Báo cáo khoa học: Inhibition of aryl acid adenylation domains involved in bacterial siderophore synthesis ppt

Inhibition of aryl acid adenylation domains involved in bacterial siderophore synthesis Marcus Miethke 1 , Philippe Bisseret 2 , Carsten L. Beckering 1 , David Vignard 2 , Jacques Eustache 2 and Mohamed A. Marahiel 1 1 Philipps-Universita ¨ t Marburg, Fachbereich Chemie ⁄ Biochemie, Marburg, Germany 2 Laboratoire de Chimie Organique et Bioorganique associe ´ au CNRS, Ecole Nationale Supe ´ rieure de Chimie de Mulhouse, Mulhouse, France Iron is an essential cofactor for many cellular proces- ses such as electron transport, synthesis of amino acids, nucleosides and DNA. However, microorgan- isms that colonize habitats in an aerobic environment or in host organisms have to deal with severe iron limi- tation. Many bacteria have developed high affinity iron chelators known as siderophores that serve as powerful tools to extract iron from extracellular sources [1,2]. Due to the fact that these iron chelators are important virulence factors of various pathogenic bacteria, the therapeutic potential of siderophore biosynthesis inhi- bition is obvious [3]. Different classes of siderophores are known, which contain either aryl, hydroxamoyl or carboxyl moieties that provide the iron coordinating ligands. They are generated by different biosynthetic pathways. Synthesis of hydroxamate and carboxylate siderophores commonly depends on a diverse spec- trum of enzymatic activities, including, for example, Keywords aryl acid adenylation domain; enzyme inhibition; iron limitation; pathogenicity; siderophore Correspondence J. Eustache, Laboratoire de Chimie Organique et Bioorganique (CNRS UMR 7015), Ecole Nationale Supe ´ rieure de Chimie de Mulhouse, 3 rue Alfred Werner, F-68093 Mulhouse-Cedex, France Fax: +33 3 89 33 6860 Tel: +33 3 89 33 6858 E-mail: jacques.eustache@uha.fr M. A. Marahiel, Philipps-Universita ¨ t Marburg, Fachbereich Chemie ⁄ Biochemie, Hans-Meerwein-Strasse, D-35032 Marburg, Germany Fax: +49 06421 2822191 Tel: +49 06421 2825722 E-mail: marahiel@chemie.uni-marburg.de (Received 5 October 2005, accepted 24 November 2005) doi:10.1111/j.1742-4658.2005.05077.x Aryl acid adenylation domains are the initial enzymes for aryl-capping of catecholic siderophores in a plethora of microorganisms. In order to over- come the problem of iron acquisition in host organisms, siderophore bio- synthesis is decisive for virulence development in numerous important human and animal pathogens. Recently, it was shown that growth of Mycobacterium tuberculosis and Yersinia pestis can be inhibited in an iron- dependent manner using the arylic acyl adenylate analogue 5¢-O-[N-(sali- cyl)-sulfamoyl] adenosine that acts on the salicylate activating domains, MbtA and YbtE [Ferreras JA, Ryu JS, Di Lello F, Tan DS, Quadri LEN (2005) Nat Chem Biol 1, 29–32]. The present study explores the behaviour of the 2,3-dihydroxybenzoate activating domain DhbE (bacillibactin syn- thesis) and compares it to that of YbtE (yersiniabactin synthesis) upon enzymatic inhibition using a set of newly synthesized aryl sulfamoyl adeno- sine derivatives. The obtained results underline the highly specific mode of inhibition for both aryl acid activating domains in accordance with their natively accepted aryl moiety. These findings are discussed regarding the structure–function based aspect of aryl substrate binding to the DhbE and YbtE active sites. Abbreviations A, adenylation; AMN, 5¢-O-[N-(aryl)-hydroxamoyl] adenosine; AMS, 5¢-O-[N-(aryl)-sulfamoyl] adenosine; DEAD, diethyl azodicarboxylate; DHB, 2,3-dihydroxybenzoate; HRMS, high resolution mass spectrometry; NRPS, nonribosomal peptide synthetase; PKS, polyketide synthase; PP i , inorganic pyrophosphate; SAL, salicylate; TFA, trifluoroacetic acid; THF, tetrahydrofluran. FEBS Journal 273 (2006) 409–419 ª 2005 The Authors Journal compilation ª 2005 FEBS 409 monooxygenases, decarboxylases, aminotransferases and aldolases [4]. In contrast, all aryl-capped sideroph- ores known so far are mainly assembled by nonribo- somal peptide synthetases (NRPSs), where polyketide synthases (PKSs) are