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• W2036388639 abstract "The chemical synthesis of new compounds designed as inhibitors of Mycobacterium tuberculosis TMP kinase (TMPK) is reported. The synthesis concerns TMP analogues modified at the 5-position of the thymine ring as well as a novel compound with a six-membered sugar ring. The binding properties of the analogues are compared with the known inhibitor azido-TMP, which is postulated here to work by excluding the TMP-bound Mg2+ ion. The crystallographic structure of the complex of one of the compounds, 5-CH2OH-dUMP, with TMPK has been determined at 2.0 Å. It reveals a major conformation for the hydroxyl group in contact with a water molecule and a minor conformation pointing toward Ser99. Looking for a role for Ser99, we have identified an unusual catalytic triad, or a proton wire, made of strictly conserved residues (including Glu6, Ser99, Arg95, and Asp9) that probably serves to protonate the transferred PO3 group. The crystallographic structure of the commercially available bisubstrate analogueP 1-(adenosine-5′)-P 5-(thymidine-5′)-pentaphosphate bound to TMPK is also reported at 2.45 Å and reveals an alternative binding pocket for the adenine moiety of the molecule compared with what is observed either in the Escherichia coli or in the yeast enzyme structures. This alternative binding pocket opens a way for the design of a new family of specific inhibitors. The chemical synthesis of new compounds designed as inhibitors of Mycobacterium tuberculosis TMP kinase (TMPK) is reported. The synthesis concerns TMP analogues modified at the 5-position of the thymine ring as well as a novel compound with a six-membered sugar ring. The binding properties of the analogues are compared with the known inhibitor azido-TMP, which is postulated here to work by excluding the TMP-bound Mg2+ ion. The crystallographic structure of the complex of one of the compounds, 5-CH2OH-dUMP, with TMPK has been determined at 2.0 Å. It reveals a major conformation for the hydroxyl group in contact with a water molecule and a minor conformation pointing toward Ser99. Looking for a role for Ser99, we have identified an unusual catalytic triad, or a proton wire, made of strictly conserved residues (including Glu6, Ser99, Arg95, and Asp9) that probably serves to protonate the transferred PO3 group. The crystallographic structure of the commercially available bisubstrate analogueP 1-(adenosine-5′)-P 5-(thymidine-5′)-pentaphosphate bound to TMPK is also reported at 2.45 Å and reveals an alternative binding pocket for the adenine moiety of the molecule compared with what is observed either in the Escherichia coli or in the yeast enzyme structures. This alternative binding pocket opens a way for the design of a new family of specific inhibitors. The incidence of tuberculosis has been increasing during the last 20 years; it is now the first cause of mortality among infectious diseases in the world (1Stokstad E. Science. 2000; 287: 2391Crossref PubMed Google Scholar). The combination of four active drugs (rifampicin, isoniazid, pyrazinamide, and ethambutol or streptomycin) is currently used in Mycobacterium tuberculosis treatment, but this has led to the appearance of resistant bacterial strains (2Cole S.T. Trends Microbiol. 1994; 2: 411-415Abstract Full Text PDF PubMed Scopus (69) Google Scholar). These resistant strains are alarming for two reasons. First, as there are only a few effective drugs available, infection with drug-resistant strains could give rise to a potentially untreatable form of the disease. Second, although only 5% of immunocompetent people infected with M. tuberculosis succumb to the disease, it is nevertheless highly contagious (3Tsuyuguchi I. Tuberculosis. 