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Mechanistic and Structural Basis for Inhibition of Thymidylate Synthase

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Mechanistic and Structural Basis for Inhibition of Thymidylate Synthase Mechanistic and structural basis for inhibition of thymidylate synthase ThyX Tamara Basta, Yap Boum, Julien Briffotaux, Hubert Becker, Isabelle Lamarre-Jouenne, Jean-Christophe Lambry, Stephane Skouloubris, Ursula Liebl, Marc Graille, Herman van Tilbeurgh, et al. To cite this version: Tamara Basta, Yap Boum, Julien Briffotaux, Hubert Becker, Isabelle Lamarre-Jouenne, et al.. Mech- anistic and structural basis for inhibition of thymidylate synthase ThyX. Open Biology, Royal Society, 2012, 2 (10), pp.120120. 10.1098/rsob.120120. hal-03298488 HAL Id: hal-03298488 https://hal.archives-ouvertes.fr/hal-03298488 Submitted on 23 Jul 2021 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. Downloaded from rsob.royalsocietypublishing.org on October 5, 2012 Mechanistic and structural basis for inhibition of thymidylate synthase ThyX rsob.royalsocietypublishing.org Tamara Basta1,†, Yap Boum2,†, Julien Briffotaux1,†, Hubert F. Becker1,3, Isabelle Lamarre-Jouenne1, Research Jean-Christophe Lambry1, Stephane Skouloubris5,1, Cite this article: Basta T, Boum Y, Briffotaux Ursula Liebl1, Marc Graille4,5, Herman van Tilbeurgh4,5 J, Becker HF, Lamarre-Jouenne I, Lambry J-C, 1 Skouloubris S, Liebl U, Graille M, van Tilbeurgh and Hannu Myllykallio H, Myllykallio H. 2012 Mechanistic and struc- 1 tural basis for inhibition of thymidylate Laboratoire d’Optique et Biosciences, INSERM U696, CNRS UMR 7645, Ecole Polytechnique, synthase ThyX. Open Biol 2: 120120. Palaiseau Cedex, Palaiseau 91228, France 2 http://dx.doi.org/10.1098/rsob.120120 Mbarara Research Base, Epicentre, PO Box 1956 Mbarara, Uganda 3UPMC Univ Paris 06, 75005 Paris, France 4Institut de Biochimie et de Biophysique Mole´culaire et Cellulaire, CNRS UMR 8619, Universite´ Paris-Sud, Orsay 91405, France Received: 16 August 2012 5Universite´ Paris-Sud, Orsay 91405, France Accepted: 11 September 2012 1. Summary Subject Area: Nature has established two mechanistically and structurally unrelated families microbiology/structural biology/biochemistry of thymidylate synthases that produce de novo thymidylate or dTMP, an essen- tial DNA precursor. Representatives of the alternative flavin-dependent thymidylate synthase family, ThyX, are found in a large number of microbial Keywords: genomes, but are absent in humans. We have exploited the nucleotide binding thymidylate synthase ThyX, inhibitor screen, pocket of ThyX proteins to identify non-substrate-based tight-binding ThyX antimicrobial compounds, naphthoquinone inhibitors that inhibited growth of genetically modified Escherichia coli cells dependent on thyX in a manner mimicking a genetic knockout of thymidylate synthase. We also solved the crystal structure of a viral ThyX bound to 2-hydroxy-3-(4-methoxybenzyl)-1,4-naphthoquinone at a resolution of 2.6 A˚ . Author for correspondence: This inhibitor was found to bind within the conserved active site of the tetra- Hannu Myllykallio meric ThyX enzyme, at the interface of two monomers, partially overlapping e-mail: [email protected] with the dUMP binding pocket. Our studies provide new chemical tools for investigating the ThyX reaction mechanism and establish a novel mechanistic and structural basis for inhibition of thymidylate synthesis. As essential ThyX proteins are found e.g. in Mycobacterium tuberculosis and Helicobacter pylori, our studies have also potential to pave the way towards the development of new anti-microbial compounds. 2. Introduction †These authors contributed equally to this De novo 20-deoxythymidine-50-monophosphate (dTMP or thymidylate) synthesis work. is a well-established target for inhibiting cellular growth [1–3]. The last step of dTMP synthesis is the methylation of 20-deoxyuridine-50-monophosphate 5 10 Electronic supplementary material is available (dUMP) to dTMP. This N ,N -methylene-5,6,7,8-tetrahydrofolate (CH2H4- at http://dx.doi.org/10.1098/rsob.120120. folate)-dependent reaction is catalysed by two distinct families of thymidylate & 2012 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0/, which permits unrestricted use, provided the original author and source are credited. Downloaded from rsob.royalsocietypublishing.org on October 5, 2012 (a)(b) 2 R90 H53 rsob.royalsocietypublishing.org Open Biol 2: 120120 O CH2THF + H4 folate O FAD NADPH NADP S88 CH3 HN FAD FADH2 HN R174 O N O N R147 O O dUMP O OH CH THF H folate HO O OH HO P 2 2 P O OH O OH R78 dUMP dTMP (c)(d) Dyospiros maritima Blume acceptable signal dynamic range 0.65 ¢ (Z = 0.62) – control 2 chemical libraries 2322 compounds 0.55 C4–C2 C8–C1 selection criterion 1: D4–C2 inhibition –75% in hibition of (nm) 0.45 G3–C2 25 hits PBCV-1 ThyX at 20 µM 340 B4–C2 OHO selection criterion 2 A 0.35 Br D3–C2 1,4-NQ H3–C2 no inhibition O 0.25 E3–C2 control