sometimes additionally involved [5,6]. Either 2,3-dihydroxybenzoate (DHB) or salicylate (SAL) serve as initial substrates for nonribosomal syn- thesis depending on enzymatic equipment of the micro- organisms to convert the chorismic acid that is used as a precursor for both substrates. Both DHB and SAL containing siderophores represent virulence factors of important human pathogens (Fig. 1A). Aryl substrate activation is catalyzed by aryl acid adenylation (A) domains, forming acyl adenylate intermediates (Fig. 1B). According to the organism-specific usage of DHB or SAL as an aryl cap, two types of aryl acid A domains are known, showing different substrate speci- ficities for both compounds. So far, aryl acid A domains are characterized as lone-standing enzymatic domains within the modular NRPS ⁄ PKS ⁄ NRPS–PKS A B C Fig. 1. Structures and synthesis of aryl- capped siderophores. (A) DHB- and SAL- capped siderophores of human pathogens; aryl moieties are shown in red; R 1 in Mycobactin T stands for a variable N-acyl chain. (B) General reaction catalyzed by aryl acid A domains; the aryl acid can be either DHB or SAL. (C) Modular organization of siderophore biosynthesis exemplified for the bacillibactin nonribosomal peptide synthe- tase (NRPS) and the yersiniabactin NRPS ⁄ nonribosomal peptide polyketide hybrid synthetase (NRPS-PKS). The lone standing aryl acid A domains DhbE and YbtE are shown in red. Abbreviation of domains: A, adenylation; ACP, acyl carrier protein; ArCP, aryl carrier protein; AT, acyltrans- ferase; C, condensation; Cy, cyclization ⁄ con- densation; ICL, isochorismate lyase; KR, a-ketoreductase; KS, ketoacyl synthase; MT, methyltransferase; PCP, peptidyl carrier pro- tein; TE, thioesterase. Inhibition of aryl acid adenylation domains M. Miethke et al. 410 FEBS Journal 273 (2006) 409–419 ª 2005 The Authors Journal compilation ª 2005 FEBS clusters (Fig. 1C). The crystal structure of Bacillus sub- tilis DhbE activating DHB for bacillibactin synthesis has been solved, and the cocrystallization with the DHB acyl adenylate revealed the binding nature of the activated aryl substrate [7]. This was the first step towards the development of acyl adenylate analogues as potential inhibitors of aryl acid A domains. Recently, the first analogue of this kind, a 5¢-O-[N-(sal- icyl)-sulfamoyl] adenosine (SAL-AMS), was found to be a potent inhibitor of several SAL A domains and was shown to inhibit growth of SAL-capped sidero- phore producing Mycobacterium tuberculosis and Yer- sinia pestis in iron-depleted medium [8]. In this context it is remarkable that the class of aryl-capped siderophores is widely distributed among pathogenic bacteria. For example, DHB-capped sider- ophores are produced by Vibrio spp., Bacillus spp. or enteric bacteria like Escherichia coli or Salmonella spp. [9–12]. Therefore, information on how a DHB A domain behaves upon inhibition with the known SAL- AMS or novel synthetic analogues would be desirable. This study shows the inhibition of the DHB activating domain DhbE with SAL-AMS and new derivatives of this inhibitor. We show that more hydrophobic ana- logues with expected higher membrane penetrating potential act as good DhbE inhibitors. Furthermore, we present a quantitative estimation of the preferential inhibition of DhbE and the SAL A domain, YbtE, by two analogues corresponding to the native products of catalysis. The relationship between substrate specificity and inhibition effectiveness is discussed based on DhbE structural information, by comparing models of the aryl acid binding pockets of the two domains. Results and Discussion Inhibition of DhbE with SAL-AMS and a set of new modified inhibitors In order to study inhibition of a DHB activating A domain, Bacillus subtilis DhbE, whose structure is known, was chosen as target enzyme. Six synthetic acyl analogues, among them SAL-AMS and five new ana- logues representing either 5¢-O-[N-(aryl)-sulfamoyl] adenosine (AMS) or 5¢-O-[N-(aryl)-hydroxamoyl] adeno- sine (AMN) derivatives were screened for inhibitory effects (Fig. 2). Enzyme activity with and without inhibitors was analyzed by adenosine