2001; 81: 221-227Crossref PubMed Scopus (5) Google Scholar). Therefore, a large effort is necessary to identify potential new targets and inhibitors. Figure FS2Reagents and conditions. a, 2-(tributylstannyl)furan, (Ph3P)2Pd(II)Cl2, dioxane or 2-(tributylstannyl)thiophene, Pd(OAc)2, Ph3P, Et3N, dioxane; b, POCl3, (MeO)3PO.View Large Image Figure ViewerDownload (PPT) An attractive potential target is thymidylate kinase (EC2.7.4.9, ATP:TMP phosphotransferase, TMPK), 1The abbreviations used are: TMPK, TMP kinase; Ap5T, P 1-(adenosine-5′)-P 5-(thymidine-5′)-pentaphosphate; HSV, herpes simplex virus; W, water; HPLC, high pressure liquid chromatography; AZTMP, azido-TMP; Mtub, M. tuberculosis; iPrOH, isopropanol 1The abbreviations used are: TMPK, TMP kinase; Ap5T, P 1-(adenosine-5′)-P 5-(thymidine-5′)-pentaphosphate; HSV, herpes simplex virus; W, water; HPLC, high pressure liquid chromatography; AZTMP, azido-TMP; Mtub, M. tuberculosis; iPrOH, isopropanol an essential enzyme that catalyzes an obligatory step in the synthesis of TTP either from thymidine via thymidine kinase (salvage pathway) or from dUMP via thymidylate synthase in all living cells (4Anderson E.P. Boyer P.D. 3rd Ed. The Enzymes. 8. Academic Press, New York1973: 49-96Google Scholar). This enzyme phosphorylates TMP into TDP using ATP as the preferred phosphoryl donor. In the case of the herpes simplex virus (HSV), the most successful antiviral drug (acyclovir) available on the market is directed against thymidine kinase. Acyclovir is phosphorylated by several viral or host kinases into acyclovir triphosphate, which terminates DNA synthesis when incorporated into the viral DNA (5Dabry G.K. Antiviral Chem. Chemother. 1995; 6: 54-63Google Scholar, 6Griffiths P.D. Antiviral Chem. Chemother. 1995; 6: 191-209Crossref Scopus (24) Google Scholar). The comparative x-ray structures of different enzyme-ligand complexes of HSV type 1 thymidine kinase (7Champness J.N. Bennett M.S. Wien F. Visse R. Summers W.C. Herdewijn P. De Clercq E. Ostrowski T. Jarvest R.L. Sanderson M.R. Proteins Struct. Funct. Genet. 1998; 32: 350-361Crossref PubMed Scopus (136) Google Scholar, 8Bennett M.S. Wien F. Champness J.N. Batuwangala T. Rutherford T. Summers W.C. Sun H. Wright G. Sanderson M.R. FEBS Lett. 1999; 443: 121-125Crossref PubMed Scopus (57) Google Scholar, 9Wild K. Bohner T. Folkers G. Schlulz G.E. Protein Sci. 1997; 6: 2097-2106Crossref PubMed Scopus (132) Google Scholar, 10Vogt J. Perozzo R. Pautsch A. Prota A. Schelling P. Pilger B. Folkers G. Scapozza L. Schulz G.E. Proteins. 2000; 41: 545-551Crossref PubMed Scopus (59) Google Scholar) revealed a number of interesting structural features and paved the way for rational structure-based drug design of antiviral compounds (11Manallack D. Pitt W.R. Herdewijn P. Balzarini J. De Clercq E. Sanderson M.R. Sohi M. Wien F. Munier-Lehmann H. Haouz A. Delarue M. J. Enzyme Inhib. Med. Chem. 2002; 17: 167-174Crossref PubMed Scopus (4) Google Scholar, 12de Winter H. Herdewijn P. J. Med. Chem. 1996; 39: 4727-4737Crossref PubMed Scopus (39) Google Scholar). A similar approach might lead to potent antituberculosis agents. TMPK from M. tuberculosis is a homodimer with 214 amino acids per monomer (13Munier-Lehmann H. Chaffotte A. Pochet S. Labesse G. Protein Sci. 2001; 10: 1195-1205Crossref PubMed Scopus (86) Google Scholar). The x-ray three-dimensional structure has been recently solved at 1.95-Å resolution (14Li de la Sierra I. Munier-Lehmann H. Gilles A.M. Bârzu O. Delarue M. Acta Crystallogr. Sect. D Biol. Crystallogr. 