‘Déjà vu GUI’ and click on ‘camera’ in the lower window. Click on ‘SW’ and then click on ‘S’.In the window that opens click on ‘specify two sets’.I the bottom ‘Molecule List’.xxxvii. xxxxvii.In it click on ‘as lines’. xxxxviii. xxxxii. xxxix.in the top list under ‘Molecule List’ click it and select macromolecule. xxxxi.Click ‘Dismiss’ in the window that opens. xxxxxi. xxxviii.In the window that opens adjust ‘bond length’ and ‘bond radius’ of the hydrogen bond by using mouse.Go to H bond and in it click on ‘Display’.Again go to ‘Display’ and click on ‘Cylinders’.Go to ‘File’ of PMV and click on ‘save as’. xxxxiv. xxxxvi. click on ‘Set Background Color’.In it click on ‘Set Parms+Build’.click it and select the ligand. xxxx. xxxxix.In it go to ‘Build’.On the window which lengthens.
2 28 .tif).xxxxxii.In the window that opens click on ‘Browse’ and go to your folder and save the picture as (protein.
RESULTS AND DISCUSSION Retrieval of protein sequence of COX2 (cyclooxygenase2). >gi|3915797|sp|P35354|PGH2_HUMAN Prostaglandin G/H synthase 2 precursor (Cyclooxygenase-2) (COX-2) (Prostaglandin-endoperoxide synthase 2) (Prostaglandin H2 synthase 2) (PGH synthase 2) (PGHS-2) (PHS II) MLARALLLCAVLALSHTANPCCSHPCQNRGVCMSVGFDQYKCDCTRTGFYGENCSTPEFLTRIKLFLKPT PNTVHYILTHFKGFWNVVNNIPFLRNAIMSYVLTSRSHLIDSPPTYNADYGYKSWEAFSNLSYYTRALPP VPDDCPTPLGVKGKKQLPDSNEIVEKLLLRRKFIPDPQGSNMMFAFFAQHFTHQFFKTDHKRGPAFTNGL GHGVDLNHIYGETLARQRKLRLFKDGKMKYQIIDGEMYPPTVKDTQAEMIYPPQVPEHLRFAVGQEVFGL VPGLMMYATIWLREHNRVCDVLKQEHPEWGDEQLFQTSRLILIGETIKIVIEDYVQHLSGYHFKLKFDPE LLFNKQFQYQNRIAAEFNTLYHWHPLLPDTFQIHDQKYNYQQFIYNNSILLEHGITQFVESFTRQIAGRV AGGRNVPPAVQKVSQASIDQSRQMKYQSFNEYRKRFMLKPYESFEELTGEKEMSAELEALYGDIDAVELY PALLVEKPRPDAIFGETMVEVGAPFSLKGLMGNVICSPAYWKPSTFGGEVGFQIINTASIQSLICNNVKG CPFTSFSVPDPELIKTVTINASSSRSGLDDINPTVLLKERSTEL 2 29 .
1: BLAST RESULT 4.BLAST Result:- Fig4.ClustalW Results: 3 30 .2.
Results of search Number of sequences Alignment score Sequence format Sequence type ClustalW version 4 20349 Pearson aa 1.aln clustalw-20070718-12352061.83) multiple sequence alignment seq2 seq4 seq3 seq1 -----------------ANPCCSNPCQNRGECMSTGFDQYKCDCTRTGFYGENCTTPEFL 43 -----------------ANPCCSNPCQNRGECMSTGFDQYKCDCTRTGFYGENCTTPEFL 43 -----------------ANPCCSNPCQNRGECMSTGFDQYKCDCTRTGFYGENCTTPEFL 43 MLARALLLCAVLALSHTANPCCSHPCQNRGVCMSVGFDQYKCDCTRTGFYGENCSTPEFL 60 seq2 seq4 seq3 seq1 seq2 seq4 TRIKLLLKPTPNTVHYILTHFKGVWNIVNNIPFLRSLIMKYVLTSRSYLIDSPPTYNVHY 103 TRIKLLLKPTPNTVHYILTHFKGVWNIVNNIPFLRSLIMKYVLTSRSYLIDSPPTYNVHY 103 TRIKLLLKPTPNTVHYILTHFKGVWNIVNNIPFLRSLIMKYVLTSRSYLIDSPPTYNVHY 103 TRIKLFLKPTPNTVHYILTHFKGFWNVVNNIPFLRNAIMSYVLTSRSHLIDSPPTYNADY 120 GYKSWEAFSNLSYYTRALPPVADDCPTPMGVKGNKELPDSKEVLEKVLLRREFIPDPQGS 163 GYKSWEAFSNLSYYTRALPPVADDCPTPMGVKGNKELPDSKEVLEKVLLRREFIPDPQGS 163 3 31 .output clustalw-20070718-12352061. Scores Table SeqA 1 1 1 2 2 3 Name seq1 seq1 seq1 seq2 seq2 seq3 Len(aa) 604 604 604 587 587 552 SeqB 2 3 4 3 4 4 Name seq2 seq3 seq4 seq3 seq4 seq4 Len(aa) 587 552 552 552 552 552 Score 86 88 87 99 99 99 CLUSTAL W (1.dnd clustalw-20070718-12352061.83 clustalw-20070718-12352061.input JalView Output file Alignment file Guide tree file Your input file .
