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Identification of Aspartic Acid-203 in Human Thymidine Phosphorylase As Identification of aspartic acid-203 in human thymidine phosphorylase as an important residue for both catalysis and non-competitive inhibition by the small molecule “crystallization chaperone” 5’–tritylinosine (KIN59) A. Bronckaers, L. Aguado, A. Negri, M.-J. Camarasa, J. Balzarini, M.-J. Pérez-Pérez, F. Gago, S. Liekens To cite this version: A. Bronckaers, L. Aguado, A. Negri, M.-J. Camarasa, J. Balzarini, et al.. Identification of aspartic acid-203 in human thymidine phosphorylase as an important residue for both catalysis and non- competitive inhibition by the small molecule “crystallization chaperone” 5’–tritylinosine (KIN59). Biochemical Pharmacology, Elsevier, 2009, 78 (3), pp.231. 10.1016/j.bcp.2009.04.011. hal-00493518 HAL Id: hal-00493518 https://hal.archives-ouvertes.fr/hal-00493518 Submitted on 19 Jun 2010 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Accepted Manuscript Title: Identification of aspartic acid-203 in human thymidine phosphorylase as an important residue for both catalysis and non-competitive inhibition by the small molecule “crystallization chaperone” 5’-O-tritylinosine (KIN59) Authors: A. Bronckaers, L. Aguado, A. Negri, M.-J. Camarasa, J. Balzarini, M.-J. Perez-P´ erez,´ F. Gago, S. Liekens PII: S0006-2952(09)00266-4 DOI: doi:10.1016/j.bcp.2009.04.011 Reference: BCP 10144 To appear in: Received date: 3-3-2009 Revised date: 3-4-2009 Accepted date: 6-4-2009 Please cite this article as: Bronckaers A, Aguado L, Negri A, Camarasa M-J, Balzarini J, Perez-P´ erez´ M-J, Gago F, Liekens S, Identification of aspartic acid-203 in human thymidine phosphorylase as an important residue for both catalysis and non-competitive inhibition by the small molecule “crystallization chaperone” 5’-O-tritylinosine (KIN59), Biochemical Pharmacology (2008), doi:10.1016/j.bcp.2009.04.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Biochem. Pharmacol., revised 1 2 3 Identification of aspartic acid-203 in human thymidine phosphorylase as an important 4 5 6 residue for both catalysis and non-competitive inhibition by the small molecule 7 8 “crystallization chaperone” 5’-O-tritylinosine (KIN59) 9 10 11 12 13 14 15 16 17 18 A. Bronckaers*1, L. Aguado*2, A. Negri3, M.-J. Camarasa2, J. Balzarini1, M.-J. Pérez-Pérez2, 19 20 F. Gago3 & S. Liekens1 21 22 23 24 25 26 27 28 29 30 31 32 1 33 Rega Institute for Medical Research, K.U.Leuven, B-3000 Leuven, Belgium 34 35 2Instituto de Química Médica, CSIC, 28006 Madrid, Spain 36 37 3 38 Departamento de Farmacología, Universidad de Alcalá, 28871 Alcalá de Henares, Spain 39 40 41 42 43 44 45 46 47 48 Accepted Manuscript 49 50 *Contributed equally. 51 52 53 54 55 56 57 58 59 60 61 62 63 1 64 Page 1 of 40 65 Abbreviations 1 2 3 6A5BU: 6-amino-5-bromouracil; 6AT: 6-aminothymine; 7-DX: 7-deazaxanthine; BVDU: 4 5 6 (E)-5-(2-bromovinyl)-2’-deoxyuridine; CAM: chicken chorio-allantoic membrane; dThd: 7 8 thymidine; EDTA: ethylenediaminetetraacetic acid; KIN59: 5’-O-tritylinosine; GST: 9 10 11 glutathione S-transferase; HPLC: high-pressure liquid chromatography; MD: molecular 12 13 dynamics; MM-GBSA: molecular mechanics-generalized Born surface area; NMA: normal 14 15 16 mode analysis; PMSF: phenylmethylsulfonylfluoride; RESP: restrained electrostatic potential; 17 18 rmsd: root mean square deviation; SASA: solvent accessible surface area; TFT: 5- 19 20 trifluorothymidine; TP: Thymidine phosphorylase ; TP65: 9-[8-phosphonooctyl]-7- 21 22 23 deazaxanthine; TPI: 5-chloro-6-[1-(2-iminopyrrolidinyl)methyl]uracil hydrochloride; TS, 24 25 transition state. 26 27 28 29 30 Keywords: 31 32 33 Thymidine phosphorylase, 5’-O-tritylinosine (KIN59), allosteric inhibitor, modeling, 34 35 mutagenesis 36 37 38 39 40 41 42 43 44 45 46 47 48 Accepted Manuscript 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 2 64 Page 2 of 40 65 ABSTRACT 1 Thymidine phosphorylase (TP) is a catabolic enzyme in thymidine metabolism that is 2 3 frequently upregulated in many solid tumors. Elevated TP levels are associated with tumor 4 5 6 angiogenesis, metastasis and poor prognosis. Therefore, the use of TP inhibitors might offer a 7 8 promising strategy for cancer treatment. The tritylated inosine derivative 5’-O-tritylinosine 9 10 11 (previously