2000; 56: 226-228Crossref PubMed Scopus (11) Google Scholar, 15Li de la Sierra I. Munier-Lehmann H. Gilles A.M. Bârzu O. Delarue M. J. Mol. Biol. 2001; 311: 87-100Crossref PubMed Scopus (108) Google Scholar) as a complex with TMP, thereby making it possible to initiate structure-based drug design studies. The global folding of the protein is similar to that of the others TMPKs and NMP kinases despite the low similarity of their amino acid sequences. The TMP kinase backbone is characterized by nine solvent-exposed α-helices surrounding a central β-sheet made of five β-strands, typical of the so-called Rossmann-fold (16Ostermann N. Lavie A. Padiyar S. Brundiers R. Veit T. Reinstein J. Goody R.S. Konrad M. Schlichting I. J. Mol. Biol. 2000; 304: 43-53Crossref PubMed Scopus (40) Google Scholar, 17Ostermann N. Schlichting I. Brundiers R. Konrad M. Reinstein J. Goody R.S. Lavie A. Structure. 2000; 8: 629-642Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 18Lavie A. Konrad M. Brundiers R. Goody R.S. Schlichting I. Reinstein J. Biochemistry. 1998; 37: 3677-3686Crossref PubMed Scopus (64) Google Scholar, 19Lavie A. Ostermann N. Brundiers R. Goody R. Reinstein J. Konrad M. Schlichting I. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14045-14050Crossref PubMed Scopus (79) Google Scholar, 20Lavie A. Vetter I. Konrad M. Goody R.S. Reinstein J. Schlichting I. Nat. Struct. Biol. 1997; 4: 601-605Crossref PubMed Scopus (88) Google Scholar). However, the dimerization mode of the M. tuberculosis enzyme differs from that reported in the yeast, human, and Escherichia coli enzymes (15Li de la Sierra I. Munier-Lehmann H. Gilles A.M. Bârzu O. Delarue M. J. Mol. Biol. 2001; 311: 87-100Crossref PubMed Scopus (108) Google Scholar). The active site of M. tuberculosis TMP kinase complexed with TMP differs from the other known TMPKs in the following ways (15Li de la Sierra I. Munier-Lehmann H. Gilles A.M. Bârzu O. Delarue M. J. Mol. Biol. 2001; 311: 87-100Crossref PubMed Scopus (108) Google Scholar). It is in a fully closed conformation with the ATP binding site being already preformed and the LID region well ordered into a α-helical conformation even though the second substrate ATP (or non-hydrolyzable ATP) is absent from the structure. In the TMP binding site, the protein-TMP interaction shows three specific features when compared with yeast, human, or E. coli enzyme structures. The first feature involves both a magnesium ion and Tyr39; they interact with two opposite non-bridging oxygens of the phosphate moiety of TMP. The second feature involves Asn100 in contact with atom N-3 of the base moiety (15Li de la Sierra I. Munier-Lehmann H. Gilles A.M. Bârzu O. Delarue M. J. Mol. Biol. 2001; 311: 87-100Crossref PubMed Scopus (108) Google Scholar). The third feature, perhaps more amenable to the design of new inhibitors, is the interaction of the 3′-OH atom of TMP with both the side chain of Asp9 and a water molecule, W9, which is a ligand of the Mg2+ ion. In addition, there is a high concentration of positively charged side chains both from the LID region and from the P-loop with arginine residues 14, 95, 149, and 160 as well as Lys13 (15Li de la Sierra I. Munier-Lehmann H. Gilles A.M. Bârzu O. Delarue M. J. Mol. Biol. 2001; 311: 87-100Crossref PubMed Scopus (108) Google Scholar). Examination of the structure therefore suggested the following targets for species-specific inhibitors. In target 1, the 3′-OH and 2′-OH groups of the ribose ring could be systematically replaced with different chemical groups; this has been recently reported (21Vanheusden V. Munier-Lehmann H. Pochet S. Herdewijn P. Van Calenbergh S. Bioorg. Med. Chem. Lett. 2002; 12: 2695-2698Crossref PubMed Scopus (53) Google Scholar). In target 2, the 5-position of the thymine