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In the previous chapter 3, we have discussed about the in vitro therapeutic potential of curcumin conjugates viz its dipiperoyl and diglycinoyl esters vis-à-vis curcumin as anticancer agents using breast cancer cell lines i.e. the anti-inflammatory, ant proliferative and antioxidant properties of curcumin are very well reported. Curcumin is well known for regulating redox potential of cells. The redox state is a measure of the activity of metabolic reactions to donate or accept electrons with the living cells. There are many redox couplers with varying potentials. The gluthione (GSH) and thioredoxin(Trx) systems have overlapping functions in thiol /disulphide redox control in both the cytoplasm and the nucleus. The literature shows that the GSH and Trx systems have unique compartmental functions in the control of transcriptional regulation by Nrf-2 a redox sensitive transcription factor that is activated by an oxidative signal in cytoplasmbut it has a specific cysteine that must be reduced to bind to DNA in the nucleus. Trx is important for later process. In the present work we have studied the effect of curcumin and other curcuminoids on the redox regulatory activity of Trx.
4.1 Thioredoxin Trx System:The thioredoxin system is the major protein disulfide reductase in cells and comprises of thioredoxin (Trx), thioredoxin reductase(TrxR) and NADPH. . Thioredoxin reductase play wide role in cellular viability by maintaining redox status of cells.
Trx, proliferation, activation of transcription factors, defense against oxidative stress, control of intracellular redox states and apoptosis. Thioredoxin is a key enzyme for nucleic acids synthesis and acts by complexing with ribonucleotide reductase and directly serving as electron donor . The major function of TrxR is reduction of ribonucleotide diphosphates (r-NDPs) to deoxyribonucleotide diphosphates (dNDPs). These dNDPs further change to corresponding triphosphates (dNTPs) by kinase action. Synthesis of dNTP's occurs under tight regulation at the active site of Ribonucleotide reductase. The activity of the thioredoxin system has been reported to be disrupted in various physiological disorders such as HIV infection, Alzheimer disease and diverse types of cancers and is therefore useful as a biological marker for several diseases. Thiordoxin reductase simultaneously present everywhere and the expression of thioredoxin reductase in tumor cells is often several fold greater than in normal tissues. The overexpression of thioredoxin reductase in tumor cells seems to be decisive for cell proliferation. Thereby thioredoxin redox system is known new potential target for anticancer drugs .
Sharma et al [ ] have shown in their Pharmacodynamic and Pharmacokinetic studies that curcumin is potent inhibitor of tumor initiation. It has been widely reported in literature that curcumin induces apoptosis in various cancers.,  Besides breast and colon cancers curcuminoids are potent chemo- preventive agents inhibit tumor growth in several epithelial origin i.e skin, oral and intestinal carcinogenesis. 
Thioredoxin reductase is irreversibly modified by curcumin binds at the active site of TrxR reductase, and inhibits the process of electron transfer from NADPH to ribonucleotide. Mammalian thioredoxin reductase ( TrxR) have a remarkably varied substrate specificity. It could be explained by their easily accessible C-terminal catalytic site redox center (SH-SeH/S-Se), which contains an essential selenocysteine residue. Fang et al. have characterized an irreversible inhibition mechanism of TrxR by curcumin due to formation of a 1:2 covalent adduct i.e. one mole of TrxR binds with two moles of curcumin. The inhibition is caused by covalent modification of the residues at the active sites CYS496 and SEC497 which destroys the Trx reduction activity. Beside this, the curcumin-modified enzyme gets converted into an NADPH oxidase with production of reactive oxygens to which cancer cells appear to be more sensitive. The IC50 value for TrxR enzyme has been reported to be 3.6 uM at room temp in vitro.