triphosphate ⁄ inorganic pyrophosphate (ATP ⁄ PP i ) exchange. At sat- urating concentrations of both DHB and ATP and an inhibitor : DHB ratio of 1 : 20, the four AMS com- pounds exhibited strong inhibition ranging from 86 to > 99% loss of DhbE activity (Table 1). DHB-AMS and SAL-AMS had the greatest inhibitory effects with A B C F E D Fig. 2. Synthetic acyl adenylate analogues for aryl acid A domain inhibition. (A) 5¢-O-[N-(benzoyl)-sulfamoyl] adenosine (BEN-AMS). (B) 5¢-O-[N-(o-fluorobenzoyl)-sulfamoyl] adenosine (F-BEN-AMS). (C) 5¢-O-[N-(salicyl)-sulfamoyl] adenosine (SAL-AMS), reported by [8]. (D) 5¢-O- [N-(2,3-dihydroxybenzoyl)-sulfamoyl] adenosine (DHB-AMS). (E) 5¢-O-[N-(benzoyl)-hydroxamoyl] adenosine (BEN-AMN). (F) 5¢-O-[N-(o-fluoro- benzoyl)-hydroxamoyl] adenosine (F-BEN-AMN). Colour code: Aryl moieties (red), sulfamoyl moieties (blue), hydroxamoyl moieties (green), adenosyl moieties (black). M. Miethke et al. Inhibition of aryl acid adenylation domains FEBS Journal 273 (2006) 409–419 ª 2005 The Authors Journal compilation ª 2005 FEBS 411 an inhibition strength of more than 99% under the chosen conditions. 5¢-O-[N-(o-fluorobenzoyl)-sulfa- moyl]adenosine (F-BEN-AMS) and 5¢-O-[N-(benzoyl)- sulfamoyl]adenosine (BEN-AMS) with more hydrophobic aryl moieties showed about 90% of DhbE inhibition, indicating that these compounds are promising candidates for in vivo studies due to their expected higher membrane penetrating abilities. In contrast, the AMN derivatives tested here showed inhi- bition only to a negligible extent and were not studied further. Compared with the a-phosphate in the native acyl adenylate, the shortened hydroxamoyl linker of the AMN compounds may impair fitting of the aryl and ⁄ or adenosine moiety to the active site, resulting in weak inhibitory properties. In contrast, the sulfamoyl linker of the AMS inhibitors shows almost no struc- tural difference to the native linker, enabling both the aryl and adenosine moieties to bind properly to their cognate pockets as shown in the model for the DHB- AMS in Fig. 3D. Determination of inhibition constants for SAL-AMS and DHB-AMS with DhbE and YbtE The DHB-AMS and SAL-AMS were selected for detailed inhibition studies. Target enzymes were Table 1. Results of inhibition studies. Acyl adenylate analogues (Fig. 2) were tested for DhbE inhibition at a concentration of 12.5 l M. ATP and DHB concentrations were at saturating levels of 2m M and 250 lM, respectively. ATP ⁄ PP i exchange reactions were carried out for 5 min at 37 °C. Acyl analogue DhbE inhibition (%) BEN-AMS 86.4 F-BEN-AMS 91.1 SAL-AMS 99.6 DHB-AMS 99.8 BEN-AMN 1.8 F-BEN-AMN 0.1 Fig. 3. Schematic presentation of aryl acid A domain binding pockets. (A) DhbE binding pocket with DHB substrate based on crystal struc- ture data [7]. (B) Suggested model of the YbtE binding pocket with SAL substrate based on sequence alignments. Colour code of depicted amino acid residues: Unpolar (blue), polar (yellow), basic (green). DHB and SAL are shown in grey; carboxy groups (dotted) and hydroxy groups (striped) are indicated. (C) Alignment of DhbE and YbtE amino acids forming the aryl substrate binding pockets. The differing amino acids are indicated by asterisks. The amino acids with the highest proposed impact for the aryl substrate specificity are shown in red. (D) Model of the DHB-AMS inhibitor bound to the DhbE active site. Polar interactions are indicated by dashed lines. Inhibition of aryl acid adenylation domains M. Miethke et al. 412 FEBS Journal 273 (2006) 409–419 ª 2005 The Authors Journal compilation ª 2005 FEBS B. subtilis DhbE and the SAL activating domain YbtE of Y. pestis. At first, specificities of the enzymes for either DHB or SAL were confirmed by ATP ⁄ PP i exchange. Michaelis–Menten constants (K m ) were K m (DHB) ¼ 1.3 ± 0.1 lm and K m (SAL) ¼ 81.8 ± 10.6 lm for DhbE and K m (DHB) ¼ 325.1 ± 10.5 lm and K m (SAL) ¼ 3.5 ± 0.5 lm for YbtE. These values were comparable with the corresponding K m values determined previously [9,13]. Then, inhibition con- stants (K i ) were determined by ATP ⁄ PP i exchange. ATP was used at saturating concentrations, the DHB and