ring is another possibility that has also been considered in the past for HSV thymidine kinase inhibitors (e.g. Refs. 11Manallack D. Pitt W.R. Herdewijn P. Balzarini J. De Clercq E. Sanderson M.R. Sohi M. Wien F. Munier-Lehmann H. Haouz A. Delarue M. J. Enzyme Inhib. Med. Chem. 2002; 17: 167-174Crossref PubMed Scopus (4) Google Scholar and 12de Winter H. Herdewijn P. J. Med. Chem. 1996; 39: 4727-4737Crossref PubMed Scopus (39) Google Scholar). Preliminary results of compounds modified at this position have already been reported (13Munier-Lehmann H. Chaffotte A. Pochet S. Labesse G. Protein Sci. 2001; 10: 1195-1205Crossref PubMed Scopus (86) Google Scholar). Here we report results on other compounds with the aim of adding an extra hydrogen bond with water molecule W12 detected in the three-dimensional structure. W12 is located close to Pro37 (which is in a cis conformation) and forms hydrogen bonds with residues building up the thymidine binding cavity, such as Phe70, Asp73, and Arg74. In target 3, the 2-position of the thymine ring could also be explored, and preliminary but encouraging results have been reported for one compound modified at this position (13Munier-Lehmann H. Chaffotte A. Pochet S. Labesse G. Protein Sci. 2001; 10: 1195-1205Crossref PubMed Scopus (86) Google Scholar). The aim here is to replace the inserted (space-filling) water molecules W2 and W3 that, if removed, give rise to a well defined cavity that can be readily materialized with computer programs such as VOIDOO (22Kleywegt G. Jones T.A. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 178-185Crossref PubMed Scopus (982) Google Scholar). In this study we have synthesized five TMP analogues (1–5, Fig. 1), modified at the thymine moiety and focused on target 2. In addition, one member of a new class of nucleotide analogues has been tested (23Ostrowski T. Wroblowski R. Busson R. Rozenski J. De Clercq E. Bennett M.S. Champness J.N. Summers W.C. Sanderson M.R. Herdewijn P. J. Med. Chem. 1998; 41: 4343-4353Crossref PubMed Scopus (57) Google Scholar, 24Vastmans K. Froeyen M. Kerremans L. Pochet S. Herdewijn P. Nucleic Acids Res. 2001; 29: 3154-3163Crossref PubMed Scopus (48) Google Scholar), namely a 1,5-anhydrohexitol analogue (6) of TMP, where the 3′-OH and/or the 5′-O-phosphate positions are expected to depend on the sugar conformation. The inhibitory effect of these TMP analogues has been measured in vitro by a novel direct specific enzymatic activity test using HPLC and compared with the reference compound AZTMP, which is a good inhibitor (13Munier-Lehmann H. Chaffotte A. Pochet S. Labesse G. Protein Sci. 2001; 10: 1195-1205Crossref PubMed Scopus (86) Google Scholar). All compounds have been subjected to co-crystallization experiments as well as soaking experiments for exchange with TMP in TMP-TMPK crystals (M. tuberculosis TMPK does not crystallize in the absence of TMP). We solved the structure of one promising enzyme-inhibitor complex by x-ray diffraction and have determined the rearrangement of side chains in the active site and the concomitant modification of the water molecule network around the thymine moiety of the TMP substrate. In addition, we have co-crystallized and solved the structure of the complex between TMP kinase and the bisubstrate analogue Ap5T. Altogether, considerable new insight into the possible mechanism of phosphoryl transfer has been gained as described at the end of the “Discussion.” The M. tuberculosis (Mtub) TMP kinase was overexpressed in E. coli and purified as described previously (13Munier-Lehmann H. Chaffotte A. Pochet S. Labesse G. Protein Sci. 2001; 10: 1195-1205Crossref PubMed Scopus (86) Google Scholar). The protein was