Figure of thioredoxin reductase enzyme
The thioredoxin reductase (TrxR) isoenzymes are homologous to glutathione reductase and contain conserved C-terminal cysteine and selenocysteine residues forming a redox center of selenosulphide and selenothiol at the active site of each chain of TrxR. The three-dimensional structure of the SEC498 mutant of rat TrxR as a complex with NADP+ has been determined to 3.0-A0 resolutions by X-ray crystallography. The overall structure of TrxR is similar to glutathione reductase (GR), including conserved amino acid residues binding the cofactors FAD and NADPH.
4.2 Curcumin & Cancer: Since curcumin is safe upto a higher dose i.e. upto 200mg and affects all three stages of cancer progression therefore design and synthesis of new synthetic analogues against the cancere targets like TrxR is highly desirable. There are several analogs has been made and in vitro tested.
Lin L etal (2006)  have synthesized a series of forty curcumin analogues for prostate cancer of both androgen dependent and androgen refractory stages. And tested against two cancer cell lines , androgent dependent LNCaP and androgen independent PC-3 they report out of forty 10 compunds were active
Liu Z et al (2008)  had evaluated the thioredoxin reductase inhibition activity of various synthetic analogs of curcumin and reported B5 is best among them. They also report that symmetrical mono keto curcumin analogues with hydroxyl groups , methoxy groups, and/ or halogen atoms at ortho position of p-phenols were more potent TrxR inhibitors than that of natural curcumin.
Qiu X et al (2008) have been synthesized series of unsymmetrical curcumin derivatives and evaluated their activity . Xu et al reported that unsymmetrical curcumin analogues having furan moiety have shown better activity than natural analogues . However very few were active in cell lines.
4.3 In present Study: In the present study natural analogues of curcumin were docked at both the active site in order to compare the potency of these naturally occurring curcuminoids viz curcumin, curcumin-I, demethoxy curcumin (curcumin-II, and bisdemethoxy curcumin curcumin-III (fig 4.1). Besides this other curcumin conjugates synthesized in our laboratory and other analogues tested against TrxR were screened at the both interface of TrxR. Receptor based pharmacophore of both sites were simulated and binding mode of curcumin in respect to Se atom was mapped.
Volume and area of both cavities was predicted and distance mapping carried out. In our docking study we report that two active sites coincide at the junction of both chains. We also report that curcumin-I at E chain and Curcumin-II at E and F chain active site is seems to be more potent as compared to the others in terms of docking energy. It is also clear that active site of TrxR occurs at the junction of E and F chain. So it is necessary to consider both chains together for any further in silico and wet lab experiments. It has also been predicted that at least one methoxy function in curcuminoids is needed for interaction with catalytic residues of thioredoxin.
4.4 Material and Methods
CASTp programme were used to measure the surface area and volume of both chains cavity, both in solvent accessible surface (SA, Richards' surface) and molecular surface (MS, Connolly's surface). Volume of cavity predicted by CASTp programme was very helpful in deciding size of Grid map in docking study.
Curcuminoids were docked at the binding site of TrxR using AutoDock-3 software.  . The target protein 1H6V was taken from protein databank. The E and F-chain of TrxR homodiameric unit was taken for our study. Cofactors FAD, NADP and hetero atoms ie water were cleaned from for docking. The docking study was performed with curcumin and two of it's naturally occurring analogs at the active site (CYS497-SeCYS498).
Thioredoxin reductase was set up for docking as follows: protonate modules were used to add polar hydrogen. Charges and salvation were added to the final protein file protein.pdbqs. To generate grid for actual docking Autogrid module were used. Dimensions of grid were decided by the cavity size, simulated previously by CastP server. The grid size for E and F chains cavity were 50.50.50 AËš with spacing of 0.35 AËš. The grid center for E chain and F chain were (30.074, 0.267 and -1.792) and (28.922, 7.243 and 42.311) respectively. Genetic algorithm was selected for all simulation, other parameters i.e number of energy evaluations, number of generation in genetic algoritham and no of GA per run were 2,500,000, 270000 and 50 respectively. Curcuminoids were also prepared for docking, all single bonds of ligands were considered flexible while targeting rigid macromolecule. Previously it has been proven that AutoDock is quite capable to successfully reproduce many crystal complexes . Genetic algoritham was used for search method for each simulation.  .