SAL substrates were used at three concentrations close to the corresponding K m values while the concen- trations of the inhibitors were varied. The observed strength of inhibition was inversely proportional to the aryl substrate concentration, indicating a substrate dependent mode of inhibition in this kinetic range. The resulting inhibition constants ranged from 29 to 106 nm revealing effective inhibition of both A domains with DHB-AMS and SAL-AMS (Table 2). Furthermore, the K i values showed significant differ- ences depending on the hydroxylation pattern of the inhibitor aryl moiety and the aryl substrate specificity of the A domain. DhbE was inhibited 1.25-fold better with DHB-AMS than with SAL-AMS and YbtE 1.8- fold better with SAL-AMS than with DHB-AMS. The observed differences clearly indicate preferential inhibi- tion by the analogue containing the natively accepted aryl moiety of the A domain. Comparison of DhbE and YbtE aryl acid binding pockets with respect to preferential A domain inhibition Comparing the K i and K m values, the observed prefer- ential inhibition of DhbE and YbtE by DHB- and SAL-based inhibitors did not reflect the larger differ- ences of the aryl substrate specificities. Indeed, the K m values for both DHB and SAL differed by approxi- mately 60-and 90-fold for DhbE and YbtE, respect- ively. The domain specificities for the aryl substrates might be explained by comparison of the amino acid residues that are critical for substrate recognition based on the DhbE crystal structure. Sequence align- ments of several DHB and SAL activating domains showed significant differences between the amino acid residues forming the aryl acid binding pockets accord- ing to the nonribosomal code of A domain specificity [7,14]. Therefore, a model of a putative binding pocket valid for SAL activating domains can be proposed and is exemplified here for YbtE in comparison with the DHB binding pocket of DhbE (Fig. 3A–C). In agree- ment with previous analyses [7,8], amino acid residues Ser240 and Val337 of DhbE and the corresponding residues Cys227 and Leu324 of YbtE, located at the bottom of the aryl acid binding pockets, seem to be involved in key interactions with the mono- or dihy- droxylated aryl substrates. Especially, the hydrophobic residues Val (which is conserved in DHB A domains) or Leu (which is conserved in SAL A domains, but can be replaced by Ile, e.g. in PchD of Pseudomonas aeruginosa) with different C-chain length in close prox- imity to the hydroxylation-variable C 3 position of the aromatic substrate ring might contribute to the observed substrate specificities of both domain types. However, considering the whole A domain, this effect seems to be overshadowed by the strong contribution of the adenylate moiety to overall binding, which prob- ably explains the smaller differences of the K i values for DHB-AMS and SAL-AMS compared to the differ- ences of the K m values for DHB and SAL. This, again, is in agreement with former observations for aminoacyl tRNA synthetases revealing higher dissociation con- stants for the substrates than for the aminoacyl adeny- lates [15]. Additionally, the linker between aryl and adenylate moieties might also contribute to effective binding of the reaction intermediates and their syn- thetic analogues. The a-phosphate of the native DHB acyl adenylate as well as the sulfamoyl linker of the DHB-AMS are in very close proximity to the His234 residue of DhbE ([7] and Fig. 3D), indicating ionic or at least polar interactions between linker and A domain. In summary, the model-based structural–func- tional relationship of aryl acid A domain inhibition supports the use of acyl adenylate analogues as effect- ive inhibitors and is in accordance with the experimen- tal data, indicating that AMS derivatives with high structural resemblance to the native intermediates can be favoured over derivatives with, for example, a shor- tened molecular linker such as AMN compounds. Conclusion The development of nonhydrolyzable analogues for inhibition of acyl adenylate forming enzymes like aminoacyl tRNA synthetases and NRPS A domains Table 2. Results