552 GFQIINTASIQSLICNNVKGCPFTSFSVPDPELIKTVTINASSSRSGLDDINPTVLLKER 600 seq2 seq4 seq3 STEL 587 ------- 3 32 .seq3 seq1 GYKSWEAFSNLSYYTRALPPVADDCPTPMGVKGNKELPDSKEVLEKVLLRREFIPDPQGS 163 GYKSWEAFSNLSYYTRALPPVPDDCPTPLGVKGKKQLPDSNEIVGKLLLRRKFIPDPQGS 180 seq2 seq4 seq3 seq1 NMMFAFFAQHFTHQFFKTDHKRGPGFTRGLGHGVDLNHIYGETLDRQHKLRLFKDGKLKY 223 NMMFAFFAQHFTAQFFKTDHKRGPGFTRGLGHGVDLNHIYGETLDRQHKLRLFKDGKLKY 223 NMMFAFFAQHFTHQFFKTDHKRGPGFTRGLGHGVDLNHIYGETLDRQHKLRLFKDGKLKY 223 NMMFAFFAQHFTHQFFKTDHKRGPAFTNGLGHGVDLNHIYGETLARQRKLRLFKDGKMKY 240 seq2 seq4 seq3 seq1 QVIGGEVYPPTVKDTQVEMIYPPHIPENLQFAVGQEVFGLVPGLMMYATIWLREHQRVCD 283 QVIGGEVYPPTVKDTQVEMIYPPHIPENLQFAVGQEVFGLVPGLMMYATIWLREHQRVCD 283 QVIGGEVYPPTVKDTQVEMIYPPHIPENLQFAVGQEVFGLVPGLMMYATIWLREHNRVCD 283 QIIDGEMYPPTVKDTQAEMIYPPQVPEHLRFAVGQEVFGLVPGLMMYATIWLREHNRVCD 300 seq2 seq4 seq3 seq1 ILKQEHPEWGDEQLFQTSKLILIGETIKIVIEDYVQHLSGYHFKLKFDPELLFNQQFQYQ 343 ILKQEHPEWGDEQLFQTSKLILIGETIKIVIEDYVQHLSGYHFKLKFDPELLFNQQFQYQ 343 ILKQEHPEWGDEQLFQTSRLILIGETIKIVIEDYVQHLSGYHFKLKFDPELLFNQQFQYQ 343 VLKQEHPEWGDEQLFQTSRLILIGETIKIVIEDYVQHLSGYHFKLKFDPELLFNKQFQYQ 360 seq2 seq4 seq3 seq1 seq2 seq4 seq3 seq1 NRIASEFNTLYHWHPLLPDTFNIEDQEYSFKQFLYNNSILLEHGLTQFVESFTRQIAGRV 403 NRIASEFNTLYHWHPLLPDTFNIEDQEYSFKQFLYNNSILLEHGLTQFVESFTRQIAGRV 403 NRIASEFNTLYHWHPLLPDTFNIEDQEYSFKQFLYNNSILLEHGLTQFVESFTRQIAGRV 403 NRIAAEFNTLYHWHPLLPDTFQIHDQKYNYQQFIYNNSILLEHGITQFVESFTRQIAGRV 420 AGGRNVPIAVQAVAKASIDQSREMKYQSLNEYRKRFSLKPYTSFEELTGEKEMAAELKAL 463 AGGRNVPIAVQAVAKASIDQSREMKYQSLNEYRKRFSLKPYTSFEELTGEKEMAAELKAL 463 AGGRNVPIAVQAVAKASIDQSREMKYQSLNEYRKRFSLKPYTSFEELTGEKEMAAELKAL 463 AGGRNVPPAVQKVSQASIDQSRQMKYQSFNEYRKRFMLKPYESFEELTGEKEMSAELEAL 480 seq2 seq4 seq3 seq1 YSDIDVMELYPALLVEKPRPDAIFGETMVELGAPFSLKGLMGNPICSPQYWKPSTFGGEV 523 YSDIDVMELYPALLVEKPRPDAIFGETMVELGAPFSLKGLMGNPICSPQYWKPSTFGGEV 523 YSDIDVMELYPALLVEKPRPDAIFGETMVELGAPFSLKGLMGNPICSPQYWKPSTFGGEV 523 YGDIDAVELYPALLVEKPRPDAIFGETMVEVGAPFSLKGLMGNVICSPAYWKPSTFGGEV 540 seq2 seq4 seq3 seq1 GFKIINTASIQSLICNNVKGCPFTSFNVQDPQPTKTATINASASHSRLDDINPTVLIKRR 583 GFKIINTASIQSLICNNVKGCPFTSFNVQ------------------------------.552 GFKIINTASIQSLICNNVKGCPFTSFNVQ------------------------------.