3 D structure of curcumin-1 was taken from Cambridge structure database while demethoxy and bis demethoxy curcumin were drawn by chemsketch and 3 D structures were obtained from CORINA server and further optimized Ligprep module using MM force filed of schrodinger software. The atomic charges were added using AutoDock empirical free energy function. [17,18]. Finally curcuminoids were setup for docking with the Autotors module of autodock and the no of flexible torsions were set to seven for all curcuminoids.
All the three naturally occurring curcuminoids were docked at both active sites of thioredoxin reductase successfully. The docking and binding energies of both curcumin-1 and curcumin-II at E and F chains active sites are given in the tables 4.1 and 4.2. We took Se atom of catalytic residues SeCys498 as a center for all simulations in both chain's active sites. Se atom has positive X, and negative Y and Z -coordinates at the E chain active site while in F chain active site all the coordinates have positive value. Since X, coordinates of Se atom of both chains active site have nearly equal positive value and negative Y and Z value, due to this both chain's active site occur in opposite directions. In our docking simulation we took both chains together because active site is residing at the junction of both chains.
Fang et.al, reported in their mass spectrometry with E- chain C-terminal residues that it forms 1:2 adduct with Cys497-SeCys498 residue. Conformation of curcumin-I molecule at the active site of E-chain is very important because Se atom of Sec498 is approaching very close to the Sp2 C11 of main chain of Curcumin-1 molecule. Curcumin-II at both E and F chain active site is showing more interesting results in comparison to Curcumin-I, which can be helpful in designing new analogs of curcumin, which can be synthesized
Structurally curcumin-2 has one methoxy group in compare to two in curcumin-1 and none in curcumin-III. In our docking simulation it is evident that Curcumin-II molecule can prove to be more active than curcumin-1 because the interacting Se atom of catalytic residue SeCys498 and ligand C13 atom is very close to each other in both E-chain and F-chain active site. While simulation at the E-chain active site with curcumin-1 and curcumin-II molecule shows it to be active while Curcumin-III is not because interacting Se atom of receptor and ligand's interacting carbon atom of curcumin-III are not as close to each other. Therefore we can conclude that for greater bioactivity one methoxy group is needed. 
Among all the simulations at E and F chain active sites Curcumin-II molecule has promised to be most bioactive molecule since it shows greatest docking energies. No additions at O25H45, O24H44 O21H40, and O22C23H3 functional groups would help in case of E-chain. However, area and volume of F chain active site is greater than E-chain active site so it provides sufficient space to add larger substituent at the O22C23H3.spc growing site.
Curcumin-1 molecule has also shown interesting result because Se atom is at a distance of 3.23Å from C11 that is very close to C13 carbon atom of curcumin-1 molecule whereas it is already proved that Se atom is forming adduct with C13 atom. The substituent OH and OCH3 attached with A17 and A18 respectively are very close to the hydrophobic point where a hydrophobic substituent could be added.
The present study shows that curcumin binding at E-chain of thioredoxin reductase (TrxR) and that of monodemethoxy curcumin at E and F chain active sites is important as Se atom approaches near either C10, C11 or C12 carbon atoms, since it is already known that Se atom forms adduct with C13 atom. Extrapolating these results through molecular modeling and receptor based drug designing new curcumin analogs can be designed to act as potent anticancer drugs at TrxR receptor. Enzyme inhibition assay with all naturally occurring curcuminoids and with some synthetic conjugates, (where are these) which, we have designed by LigBuilder program, can confirm our simulations. Thus, it is possible to design synthetic curcumin analogs that may become more potent anti-cancer drugs, in addition to their current anticancerous profile.