of inhibition studies. Inhibition constants (K i ) were determined with DHB-AMS and SAL-AMS using DhbE and YbtE (each 300 n M) as target enzymes. ATP ⁄ PP i exchange reactions were carried out for 30 s at 37 °C. Acyl analogue Inhibition constants (nM) DhbE YbtE SAL-AMS 106 29 DHB-AMS 85 54 M. Miethke et al. Inhibition of aryl acid adenylation domains FEBS Journal 273 (2006) 409–419 ª 2005 The Authors Journal compilation ª 2005 FEBS 413 emerged in the last years [16–18]. A novel contribution to pathogenicity control was the development of the SAL-AMS compound for aryl acid A domain inhibi- tion of SAL-capped siderophore producing pathogens [8]. Inhibition of aryl acid A domains bears the advantage that higher vertebrates, including humans, do not possess such class of enzymes, making cross reactions unlikely. Now, another type of aryl acid A domain, activating DHB for siderophore synthesis, has been inhibited for the first time with SAL-AMS and new AMS derivatives. Testing the SAL-AMS and the herein presented DHB-AMS with both a DHB and a SAL activating domain resulted in preferential inhibi- tion with the analogue containing the aryl moiety of the native reaction intermediate, indicating that DHB- AMS might be more suitable for inhibition of DHB- capped siderophore producing bacteria. The presented in vitro results and the discussed model for substrate specificity will lead to a deeper understanding of aryl acid A domain inhibition mechanisms and may help to extend the possibilities of pathogen inhibition based on iron limitation. We now intend to confirm the effect- iveness of the new AMS compounds by growth inhi- bition (in vivo studies) using a broad range of microorganisms producing aryl-capped siderophores. We will also study in detail the relationship between inhibitor structure and uptake with the aim of scaling- down in vivo effective doses to levels enabling potential therapeutic applications. Experimental procedures Synthesis of acyl adenylate analogues Description of chemical synthesis strategies Salicyl-derived 5¢-O-sulfamoyladenosine derivatives 8–10 were synthesized from 2¢,3¢-O-isopropylidene-5¢-sulfamoyl- adenosine 1, which was prepared as already reported (Scheme 1) [19] . For the key coupling reaction between 1 and benzoic acid derivatives, we initially tested, without success, the coupling conditions previously used for the pre- paration of amino acid derived sulfamates [20,21]. In par- ticular, the condensation between 1 and benzoic acid using carbonyldiimidazole as a coupling agent in the presence of DBU was not satisfactory. The desired sulfamate 5 was formed in low yield under these conditions together with unidentified products of similar polarity. (While this manu- script was in preparation, the synthesis of the sulfamoyl derivative 10 was reported [8]. This study showed that coupling worked well when starting from 2¢,3¢-O-silylated- 5¢-O-sulfamoyl-adenosine instead of the isopropylidene derivative 1.) Finally, we found that the couplings with 1 could be realized, albeit in modest yields, using the succin- imidyl-derivatives 2–4 [22–24] and cesium carbonate as a base. Using other bases like DBU or Et 3 N yielded complex mixtures containing only traces of the desired coupling product. Deprotection of the isopropylidene was best real- ized using 50% aqueous trifluoroacetic acid (TFA) and the final compounds were purified by thin-layer chroma- tography (TLC). The hydrolysis step was effective to obtain Scheme 1. Preparation of salicyl-5¢-O-sulfamoyladenosine derivatives 8–10. Reagents and conditions: (a) Cs 2 CO 3 (2 equiv), room tempera- ture, 20 h (5, 16%; 6, 31%; 7, 23%); (b) TFA : H 2 O(1:1,v⁄ v), room temperature, 4 h (8, 16%; 9, 63%; 10, 92%). Inhibition of aryl acid adenylation domains M. Miethke et al. 414 FEBS Journal 273 (2006) 409–419 ª 2005 The Authors Journal compilation ª 2005 FEBS 9 and 10 but, however, more problematic in the case of the nonsubstituted benzoyl compound 8, which appeared to be more sensitive to acidic conditions and could be obtained only in low yield. Extension of this procedure for preparation of the dihydroxybenzoyl derivative 