1 micro moles /L 4. Structure Inhibitor Name Sodium Parecoxib sodium N-[4-(5methyl-3phenyl-1.3 SWISS MODEL Result: _________________ 4.2oxazol-4yl)benzenesulfon amide Phase III Valdecoxi b 0.3mi croM Phase III 3 33 .005 +/-0.seq1 STEL 604 4.ulc ers Hyper algesia :increa sed sensiti vity to pain Clinical phase Phase IV celecoxib 4-[5-(4methylphenyl)3(trifluoromethyl) pyrazol-1yl]benzenesulfon amide 4-(5-methyl-3phenyl-1.4.001 +/0.8 +/-0.2oxazol-4yl)phenyl]sulfon ylpropanimidate IUPAC Name IC50 Value 0.4 nmol/ L Side effects Gastro intesti nal infecti on Gastro intesti nal infecti on. INHIBITOR TABLE: Table 4.1 : List of inhibitors against COX2(cyclooxygenase2).
10dihydro-7Htetracene-5.9R)-7[(2S.7+/ -0.6S)4-amino-5hydroxy-6methyl-oxan-2yl]oxy-6.12dione 5-chloro-2-(6methylpyridin-3yl)-3-(4methylsulfonylp henyl)pyridine 0.5) nmol/ L headac Phase III he.1 micro M Fatigu e and dizines s Phase III Lumiracox ib 2-[2-[(2-chloro6-fluorophenyl)amino]5-methylphenyl]acetic acid 0.ble eding Doxorubic in (7S.4S.5S.42+/ Hair -0.fa tigue Phase II Etoricoxib 1.06+/ dizzine -0.3 micro M ss ess or sleepin Phase III 3 34 .11trihydroxy-9-(2hydroxyacetyl)4-methoxy-8.9.Rofecoxib 4-(4methylsulfonylp henyl)-3-phenyl5H-furan-2-one (4.1+/0.3 micro M loss.
4.4 micro M -0.9+/0.5- 4.8 micro M allergi c reactio n Phase II 4.6+/0. dizzines s Phase III Etodolac 1.4.3thiazol-2yl)amino]methyl idene]-9-methyl10. fatigue related to anemia Phase III 3 35 .3 micro M constipa tion.071 +/-0.3.8-diethyl1.6-dioxo-3piperidyl)isoindo le-1.3 micro M agranulo cytosis Phase II dimethyl-3-oxopyrazol-4-yl)methylamino]methanes ulfonate 2-(2.3-dione Thalidomi de 3.1+/0.10-dioxo10λ6-thia-9azabicyclo[4.Nimesulid e N-(4-nitro-2phenoxyphenyl)methanes ulfonamide 0. liver toxific ation Phase III Dipyrone sodium 2-phenyl- [(1.51+/ headach e.4-b]indole-1acetic acid.3. (8E)-8[hydroxy-[(5methyl-1.5 micro M Liver enlarg ement.0 ]deca-1.5trien-7-one 2.9tetrahydropyrano -[3.
2: Docked energies and other parameters of the inhibitors using Auto Dock docking program.5 Docking of ligand to receptor 4.4 +/-0. s and dizzines Phase II methoxynaphtha len-2yl)propanoate Table4. chemical structure and IUPAC name of different inhibitors which show interaction with COX2(cyclooxygenase2). 3 36 .4 micro M headach e. abdomi nal pain Phase III Naproxen sodium (6- (2S)-2- 3.4 shows the chemical formula. protein. molecular weight.5.1 AUTODOCK RESULTS Table 4.8 +/-0.7 micro M ulcerati ons.ibuprofen 2-[4-(2methylpropyl)ph enyl]propanoic acid 3. 4. The IUPAC name of the inhibitor is further used in making pdb file of that inhibitor.