designated KIN59) is a non-competitive inhibitor of TP which was previously 12 13 found to be instrumental for the crystallization of human TP. A combination of computational 14 15 16 studies including normal mode analysis, automated ligand docking and molecular dynamics 17 18 simulations were performed to define a plausible binding site for 5’-O-tritylinosine on human 19 20 TP. A cavity in which 5’-O-tritylinosine could fit was identified in the vicinity of the Gly405- 21 22 23 Val419 loop at a distance of about 11 Å from the substrate-binding site. In the X-ray crystal 24 25 structure, this pocket is characterized by an intricate hydrogen-bonding network in which 26 27 28 Asp203 was found to play an important role to afford the loop stabilization that is required for 29 30 efficient enzyme catalysis. Site-directed mutagenesis of this amino acid residue afforded a 31 32 33 mutant enzyme with a severely compromised catalytic efficiency (Vmax/Km of mutant enzyme 34 35 ~ 50-fold lower than for wild-type TP) and pronounced resistance to the inhibitory effect of 36 37 38 5’-O-tritylinosine. In contrast, the D203A mutant enzyme kept full sensitivity to the 39 40 competitive inhibitors 6-aminothymine and 6-amino-5-bromouracil, which is in line with the 41 42 kinetic properties of these inhibitors. Our findings reveal the existence of a previously 43 44 45 unrecognized site of TP that can be targeted by small molecules to inhibit the catalytic activity 46 47 of TP. 48 Accepted Manuscript 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 3 64 Page 3 of 40 65 1. Introduction 1 2 3 Thymidine phosphorylase (TP) is a key enzyme of the nucleoside salvage pathway [1] that 4 5 6 catalyzes the reversible conversion of thymidine and 2’-deoxyuridine to their respective bases 7 8 and α-D-2-deoxyribose-1-phosphate. TP not only recognizes natural 2’-deoxynucleosides but 9 10 11 is also able to catalyze the phosphorolysis of several nucleoside analogues endowed with 12 13 antiviral or antitumor activity, such as 5-trifluorothymidine (TFT), 5-(E)-(2-bromovinyl)-2’- 14 15 16 deoxyuridine (BVDU) and 5-fluoro-2’-deoxyuridine [2-4]. In addition to its catabolic role in 17 18 the nucleoside salvage pathway, TP is also implicated in tumor progression as it has been 19 20 shown to stimulate angiogenesis [5-8] and protect tumor cells from apoptosis [9-11]. A wide 21 22 23 range of tumors, including breast, colon and oesophageal carcinomas overexpress TP, and 24 25 high levels of TP have been correlated with microvessel density, metastasis and poor patient 26 27 28 survival [10]. 29 30 Thus, TP represents a promising chemotherapeutic target and TP inhibitors may (i) 31 32 33 improve the antiviral and antitumor activity of certain nucleoside analogues, and (ii) inhibit 34 35 the angiogenic and anti-apoptotic activities of TP. During the last decade, several classes of 36 37 38 novel TP inhibitors have been developed [12]. For more than 30 years, the reference TP 39 40 inhibitors were 6-aminouracil derivatives including 6-aminothymine (6AT) and 6-amino-5- 41 42 bromouracil (6A5BU)[13]. In 2000, Fukushima et al. reported on TPI (5-chloro-6-[1-(2- 43 44 45 iminopyrrolidinyl)methyl]uracil hydrochloride) as one of the most effective TP inhibitors to 46 47 date, with an IC50 value of 35 nM [14]. In fact, a combination of TPI and TFT is designated 48 Accepted Manuscript 49 50 TAS-102 which is currently being evaluated in clinical trials for the treatment of various solid 51 52 tumors, including metastatic colon and breast carcinomas [15, 16]. 53 54 55 Our research groups have described 7-deazaxanthine (7-DX), the first purine 56 57 derivative with inhibitory activity against TP and angiogenesis in the ‘chicken chorioallantoic 58 59 membrane’ (CAM) assay [17], and also TP65 (9-[8-phosphonooctyl]-7-deazaxanthine), the 60 61 62 63 4 64 Page 4 of 40 65 first multisubstrate inhibitor of TP that was shown to interact with both the thymidine and the 1 phosphate binding site of TP [18, 19]. More recently, we identified the purine nucleoside 5’- 2 3 O-tritylinosine as a novel TP inhibitor [20, 21], in which the presence of the trityl moiety was 4 5 6 proven to be crucial for both its inhibitory activity against TP and its anti-angiogenic effect in 7 8 the CAM assay.
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