13 starting from the succini- mide derivative of dihydroxybenzoic acid was unsuccessful. The desired coupling could be achieved, however, using the benzylidene protected equivalent 11. Both the ispopropylid- ene and benzylidene groups were removed by TFA treat- ment as shown above to afford 13 in fair yield (Scheme 2). We also prepared the hydroxamoyl derivatives 18 and 19 by Mitsunobu coupling of the O-protected N-hydroxy- amides 14 and 15 (Scheme 3). This was best realized using polymer-supported triphenylphosphine [25]. Preparation of 5¢-O-sulfamoyladenosine derivatives 8–10 and 13: general procedure To a solution of 2¢,3¢-O-isopropyliden-5¢-O -sulfamoyladeno- sine 1 in CH 2 Cl 2 : DMF (7 : 3, v ⁄ v) were added the succin- imide derivative (2–4 and 11, 1.1 equiv) followed by cesium Scheme 2. Preparation of 2,3-dihydroxybenzoyl-5¢-O-sulfamoyladenosine 13. Reagents and conditions: (a) Cs 2 CO 3 (2 equiv), room tempera- ture, 20 h, 30%; (b) TFA : H 2 O (1 : 1, v ⁄ v), room temperature, 4 h, 40%. Scheme 3. Preparation of hydroxamoyl derivatives 18 and 19. Reagents and conditions: (a) polymer-supported triphenylphosphine (4 equiv), Diethyl azocarboxylate (DEAD) (4 equiv), room temperature, tetrahydrofluran (THF), 1 h, 80%; (b) TFA : H 2 O(2:1,v⁄ v), room temperature, 3 h, 70%. M. Miethke et al. Inhibition of aryl acid adenylation domains FEBS Journal 273 (2006) 409–419 ª 2005 The Authors Journal compilation ª 2005 FEBS 415 carbonate (2 equiv). After stirring for 20 h at room tem- perature, methanol was added and the mixture was concen- trated under reduced pressure. The residue was purified on a SiO 2 column [EtOAc : MeOH (9 : 1)] to afford the pro- tected salicyl derivatives 5–7 and 12 as white solids in the following yields: 5, 16%; 6, 31%; 7, 23%; 12, 30%. The protected sulfamates (5–7 and 12) were then stirred for 4 h at room temperature in a mixture of TFA : H 2 O (1 : 1). After addition of MeOH and toluene ( 1 : 1), the reaction medium was concentrated under reduced pressure and purified by TLC using CHCl 3 : MeOH (2 : 1) as eluent to afford the free salicyl derivatives 8–10 and 13 in the fol- lowing yields: 8, 16%; 9, 63%; 10, 92%; 13, 40%. Preparation of hydroxamoyl derivatives 18 and 19: general procedure A 40% solution of diethyl azodicarboxylate (DEAD) in toluene (4 equiv) was added dropwise at room temperature to polymer-supported triphenylphosphine (4 equiv), 2¢,3¢-O- isopropyliden-adenosine (1 equiv) and 14 or 15 (1.2 equiv) in suspension in tetrahydrofluran (THF). After 1 h of stir- ring at room temperature, the polymer was filtered-off and washed with CHCl 3 . The filtrate was concentrated under reduced pressure and the residue was purified by TLC using EtOAc : MeOH (9 : 1) as an eluent, yielding the protected hydroxamoyl derivative 16 or 17 (80% yield), that were then dissolved into a solution of TFA : H 2 O (2 : 1). After 3 h of stirring at room temperature, MeOH and toluene (1 : 1) were added and the medium was concentrated under reduced pressure yielding, after TLC purification [EtO- Ac : MeOH (9 : 1)], the deprotected derivative 18 or 19 in 70% yield. Analytical chemistry Analytical TLC was performed on Merck 60F 254 silica gel plates (Merck KGaA, Darmstadt, Germany) (spots visual- ized with UV light at 254 nm and at 366 nm after aspersion of a 0.1% ethanolic solution of berberine chlorhydrate and ⁄ or with a vanillin-MeOH ⁄ H 2 SO 4 solution followed by heating). Column chromatography was carried out on MN Kieselgel 60 (30–270 mesh) (Macherey-Nagel GmbH & Co. KG, Du ¨ ren, Germany). NMR spectra were recorded using a Bruker AVANCE 400 spectrometer ( 1 H, 400 MHz; 13 C, 100.69 MHz) (Bruker BioSpin GmbH, Rheinstein, Ger- many) in CDCl 3 or CD 3 OD as solvent. High resolution MS were performed under electrospray. 5:R f ¼ 0.34 (EtOAc : MeOH: 4 : 1, v ⁄ v); [a] 25 D ¼ )33 (c ¼ 0.25; MeOH) 1 H NMR (CD 3 OD, 300 K): d ¼ 8.47 (1H, s), 8.12 (1H, s), 7.96 (2H, d, J ¼ 7.8 Hz), 7.40 (2H, t, J ¼ 7.5 Hz), 7.31 (1H, t, J ¼ 7.5 Hz), 6.19 (1H, d, J ¼ 3.3 Hz), 5.35 (1H, dd, J ¼ 3.3 & 6 Hz), 5.13 (1H, dd, J ¼ 2 & 6 Hz), 4.57 (1H, m), 4.27 (2H, d, J ¼ 3.8 Hz), 1.59 (3H, s), 1.35 (3H, s). 