79 -19.3 35.96 2.9 -11.18 24.0 1.12 0.67 -18.74 -19.67 -2. Internal-energy and finally Docked energy of the COX2 (cyclooxygenase2) with its inhibitor.22 -9.7 -16.53 28.61 25.49 -19.3 -16.0 3.1 -2.56 Free Energy -16.87 0.84 -10.3 55.47 31.SN 1 2 3 4 5 6 7 8 9 10 Inhibitor Name Etodolac Nimesulide Etoricoxib Melocoxib Valdecoxib Doxorubicin Thalidomide Ibuprofen Naproxene Rofecoxib Docked Energy -15.57 0.2 shows the results displayed by Autodock docking program displaying Freeenergy.01 -15.0 0.92 Intermolecular Energy -17.79.57 0.98 -17.56 -11. 4. Autodock docking results show that Bellnamine inhibitor of COX2 (cyclooxygenase2) shows best interaction with the COX2(cyclooxygenase2). Intermolecular-energy.05 36.84 -8.0 Table 4.04 -18.49 -11.4 -1.6 Python Molecular Viewer (PMV) Results: 3 37 .3 37.1 Internal Energy 2.63 0.87 -19. with its docked energy of –19.73 -16.1 -17.74 -16.97 -11.1 Ref RMS 37.79 -19.13 -19.12 -19.68 32.78 -11.
6.Fig 4.: Hydrogen Bond Formed Between Protein’s Active Site and Inhibitor Etorocoxib 3 38 .1.
2.6.: Hydrogen Bond Formed Between Protein’s Active Site and Inhibitor Etodolac 3 39 .Fig 4.
Fig 4.6.: Nimesulide Hydrogen Bond Formed Between Protein’s Active Site and Inhibitor 4 40 .3.
6.: Hydrogen Bond Formed Between protein’s Active Site and Inhibitor Doxorubicin 4 41 .Fig4.4.
: Hydrogen Bond Formed Between Protein’s Active Site and Inhibitor Melocoxib 4 42 .6.Fig4.5.
Fig 4.6.: Hydrogen Bond Formed Between Protein’s Active Site and Inhibitor Thalidomide 4 43 .6.
Fig 4.6.: Hydrogen Bond Formed Between Protein’s Active Site and Inhibitor Rofecoxib 4 44 .7.
6.Fig 4.: Representation Of The Interaction between the protein and Valdecoxib 4 45 .8.
: Representation Of The Interaction between the protein and Ibuprofen 4 46 .9.6.Fig 4.
money and energy as compared to other hit and trial methods.helping furthur development in research and development in this field.: Representation Of The Interaction between the protein and Naproxene DISCUSSION Rational Drug Designing Strategies reduce a lot of time .The use of sophasticated softwares and tools greatly help in this process.10.6.According to recent trends mathematical modelling has become very valuable. The 4 47 .Fig 4.
the better it is. From the figure we can conclude that ‘Naproxene’ has the minimum docked energy.The more negative the docking energy.7.Shows the relative docked energies of various inhibitors with the target protein. Fig 4.which essentially should be less than zero.hence the best inhibitor 4 48 .main concern in AutoDock is computation of docking energy.
Rational Drug Designing Strategies reduce a lot of time .money and energy as compared to other hit and trial methods.Basically COX 2 is an enzyme present in our body which is an integral part of the inflamatory responses of our immune system.it can cause tumor formation.The threat if cancer bein cause by COX 2 has spread globally and many bio-pharma giants have launched several medicines eg:Celebrex.Hence the task was completed successfully.as stated earlier in laoratories.Nevertheless we try to reduce the burden on the general health of the patient to the maximum extent possible.its furthur verification is done.After a drug has been designined in-silico.There are many reasons and factors responsible for induction of cancer.Hence the conclusion is that naproxene is the best inhibitor.The precise reason for it was its docking energy which was the lowest(docked energy=-19.This method has greately reduced the time .According to recent trends mathematical modelling has 4 49 .for COX 2.as gastric ulcer in case of aspirin.naproxene emerged to be be the best inhibitor for the protein COX 2.This method of using computer to design the drugs has indeed hastened the process of drug discovery.COX 2 is also one of those factors responsible for the induction of cancer.If due to any reason the secretion of this enzyme crosses a perticular threshold.CONCLUSION After the project work on rational drug design for COX 2.among all other inhibitors used.some are rejected due to their side effects.There are many inhibitors which are used for the inhibition of over secretion of COX 2.This tumor may under the influence of mitogens and other carcinogens can cause cancer.However there is no inhibihitor which is fully perfect and without any sideeffects.The main concern is to reduce the over-expression of COX 2 and not to terminate its secretion completely.The intial phase of discovering a new drug nowadays is by using CADD. Cancer is a major threat to the world’s health.Hence .the conclusion is that out of all the inhibitors chosen for the docking.newer drugs are required which have the same efficacy as the older one but are having fewer side effects.Vioxx etc.Out of many inhibitors.energy and money involved in the traditional methods.79).