stored at −20 °C in aliquots of 100 μl at 4 mg/ml in a buffer containing 20 mm Tris-HCl, pH 7.5, 0.5 mm dithiothreitol, and 1 mm EDTA, conditions at which it is stable over several months. Ap5T was purchased from Jena Bioscience (Germany). All other reagents used were purchased from Sigma, including the reference inhibitor AZTMP. All solutions were made with pyrolyzed water. Several methods have already been reported for the synthesis of 5-hydroxymethyl-2′-deoxyuridine. We found that hydroxymethylation of 2′-deoxyuridine with formaldehyde under acidic (25Cline R.E. Fink R.M. Fink K. J. Am. Chem. Soc. 1958; 80: 2521-2525Google Scholar) or basic (26Scheit K.H. Chem. Ber. 1966; 99: 3884-3891Crossref Scopus (38) Google Scholar) catalysis gave only low yields. Therefore, we decided to couple 5-benzyloxymethyluracil with an appropriate sugar. In Ref. 27Tona R. Bertolini R. Hunziker J. Org. Lett. 2000; 2: 1693-1696Crossref PubMed Scopus (28) Google Scholar, the base is coupled with 1,2,3,5-tetracetylribose. Since this method requires subsequent 2′-deoxygenation, we chose to glycosylate the silylated base with 2-deoxy-3,5-O-di-(toluoyl)-α-d-erythro-pentafuranosylchloride (28Gupta V.S. Bubbar G.L. Can. J. Chem. 1971; 49: 719-722Crossref Google Scholar). Unfortunately racemization of the sugar prior to coupling led to a hardly separable mixture of the α- and β-anomers of the protected nucleoside. After alkaline removal of the acyl groups, however, the anomeric mixture could be separated via column chromatography to give the α- and β-anomers 10 and 11 as white foams. Both isomers were then phosphorylated. We noticed that the 5-O-benzyl group got partly removed during this step. Thus phosphorylation of the β-nucleoside gave three compounds: 2′-deoxy-5-hydroxymethyl-5′-O-phosphoryluridine (1), its benzyl-protected analogue (2), and 2′-deoxy-5-hydroxymethyluridine (12). Phosphorylation of the α-nucleoside gave the corresponding benzylated and non-benzylated nucleotides 13 and 3 (Scheme 1). 2′-Deoxy-5-(2-furyl)uridine (15) and 2′-deoxy-5-(thien-2-yl)uridine (16) were synthesized according to a published procedure from unprotected 2′-deoxy-5-iodouridine (14) (29Wigerinck P. Pannecouque C. Snoeck R. Claes P. De Clercq E. Herdewijn P. J. Med. Chem. 1991; 34: 2383-2389Crossref PubMed Scopus (97) Google Scholar). Phosphorylation of these two nucleosides yielded the two desired 5-heteroaryl-substituted nucleotides 4 and 5 (Scheme 2). NMR spectra were obtained with a Varian Mercury 300 or 500 spectrometer using the solvent signal of Me2SO-d 6 as a secondary reference. All signals assigned to amino and hydroxyl groups were exchangeable with D2O. Mass spectra and exact mass measurements were performed on a quadrupole/orthogonal-acceleration time-of-flight tandem mass spectrometer (qTof 2, Micromass, Manchester, UK) equipped with a standard electrospray ionization interface. Samples were infused in a 2-propanol:water (1:1) mixture at 3 μl/min. If necessary, nucleoside 5-O-monophosphates were ultimately purified using a Gilson HPLC system with a Gilson 322 pump, a UV/VIS-156 detector on a C18 column (10 μm, Altech, Altima, 250 × 22 mm). Precoated Merck silica gel F254plates were used for TLC, and spots were examined with UV light at 254 nm and sulfuric acid-anisaldehyde spray or phosphomolybdic acid (0.5% in EtOH) solution. Column chromatography was performed on Uetikon 560 silica (0.2–0.06 mm) and Amersham Biosciences DEAE-SephadexTM A-25. The 1H (and 31P, if appropriate) NMR spectra allowed the characterization of all purified intermediates in the synthesis and final products and are available from the authors upon request. In all instances, mass spectra were found to give the calculated mass within experimental error. 