13 C NMR (CD 3 OD, 300 K): d ¼ 175.2, 157.3, 153.9, 150.5, 141.4, 138.9, 132.1, 129.9, 129.5, 128.8, 120.1, 115.2, 91.9, 85.8, 85.7, 83.4, 69.7, 27.5, 25.4. High resolution mass spectrometry (HRMS) calculated for C 20 H 23 N 6 O 7 S (MH + ): 491.1349; found: 491.1350. 6:R f ¼ 0.37 (EtOAc : MeOH, 8 : 2, v ⁄ v); [a] 25 D ¼ )37 (c ¼ 0.3; MeOH) 1 H NMR (CD 3 OD, 300 K): d ¼ 8.51 (1H, s), 8.18 (1H, s), 7.70 (1H, t, J ¼ 7.5 Hz), 7.39 (1H, m) 7.14 (1H, t, J ¼ 7.5 Hz), 7.05 (1H, dd, J ¼ 8.6; 10.5 Hz), 6.24 (1H, d, 3.3 Hz), 5.39 (1H, dd, J ¼ 3.3; 6 Hz), 5.19 (1H, dd, J ¼ 2; 6 Hz), 4.60 (1H, m), 4.33 (2H, d, J ¼ 3.5 Hz), 1.60 (3H, s), 1.38 (3H, s). 13 C NMR (CD 3 OD, 300 K): d ¼ 173.6, 161.9 (d, J ¼ 257 Hz), 157.3, 153.9, 150.5, 141.4, 134.5, 132.7 (d, J ¼ 8.4 Hz), 131.8, 124.7, 117.1 (d, J ¼ 13.4 Hz), 115.2, 91.9, 85.8, 85.6, 83.7, 69.9, 27.5, 25.5. HRMS calculated for C 20 H 22 N 6 O 7 FS (MH + ): 509.1255; found: 509.1243. 7:R f ¼ 0.52 (EtOAc : MeOH, 4 : 1, v ⁄ v) 1 H NMR (CD 3 OD, 300 K): d ¼ 8.47 (1H, s), 8.11 (1H, s), 7.90 (1H, dd, J ¼ 7.8; 1.8 Hz), 7.28 (1H, dt, J ¼ 1.8; 6.8 Hz), 6.74 (2H, m), 6.23 (1H, d, J ¼ 3.1 Hz), 5.38 (1H, dd, J ¼ 3.1 & 6 Hz), 5.13 (1H, dd, J ¼ 2.3 & 6 Hz), 4.57 (1H, m), 4.33 (2H, d, J ¼ 3.8 Hz), 1.55 (3H, s), 1.31 (3H, s). 13 C NMR (CD 3 OD, 300 K): d ¼ 175.3, 162.0, 157.2, 153.9, 150.3, 141.5, 134.4, 131.4, 120.3, 120.2, 119.3, 117.9, 115.3, 91.9, 85.7, 85.6, 83.2, 69.9, 27.4, 25.4. HRMS calculated for C 20 H 23 N 6 O 8 S (MH + ): 509.1298; found: 509.1286. 8:R f ¼ 0.40 (CHCl 3 : MeOH, 2 : 1, v⁄ v); [a] 25 D ¼ )18 (c ¼ 0.5; MeOH) 1 H NMR (CD 3 OD): d ¼ 8.55 (1H, s), 8.17 (H, s), 8.02 (2H, d, J ¼ 7.1 Hz), 7.43 (1H, t, J ¼ 7.1 Hz), 7.35 (2H, t, J ¼ 7.1 Hz), 6.10 (1H, d, J ¼ 6.0 Hz), 4.72 (1H, t, J ¼ 6.0 Hz), 4.38 (4H, m). 13 C NMR (CD 3 OD): d ¼ 175.3, 157.2, 153.8, 150.9, 141.2, 138.9, 132.1, 129.9, 128.8, 120.1, 92.5, 89.2, 76.1, 72.4, 69.2. HRMS calculated for C 17 H 19 N 6 O 7 S (MH + ): 451.1036; found: 451.1029. 9:R f ¼ 0.41 (CHCl 3 : MeOH, 2 : 1, v⁄ v); [a] 25 D ¼ )13 (c ¼ 0.5; MeOH) 1 H NMR (CD 3 OD): d ¼ 8.53 (1H, s), 8.19 (1H, s), 7.73 (1H, t, J ¼ 7.6 Hz), 7.38 (1H, m), 7.13 (1H, t, J ¼ 7.6 Hz), 7.07 (1H, dd, J ¼ 8.3; 10,6 Hz), 6.10 (1H, d, J ¼ 5.7 Hz), 4.73 (1H, t, J ¼ 5.7 Hz), 4.43 (4H, m). 13 C NMR (CD 3 OD): d ¼ 174.5, 163.8, 162.9 (d, J ¼ 250 Hz), 158.1, 154.7, 151.7, 141.9, 133.8 (d, J ¼ 8.4 Hz), 132.8, 129.2, 121.0, 118.0 (d, J ¼ 29.7 Hz), 90.3, 85.4, 76.8, 73.2, 70.4. HRMS calculated for C 17 H 18 N 6 O 7 FS (MH + ): 469.0942; found: 469.0952. 10:R f ¼ 0.44 (CHCl 3 : MeOH, 2 : 1, v ⁄ v); [a] 25 D ¼ )14 (c ¼ 0.3; MeOH) Inhibition of aryl acid adenylation domains M. Miethke et al. 416 FEBS Journal 273 (2006) 409–419 ª 2005 The Authors Journal compilation ª 2005 FEBS 1 H NMR (CD 3 OD): d ¼ 8.49 (1H, s), 8.13 (1H, s), 7.90 (1H, d, J ¼ 8 Hz), 7.23 (1H, t, J ¼ 8 Hz), 6.74 (2H, m), 6.05 (1H, d, J ¼ 5.8 Hz), 4.68 (1H, t, J ¼ 5.3 Hz), 4.68 (4H, m). 13 C NMR (CD 3 OD): d ¼ 175.1, 162.1, 157.2, 153.8, 150.9, 141.1, 134.3, 131.4, 120.6, 120.1, 119.2, 117.8, 89.1, 84.6, 76.1, 72.4, 69.5, 69.0. HRMS calculated for C 17 H 19 N 6 O 8 S (MH + ): 467.0985; found: 467.0994. 12 (mixture of diastereomers): R f ¼ 0.27 (EtO- Ac : MeOH, 4 : 1, v ⁄ v) 1 H NMR (CD 3 OD, 300 K): d ¼ 8.4 (1H, s), 8.05 (1H, m), 6.7–7.5 (9H, m), 6.08 (1H, broad s), 5.18 (1H, broad s), 5.02 (1H, broad s), 4.42 (1H, broad s), 4.21 (2H, broad s), 1.47 (3H, s), 1.19 (3H, s). 13 C NMR (CD 3 OD, 300 K): although the purity of the corresponding final deprotected compound 13 was at the end excellent, 12 itself was difficult to purify and did not give a good 13 C NMR. 13:R f ¼ 0.21 (CHCl 3 : MeOH, 2 : 1, v ⁄ v) 1 H NMR (CD 3 OD): d ¼ 8.43 (1H, s), 8.06 (1H, s), 7.33 (1H, d, J ¼ 8.0 Hz), 6.74 (1H, d, J ¼ 8.0 Hz), 6.51 (1H, t, J ¼ 8.0 Hz), 5.98 (1H, d, J ¼ 5.8 Hz), 4.61 (1H, t, J ¼ 5.8 Hz), 4.3 (4H, m). 13 C NMR (CD 3 OD): d ¼ 175.2, 164.3, 157.3, 153.8, 150.6, 146.8, 141.1, 121.9, 120.9, 119.3, 118.5, 89.1, 84.7, 76.1, 72.5, 69.5. 