simpler the drug more easiliy the molecule will be docked with the inhibitor.which I had chosen.energy and money involved in drug discovery. In my project I found naproxene(dockin energy= -19. Hence we saw how rational drug design strategies reduce time .It also shows the hygrogen bonds formed between proteins active site and the inhibitor.79) as to be the best inhibitor among the rest of the inhibitors.The dock energy table and the docked energy graph indeed confirm it.e.become very valuable recently.The use of sophasticated softwares and tools greatly help in this process. 5 50 .laboratories) for the furthur verification of the inhibitor.helping furthur development in research and development in this field.The complexity of the protein plays a key role in designing of the drug.The result can act as a guideline for the carrying of furthur experimentation to be carried out in wet lab(i.The PMV showed us the sites where the inhibitor attacks the target protein. Through my endeavour I came to to the conclusion that selective inhibitors are the best suited for inhibiting the action of COX 2.
... Melanoma Research.D.S. Aspirin and the Risk of Colorectal Cancer in Relation to the Expression of COX-2.A. Taddei. Robert. COX-2 and ppRb expression in neurons occurs during early Braak stages prior to the maximal activation of astrocytes and microglia in Alzheimer's disease. Arganini. 9:41-47... Jana..F. Charles.A. Journal of Neuroinflammation. 4:22-25.A. Learning Memory.. and Nicolas. cyclooxygenase-2 (COX-2) inhibitor compared with dexamethasone in a survival study of rats with intracerebral 9L gliosarcomas.S. (2007). Anna.A.M. Shuji. 5 51 .P. Cyclooxygenase-2 (COX-2) Overexpression in Meningiomas: Real Time PCR and Immunohistochemistry.H. and Maximal (2005).A(2002). and Lukasz. Applied Immunohistochemistry & Molecular Morphology. Francesca.Nuero Oncology..R.V.(2002). Elise S.. Mark.G.. Barbara.and Mennonna.W. and Kathryn..V.Thomas.M.E... Duccio.T. F.. The New England Journal Of Medicine.L. Jeroen.REFERENCES Andrew. Susan. 2:27-28.H..G.. Kuzbicki. Stuart..C.B. 15:187-192.C.. Cyclooxygenase-2 (COX-2): first immunohistochemical marker distinguishing early cutaneous melanomas from benign melanocytic skin tumours.P.G. Lisa... 17:139-145. Samia. Post-Training Cyclooxygenase-2 (COX-2) Inhibition Impairs Memory Consolidation.T. 5:685693. (2007).O...(2005).
ncbi.pharmacy.P.expasy.T. Cardiovascular events associated with rofecoxib in a colorectal adenoma chemoprevention trial.. Inhibition of Interleukin1ß-Induced COX-2 and EP3 Gene Expression by Sodium Salicylate Enhances Pancreatic Islet ß-Cell Function..G.D. New England Journal of Medicine.org/workspace Journals: · · · · · · · · Applied Immunochemistry And Molecular Morphology.T..nlm.R.S.gov/ www. (2005). 11:1092-1102. 6:880-888 .T. . Sonia.(2005).Marco..L. http//:Swissmodel. and Ross P....pdb. Diabetes. Commonly used websites: · · · · www. Robert.ca/drugbank. Phuong. Cyclooxygenase-2 (COX-2) is frequently expressed in multiple myeloma and is an independent predictor of poor outcome. and Robertson.B. Catherine. Blood Cancer Biology Diabetes Health Affairs Journal Of Neuroinfllammation. 51:112-118. Michael.L..org http://redpoll. and Maria.ualbarta.nih... Learning Memory Melanoma Research 5 52 .E. Andreas.(2002).Blood.V.O.
· · · · · Nuero Oncology The New England Journal of Medicine Neuro Oncology The New England Journal Of Medicine Thieme Docking Abbreviations: NCBI-National Center Of Biotechnology Information. BLAST-Basic Local Alignment Search Tool. COX-Cyclooxygenase. PDB-Protein Data Bank. CADD-Computer Aided Drug Designing. 5 53 .
5 54 ..
5 55 .