5-Benzyloxymethyluracil (680 mg, 2.93 mmol) was suspended in a mixture of hexamethyldisilazane (62 ml), trimethylsilyl chloride (0.5 ml, 3.94 mmol), and pyridine (5 ml). The mixture was refluxed overnight. The resulting solution was evaporated and co-evaporated with toluene. The obtained residue was suspended in anhydrous CH3CN (3.5 ml), and 2-deoxy-3,5-O-di-(toluoyl)-α-d-erythro-pentofuranosyl chloride (1 g, 2.58 mmol) was added. The reaction mixture was stirred for 3 h at room temperature. CH2Cl2 (25 ml) was added, and the organic layer was washed with a 7% solution of NaHCO3 (25 ml). The water layer was washed twice with CH2Cl2 (25 ml). The combined organic layers were dried over MgSO4 and evaporated. The residue was purified by column chromatography (silica, CH2Cl2:MeOH 98:2) to give a mixture of α- and β-anomers (0.932 g, 62%). The anomers were partly separated by a combination of precipitation (ether:MeOH 13:8) and column chromatography (silica, CH2Cl2:MeOH 100:0 → 99:1 → 98:2). The two mixtures, enriched in either anomer, were used without further purification in the next step. A mixture of 8 and 9 (932 mg, 1.70 mmol) was dissolved in EtOH (60 ml), and 2 n NaOH (37 ml) was added. The mixture was stirred at room temperature for 15 min and evaporated. The obtained residue was dissolved in 5 ml of H2O and neutralized with HCl. The precipitate was filtered, and the filtrate was evaporated and co-evaporated with EtOH. The obtained residue was purified by column chromatography (silica, CH2Cl2:MeOH 93:7) to give 10 (261 mg, 44%) and 11 (284 mg, 48%) as white foams. A solution of 10 (261 mg, 0.75 mmol) in trimethyl phosphate (3.5 ml) was cooled to 0 °C, POCl3 (0.22 ml, 2.4 mmol) was added dropwise, and the mixture was stirred for 4 h at 0 °C. The mixture was poured into crushed ice-water (20 ml), neutralized with concentrated NH4OH, and evaporated to dryness. The residue was subjected to column chromatography (silica, iPrOH:NH4OH:H2O 77.5:15:2.5 → 60:30:5) yielding 12 (38.7 mg, 20%) as a white foam as well as an oily mixture of 1 and 2. This mixture was further purified by HPLC (C18, CH3CN:MeOH:0.05% HCOOH in H2O 45:45:10, 3 ml/min), and the fractions containing the nucleotides were lyophilized yielding 2 (47 mg, 14%) and1 (109 mg, 41%) as white powders. A solution of 11 (248 mg, 0.71 mmol) in trimethyl phosphate (3.5 ml) was cooled to 0 °C, POCl3 (0.21 ml, 2.3 mmol) was added dropwise, and the mixture was stirred for 4 h at 0 °C. The mixture was poured into crushed ice-water (20 ml), neutralized with NH4OH, and evaporated to dryness. 3 and 13 were separated by column chromatography (silica, iPrOH:NH4OH:H2O 77.5:15:2.5 → 60:30:5). Further purification was accomplished by HPLC (C18, CH3CN:MeOH:0.05% HCOOH in H2O 45:45:10, 3 ml/min). The fractions containing the principal nucleotides were lyophilized yielding 3 (106 mg, 42%) and 13(47 mg, 15%) as white powders. A solution of 15 (382 mg, 1.22 mmol) in trimethyl phosphate (5.8 ml) was cooled to 0 °C. POCl3 (0.4 ml, 4.29 mmol) was added dropwise, and the mixture was stirred for 4 h at 0 °C. It was poured into crushed ice-water (40 ml), neutralized with NH4OH, and evaporated to dryness. The resulting residue was purified by column chromatography (silica, iPrOH:NH4OH:H2O 77.5:15:2.5 → 60:30:5). The obtained white powder was further purified on DEAE-Sephadex A-25 (triethylammoniumbicarbonate 0 → 0.5m) yielding the triethylammonium salt of 4. This was converted to its corresponding sodium salt (NaI, acetone) (248 mg, 51%) as a white powder. A solution of 16 (320 mg, 1.03 mmol) in trimethyl phosphate (4.6 ml) was cooled to 0 °C, POCl3 (0.31 