16:R f ¼ 0.46 (EtOAc : MeOH, 9 : 1, v ⁄ v); [a] 25 D ¼ )20 (c ¼ 0.5; CHCl 3 ) 1 H NMR (CDCl 3 ): d ¼ 8.29 (1H, s), 8.00 (1H, s), 7.55 (2H, d, J ¼ 7.5 Hz), 7.30 (5H, m), 6.85 (2H, d, J ¼ 8.6 Hz), 6.14 (1H, d, J ¼ 2.5 Hz), 5.62 (1H, broad s), 5.29 (1H, dd, J ¼ 2.5 & 6.3 Hz), 5.12 (1H, dd, J ¼ 2.8 & 6.3 Hz), 5.02 (2H, s), 4.52 (1H, m), 4.43 (1H, dd, J ¼ 3.8 & 11.0 Hz), 4.32 (1H, dd, J ¼ 5.0 & 11.0 Hz), 3.79 (3H, s), 1.61 (3H, s), 1.34 (3H, s). 13 C NMR (CDCl 3 ): d ¼ 159.4, 155.6, 153.9, 153.0, 149.4, 139.5, 130.3, 130.2, 130.0, 129.3, 128.3, 126.9, 120.0, 114.4, 113.7, 90.6, 85.6, 84.2, 81.4, 76.5, 71.0, 55.2, 27.1, 25.2. 17:R f ¼ 0.45 (EtOAc : MeOH, 9 : 1, v ⁄ v) 1 H NMR (CDCl 3 ): d ¼ 8.23 (1H, s), 8.08 (1H, s), 7.30 (7H, m), 7.02 (2H, dd, J ¼ 7.6 & 10.3 Hz), 6.15 (1H, d, J ¼ 2.8 Hz), 5.84 (1H, broad s), 5.20 (1H, dd, J ¼ 2.8 Hz & 6.3 Hz), 5.09 (2H, s), 5.06 (1H, m), 4.51 (1H, m), 4.28 (1H, dd, J ¼ 3.3 Hz & 11.3 Hz), 4.18 (1H, dd, J ¼ 4.6 Hz & 11.3 Hz), 1.61 (3H, s), 1.34 (3H, s). 13 C NMR (CDCl 3 ): d ¼ 161.5, 158.9, 155.2, 152.8, 150.6 (d, J ¼ 234 Hz), 139.6, 137.3, 132.2, 131.2, 128, 4, 128.3, 127.9, 124.3, 119.9, 116.0, 114.4, 90.7, 85.3, 84.6, 81.4, 70.6, 27.2, 25.3. HRMS calculated for C 27 H 28 N 6 O 5 F (MH + ): 535.2105; found: 535.2083. 18:R f ¼ 0.30 (EtOAc : MeOH, 9 : 1, v ⁄ v); [a] 25 D ¼ )17 (c ¼ 0.2; MeOH) 1 H NMR (CD 3 OD): d ¼ 8.20 (1H, s), 8.10 (1H, s), 7.96 (2H, d, J ¼ 7.1 Hz), 7.60 (1H, t, J ¼ 7.7 Hz), 7.46 (2H, t, J ¼ 7.7 Hz), 6.02 (1H, d, J ¼ 4.3 Hz), 4.71 (1H, dd, J ¼ 3.5 & 12.1 Hz), 4.58 (2H, m), 4.37 (1H, dd, J ¼ 4.8 & 8.6 Hz). 13 C NMR (CDCl 3 ): d ¼ 167.7, 157.3, 153.9, 150.5, 141.4, 134.4, 131.0, 130.6, 129.6, 90.7, 83.5, 74.9, 71.8, 64.9. 19:R f ¼ 0.30 (AcOEt : MeOH, 9 : 1, v ⁄ v); [a] 25 D ¼ )42 (c ¼ 0.3; MeOH) 1 H NMR (CDCl 3 ): d ¼ 8,19 (1H, s), 8,09 (1H, s), 7.89 (1H, t, J ¼ 7.3 Hz), 7.63 (1H, m), 7.23 (2H, m), 6.03 (1H, d, J ¼ 4.3 Hz), 4.82 (1H, m, H-2), 4.72 (1H, dd, J ¼ 3.2 & 12 Hz), 4.58 (2H, m), 4.38 (1H, m) 13 C NMR (CDCl 3 ): d ¼ 165.4, 163.2 (d, J ¼ 259 Hz), 157.3, 153.9, 153.5, 150.5, 141.1, 136.3, 136.2, 133.2, 125.4, 120.5, 118.1, 117.9, 90.5, 83.5, 75.1, 71.7, 65.2. Overproduction and purification of proteins The His6-tagged fusions of B. subtilis DhbE and Y. pestis YbtE were produced and purified according to described procedures [9,13]. Proteins were stocked with and without 10% glycerol at )20 °C without observed differences in activity. ATP-pyrophosphate exchange assays and determination of kinetic constants The ATP ⁄ PP i exchange reaction [26] was used to determine substrate specificity of DhbE and YbtE for DHB and SAL and the quality of inhibition for the A domain inhibitors. For all assays, the enzyme concentration was 300 nm and ATP concentration was at a saturating level of 2 mm. All reactions were performed at 37 °C. To confirm the aryl substrate specificities, DHB and SAL concentrations were varied from 0 to 250 lm for testing DhbE and 0–500 lm for testing YbtE. ATP ⁄ PP i exchange reactions were carried out for 30 s for each substrate concentration. K m values were determined by using the nonlinear fit modeling option of Microcal tm origin tm 5.0 software (Microcal Software Inc., Northampton, MA, USA). The inhibitory effect of the six acyl adenylate analogues on DhbE was tested using 12.5 lm of inhibitor and 250 lm of DHB. The ATP ⁄ PP i exchange reactions were stopped after 5 min. To deter- mine the K i values for DhbE with both DHB-AMS and SAL-AMS, the DHB concentration was set around the K m value at concentrations of either 0.5, 1 or 2 lm, while the concentration of the inhibitors was varied from 0 to 50 nm. In the case of YbtE, the SAL concentration was set at 2.25, 4.5 or 9 lm and the concentration of the inhibitors was varied from 0 to 25 nm. The ATP ⁄ PP i exchange reactions were carried out for 30 s. The inhibition constants were cal- culated using the Dixon plot method. M. Miethke et al. 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Inhibition of aryl acid adenylation domains involved in bacterial siderophore synthesis Marcus Miethke 1 , Philippe Bisseret 2 , Carsten L. Beckering 1 ,. 2005) doi:10.1111/j.1742-4658.2005.05077.x Aryl acid adenylation domains are the initial enzymes for aryl- capping of catecholic siderophores in a plethora of microorganisms. In order to