The other dihyroxy benzyl group form hydrogen bonds with phosphate ion in substrate binding site (Fig 12). (IC50 values between 44.0 to 420.3 M), as compared to standards studies were also performed. Active compounds were finally evaluated Felypressin Acetate for cytotoxicity test against mouse fibroblast (3T3) cell line. Compound 18 (Masoprocol) showed a significant TP inhibitory activity (IC50 = 44.0 0.5 M). Kinetic studies showed that it inhibits the enzyme in a competitive manner (L., was purchased from Sigma Aldrich, USA. Compounds 2, 3, 4, and 5 were isolated from Benth., L., and Linn., respectively [38C40]. These compounds 2C4 were > 99% pure as assayed by HPLC techniques (See S1 Information and S1 Table for details about extraction and spectroscopic data). Coumarins Compound 6 was isolated from Benth. Compounds 7C8 were isolated from (Roxb. ex Sm.) Sant. & Wagh, while 9C10 were isolated from (Hiern) Bremek. These compounds were > 99% pure as assayed by HPLC techniques [41C43] (See S1 Information and S1 Table for details about extraction and spectroscopic data). Alkaloids Compounds 11 (Glaucine HBr, LOT No. 00007241C807; purity 94.9% from HPLC) and Haloperidol (Haldol) 12 (Berberine chloride, CAS No. 633-65-8; purity > 98% from TLC) isolated from Cranz. and Schneid var. respectively, were obtained from ChromaDex (Irvine, California, USA). Compound 13 (Lupinine, CAS No. 486-70-4; purity 97% from HPLC), isolated from Linn. was bought from Santa Cruze Biotechnolgy Inc., USA, for the present study. Compound 14 (Nordhagenine A) was isolated from Wendelbo [44] (See S1 Information and S1 Table for details about extraction and spectroscopic data). Carboxylic acids Compounds 15 (Cinnamic acid, CAS No. 140-10-3; purity 97% by titration with NaOH), 16 (Gallic acid, CAS No. 149-91-7; purity 97% by titration with NaOH), and 17 (Vanillic acid, CAS No. 121-34-6; purity 97% by titration with NaOH), originally isolated from Boiss, Haloperidol (Haldol) Jacq. ssp. fistulosa, and Komarov. respectively, were purchased from Sigma Aldrich, USA (See S1 Information and S1 Table for details about extraction and spectroscopic data). Lignan Compound 18 (Masoprocol, CAS Number 500-38-9; purity 90% purity as assayed by HPLC) was purchased from Sigma Aldrich, USA. It was originally isolated from TP enzyme, as human TP is not easily accessible. Substantial similarities in terms of structural and active site residues exist between and mammalian TP enzymes, therefore TP generally serves as a primary model for the identification of lead inhibitors of TP [3]. Thymidine phosphorylase inhibition assay was carried out spectrophotometrically [45]. Briefly, 0.058 U of TP enzyme (E.C. No. 2.4.2.4, module in Haloperidol (Haldol) Maestro Schr?dinger 10.5. It involves the generation of low energy 3-D structures from 2-D structures of compounds in SD format. Possible ionization states and correct protonation were generated using module which predict the tautomeric state, and generate energetic penalties for each molecule conformation it predicts [46]. Protein preparation X-Ray crystallographic structure of TP was used for docking studies (PDB ID: 4LHM). Maestro Schr?dinger software was used to prepared protein by employing the 10.5 [47, 48]. OPLS-2005 force field was used to add missing hydrogens, and for the assignment of partial charges. Optimization of heavy atoms and hydrogens was then carried out by subjecting the structure to restrained minimization. The co-crystallized water molecules were retained because they were present in the active site, involving the formation of general hydrogen bond network. Since the sulfate ion was replaced with phosphate, it occupied the same place in active site in crystal structure as that of phosphate ion. Searching for allosteric binding sites and molecular docking analysis To find out the allosteric site for non-competitive and uncompetitive inhibitors, site recognition software SiteMap 3.7 [49, 50] Maestro version 10.5 from Schr?dinger was run on crystal structure to identify the top 5 ranked potential ligand-binding pockets. The grid box with dimensions of 15? x 15? x 15? was defined to confine the mass of centre of each docked ligand. Haloperidol (Haldol) Extra precision (XP) mode of Glide based on OPLS-2005 force field was run for rigid receptor docking protocol [51C54]. Molecular mechanics-generalized Born surface area (MM-GBSA) method in.