ml, 3.3 mmol) was added dropwise, and the mixture was stirred for 4 h at 0 °C. The mixture was poured into crushed ice-water (20 ml), neutralized with 28% ammonia, and evaporated to dryness. The resulting residue was purified by column chromatography (silica, iPrOH:NH4OH:H2O 77.5:15:2.5 → 60:30:5). The obtained white powder was further purified on DEAE-Sephadex A-25 (triethylammonium bicarbonate 0 → 0.5m) yielding the triethylammonium salt of 5. This was converted to its corresponding sodium salt (NaI, acetone) (240 mg, 57%) as a white powder. The TMP kinase activity was measured by HPLC separation of nucleotide substrates and products as described below. The major reason for using this test instead of the more rapid coupled spectrophotometric assay (30Blondin C. Serina L. Wiesmuller L. Gilles A.M. Bârzu O. Anal. Biochem. 1994; 220: 219-221Crossref PubMed Scopus (113) Google Scholar) is that some inhibitors absorb light at 340 nm (4 and 5), thereby rendering difficult the evaluation of NADH concentration in the coupled reaction. Also, coupled enzymatic tests might be error-prone in the determination of the true value of K i and K m of inhibitors and substrate, respectively, through the recycling of some substrate or product during the reaction coupling (31Haouz A. Geleso-Meyer A. Burstein C. Enzyme Microb. Technol. 1994; 16: 292-297Crossref PubMed Scopus (20) Google Scholar). The reaction is carried out in a 1-ml final volume of a solution of 50 mmTris-HCl, pH 7.5, 20 mm magnesium acetate, 100 mm KCl, 1 mm EDTA, 0.5 mmdithiothreitol using different initial concentrations of ATP and TMP in the presence or absence of various inhibitors. To follow the enzymatic kinetics an aliquot of 100 μl is taken at different times after enzyme addition, and the reaction is quenched by adding 900 μl of 100 mm sodium phosphate, pH 7. The concentration of each nucleotide at different times during the reaction is measured at 260 nm by HPLC (isocratic mode) using a SephasilTM C18, 5-μm SC 2.1/10 (Amersham Biosciences) column. The buffer used for elution is 50 mm sodium phosphate, pH 6.5, 2.5% (v/v) ethanol, 40 mm tetrabutylammonium bromide with a flow rate of 250 μl/min. All the reaction velocities are calculated by monitoring the production of TDP expressed in terms of optical absorbance per minute at 260 nm. It was checked that all four sources of information (appearance of TDP or ADP, disappearance of ATP or TMP) can be used and lead to the same results (see also Ref. 11Manallack D. Pitt W.R. Herdewijn P. Balzarini J. De Clercq E. Sanderson M.R. Sohi M. Wien F. Munier-Lehmann H. Haouz A. Delarue M. J. Enzyme Inhib. Med. Chem. 2002; 17: 167-174Crossref PubMed Scopus (4) Google Scholar). Co-crystals of M. tuberculosis TMPK in complex with compound 1 (see Fig. 1) were obtained as described for crystals with the TMP substrate (14Li de la Sierra I. Munier-Lehmann H. Gilles A.M. Bârzu O. Delarue M. Acta Crystallogr. Sect. D Biol. Crystallogr. 2000; 56: 226-228Crossref PubMed Scopus (11) Google Scholar). Briefly a 6-μl drop of a 1:1 mixture of the protein solution (3.5 mg/ml) incubated overnight with 5 mm analogue 1 and the reservoir solution was equilibrated with 34% (w/v) ammonium sulfate solution, 100 mm HEPES, pH 6.0, containing 2% (w/v) polyethylene glycol 2000, 20 mm magnesium acetate, and 0.5 mmβ-mercaptoethanol. Crystals grew in 1–3 weeks to bipyramids of 400 × 200 × 200 μm3. X-ray data were collected from cryo-cooled crystals using 25% (w/v) glycerol as cryoprotectant. Soaking experiments were performed by first transferring TMP-TMPK co-crystals to a stabilizing solution containing 70% ammonium sulfate and 1 mm TMP and then to a fresh solution containing 