library + control 2E04 80 1,2-NQ 0 100 200 300 dedicated library time (s) 160 1,4-NQ no hits O R OH O 2-hydroxy-1,4-NQ Figure 1. Screening process for identification of ThyX inhibitors. (a) The last step in the de novo synthesis of thymidylate is catalysed by two unrelated families of thymidylate synthases, ThyX (top) and ThyA (bottom). (b) Unique stacking interaction of the pyrimidine ring of dUMP (magenta) with the isoalloxazine ring of the FAD cofactor at the active site pocket of T. maritima ThyX. Conserved active-site residues of ThyX proteins are shown. (c) Validation of the ThyX activity assay based upon detection of the NADPH oxidation, followed by decrease of A340. Negative control without enzyme and positive control with PBCV-1 ThyX are shown. Also shown are representative data indicating inhibition of ThyX activity or lack thereof. Inhibition parameters for specific molecules are given in table 1 and the main text. (d) Flow chart describing distinct steps leading to the identification of 2-hydroxy-1,4-naphthoquinones as specific ThyX inhibitors. Selection criteria 1 and 2 are described in the text. The lead compound 2-bromo-5-hydroxy-1,4-naphthoquinone (2E04) was isolated from the fruit of the Malaysian persimmon (Dyospiros maritima Blume); NQ, naphthoquinone. synthases, ThyA (EC 2.1.1.45) and ThyX (EC 2.1.1.148; flavin- On the other hand, the different ThyX proteins share a high dependent thymidylate synthase (FDTS)), without detectable level of structural similarity, as exemplified by a Z-value sequence [4] or structural [5–8] similarity (figure 1a). Whereas of 18.0 between the Paramecium bursaria chlorella virus (PBCV- human and bacterial ThyA proteins use tetrahydrofolate 1) and Mycobacterium tuberculosis ThyX proteins. Site-directed (H4folate) to reduce the methylene moiety after its transfer mutagenesis studies together with several ThyX crystal struc- to the uracil ring, ThyX proteins use a non-covalently bound tures revealed that the active site of tetrameric ThyX proteins flavin adenine dinucleotide (FAD) cofactor to facilitate (figure 1b) is located at the interface of three monomers, allow- hydride transfer from NAD(P)H [9–11]. Recent studies have ing surface exposure of the N5 atom of the isoalloxazine ring demonstrated accumulation of a 5-hydroxymethyl-dUMP participating in hydride transfer [5,6,8,15]. The large active site (5hmdUMP) as an acid-trapped intermediate during ThyX cat- cavity is formed at the centre of the tetramer that contains the alysis [10]. Interestingly, 5hmdUMP stoichiometrically replaces FAD cofactor. ThyX proteins do not carry a specific domain, dTMP in the genome of the bacteriophage SPO1 of Bacillus such as a Rossman fold, for fixation of nicotine adenine dinu- subtilis [12]. Strikingly, this modified nucleotide is synthetized cleotide phosphate, reduced form (NADPH). As particular by a viral protein homologous to thymidylate synthase microaerophilic conditions are necessary for observing genetic ThyA [13,14]. complementation of an E. coli DthyA strain by Campylobacter Although details of the ThyX reaction mechanism are not jejuni thyX [16], this hydride transfer is likely inhibited by the fully established, the pronounced structural and mechanistic presence of molecular oxygen. differences between ThyA and ThyX proteins provide an excel- ThyX has a complex fold with a central a/b domain flanked lent starting point for investigating how two distinct ways of by two helical domains. It forms a tetramer with a 222 sym- producing thymidylate have evolved and how the activities of metry. For most of the documented structures, ThyX has these enzymes may be regulated and controlled in a cellular set- FAD bound in an extended conformation and with the adenine ting. The fact that the ThyX homotetramer does not show ring buried in a deep binding pocket in the enzyme. The key significant structural similarity to any other protein structure feature of the active site of ThyX proteins is the stacking of currently known reveals the uniqueness of ThyX proteins. the pyrimidine ring of dUMP against the isoalloxazine ring Downloaded from rsob.royalsocietypublishing.org on October 5, 2012 Table 1. Hits issued from screening the 1,4-naphthoquinone libraries. 3 rsob.royalsocietypublishing.org Open Biol 2: 120120 % inhibition at 20 mM IC (mM) deprotonation 50 NADPH compound structure PBCV-1 ThyX oxidation C8-C1 O 78 + 4 4.5 2-hydroxy-3-(4-methoxybenzyl)-1,4- naphthoquinone OH O O E11-C1 36 + 5 11.0 O 2-hydroxy-3-(2-oxo-2-phenylethyl)-1,4- O naphthoquinone OH O H3-C1 S 38 + 37 O 2-hydroxy-3-(2-oxo-2-(thiophen-2-yl)ethyl)- O 1,4-naphthoquinone OH O F3-C1 38 + 77 O 2-hydroxy-3-(2-oxo-2-p-tolylethyl)-1,4- O naphthoquinone OH O G3-C1 O 22 + 710 O 2-hydroxy-3-(2-(4-methoxyphenyl)-2- O oxoethyl)-1,4-naphthoquinone OH O B3-C1 OH O 12 + 6 .10 2-(5-hydroxy-1,4-dioxo-1,4-dihydronaphthalen- 2-ylamino)benzoic acid NH O O OH of the FAD cofactor [5,6,8].
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