70% ammonium sulfate and 10 mm inhibitor (but no TMP). This last solution was replaced three times, each soaking time lasting 24 h. Diffraction data were processed using the DENZO/SCALEPACK (32Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38526) Google Scholar) package. The CCP4 package (33CCP4 Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19748) Google Scholar) was used to calculate structure factors from the observed intensities (TRUNCATE). Reflections in the resolution ranges 2.28–2.22 and 2.70–2.64 Å had to be suppressed due to the presence of ice rings during data collection for the 1-TMPK complex. Refinement was performed up to 2.0-Å resolution with CNS (34Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16957) Google Scholar). Standards protocols, including maximum likelihood target, bulk solvent correction, and isotropic B-factors, were used (34Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16957) Google Scholar, 35Murshudov G. Vagin A. Dodson E. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13853) Google Scholar). The model was inspected manually with SIGMAA-weighted 2Fo − Fc andFo − Fc maps (36Read R.J. Acta Crystallogr. Sect. A. 1986; 42: 140-149Crossref Scopus (2035) Google Scholar), and progress in the model refinement was evaluated by the decrease in the free R-factor. Manual rebuilding in the electron density maps was done with O (37Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13009) Google Scholar). Stereochemistry of the final model was assessed using PROCHECK (38Laskowski R.A. McArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 21: 283-291Crossref Google Scholar). Coordinates of the1-TMPKMtub binary complex have been deposited in the Research Collaboratory for Structural Bioinformatics (RSCB) Protein Data Bank (accession code 1MRS). Similar structural details apply to the Ap5T-TMPK complex where all the dictionaries necessary for O and CNS come from the data base of G. Kleywegt. 2HIC-Up, available at alpha2.bmc.uu.se/hicup/t5a. The coordinates have been deposited in the RSCB Protein Data Bank (accession code1MRN). In all experiments, the enzyme concentration was set to a value at least 200 times lower than the substrates concentrations, i.e.typically 5 × 10−8m. The reaction catalyzed by TMP kinase was found to follow the Michaelis model (39Segel I.H. Enzyme Kinetics. Wiley-Interscience Publications, John Wiley, New York1993Google Scholar). The initial velocity of the reaction at different fixed (saturating) concentrations of ATP and different concentrations of TMP allows for the determination of the apparent K m for TMP and for ATP. With this protocol we find an apparentK m = 40 μm TMP. For ATP, we measuredK m = 100 μm. Both values compare well with the values obtained with the coupled assay (13Munier-Lehmann H. Chaffotte A. Pochet S. Labesse G. Protein Sci. 2001; 10: 1195-1205Crossref PubMed Scopus (86) Google Scholar). TheK m of 40 μm for TMP has been measured at the fixed concentration of ATP of 0.5 mm and shows some dependence upon ATP concentration. The K m value of TMP extrapolated at zero ATP concentration is 4–5 μm in agreement with results reported earlier (13Munier-Lehmann H. Chaffotte A. Pochet S. Labesse G. Protein Sci. 2001; 10: 1195-1205Crossref PubMed Scopus (86) Google Scholar). The role of ATP as the phosphoryl donor has been explored in Ref. 13Munier-Lehmann H. Chaffotte A. Pochet S. Labesse G. Protein S" @default.
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• W2036388639 title "Enzymatic and Structural Analysis of Inhibitors Designed against Mycobacterium tuberculosis Thymidylate Kinase" @default.
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