MINIREVIEW Thiamin diphosphate in biological chemistry: analogues of thiamin diphosphate in studies of enzymes and riboswitches Kwasi Agyei-Owusu and Finian J. Leeper Department of Chemistry, University of Cambridge, UK Thiamin diphosphate 1 (ThDP) (Fig. 1) is a coenzyme that assists in the catalysis of carbon–carbon bond- forming and bond-breaking reactions adjacent to a carbonyl group. A large and diverse collection of enzymes require ThDP, which acts as an electron sink during catalysis, stabilizing what would otherwise be an acyl carbanion in the form of an enamine interme- diate (Fig. 2). These enzymes include pyruvate decar- boxylase (PDC; EC 4.1.1.1) which is involved in the formation of alcohol in anaerobic fermentation [1], transketolase (TK; EC 2.2.1.1) which transfers a two- carbon unit from a ketose to an aldose [2] and pyruvate dehydrogenase (PDH; EC 1.2.4.1), a highly complex enzyme that links the glycolytic pathway to the citric acid cycle through the formation of acetyl CoA [3]. The general catalytic cycle for ThDP-dependent enzymes, as illustrated by the cycle for PDC (Fig. 2), was first elucidated by Breslow [4] and begins with deprotonation of the C-2 carbon of the thiazolium ring (now believed to be effected by the 4¢-N atom of the aminopyrimidine ring in its imino tautomer). The resulting ylid subsequently carries out a nucleophilic attack on the keto group of pyruvate, followed by decarboxylation which leads to formation of the enam- ine intermediate (which can also be drawn as an Keywords acetohydroxyacid synthase; enzyme crystal structure; enzyme inhibition; pyruvate decarboxylase; pyruvate dehydrogenase; reaction mechanism; riboswitch; thiamin pyrophosphate; thiamine diphosphate; transketolase Correspondence F. J. Leeper, Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK Fax: +44 1223 336362 Tel: +44 1223 336403 E-mail: FJL1@cam.ac.uk Website: http://www.ch.cam.ac.uk/staff/ fjl.html (Received 9 October 2008, revised 9 March 2009, accepted 12 March 2009) doi:10.1111/j.1742-4658.2009.07018.x The role of thiamin diphosphate (ThDP) as a cofactor for enzymes has been known for many decades. This minireview covers the progress made in understanding the catalytic mechanism of ThDP-dependent enzymes through the use of ThDP analogues. Many such analogues have been syn- thesized and have provided information on the functional groups necessary for the binding and catalytic activity of the cofactor. Through these studies, the important role of hydrophobic interactions in stabilizing reaction inter- mediates in the catalytic cycle has been recognized. Stable analogues of intermediates in the ThDP-catalysed reaction mechanism have also been synthesized and crystallographic studies using these analogues have allowed enzyme structures to be solved that represent snapshots of the reaction in progress. As well as providing mechanistic information about ThDP-depen- dent enzymes, many analogues are potent inhibitors of these enzymes. The potential of these compounds as therapeutic targets and as important her- bicidal agents is discussed. More recently, the way that ThDP regulates the genes for its own biosynthesis through the action of riboswitches has been discovered. This opens a new branch of thiamin research with the potential to provide new therapeutic targets in the fight against infection. Abbreviations 3-deazaThDP, 3-deazathiamin diphosphate; PDB, Protein Database; PDC, pyruvate decarboxylase; PDH, pyruvate dehydrogenase; PDHc, pyruvate dehydrogenase complex; ThDP, thiamin diphosphate; ThTDP, thiamin 2-thiazolone diphosphate; ThTTDP, thiamin 2-thiothiazolone diphosphate; TK, transketolase; TPK, thiamin pyrophosphokinase. FEBS Journal 276 (2009) 2905–2916 ª 2009 The Authors Journal compilation ª 2009 FEBS 2905 a-carbanion resonance structure). Depending on the ThDP-dependent enzyme involved, the enamine can attack a number of electrophilic species. In the case of PDC, protonation of the enamine occurs and finally release of acetaldehyde regenerates the ThDP ylid. Over the course of the last 70 years, numerous ana- logues of ThDP have been synthesized by various research groups and this has afforded us an insight into the catalytic mechanism of ThDP-dependent enzymes [4]. Binding and inhibition studies, as well as X-ray crystallography involving ThDP analogues, have provided us with information on enzyme ⁄ coen- zyme ⁄ substrate interactions and how reaction interme- diates are stabilized by the enzyme. More recently, work on various enzymes as targets for antitumour and antimalarial drugs based on ThDP has been pur- sued with some promise [5,6]. With the discovery of ThDP-reponsive riboswitches [7], an exciting field for exploration has recently been opened up: the regula- tion of thiamin biosynthesis in some organisms by ThDP riboswitches offers a novel target for drugs based on thiamin. This minireview covers the key enzymatic studies of ThDP analogues that have aided our mechanistic understanding of ThDP-dependent enzymes, as well as studies of thiamin analogues as drug molecules and in the field of riboswitches. Modifications to the aminopyrimidine moiety Analogues of ThDP with a modified aminopyrimidine moiety (Fig. 3) provided a key insight into binding of the coenzyme as well as its catalytic activity. Schellen- berger et al. synthesized a number of pyridyl and dea- minopyrimidine species for testing on the apoenzymes of PDC, PDH and TK [8,9]. The N 1 0 -pyridyl analogue (in which the 3¢-nitrogen has been replaced by carbon) was found not only to bind to the apoenzymes tested, but also to be catalytically active. It was concluded that the 3¢-nitrogen was not crucial for binding or cat- alytic activity. The N 3 0 -pyridyl analogue (in which the 1¢-nitrogen has been replaced by carbon), however, was found to bind relatively weakly and produce no discernible catalytic activity. From this result, it could Fig. 2. Catalytic mechanism for pyruvate decarboxylase. Fig. 1. Thiamin diphosphate. Studies with analogues of thiamin diphosphate K. Agyei-Owusu and F. J. Leeper 2906 FEBS Journal 276 (2009) 2905–2916 ª 2009 The Authors Journal compilation ª 2009 FEBS be concluded that hydrogen bonding between a suit- able amino acid residue and the N 1 0 nitrogen was not only required for tight binding, but also was critical for the catalytic activity of the coenzyme. It was subse- quently discovered by X-ray crystallography, site-spe- cific mutagenesis and NMR studies of coenzyme analogues that there is a conserved glutamate residue in the active site of thiamin-dependent enzymes that hydrogen bonds to the N 1 0 nitrogen (Fig. 4) [10] and the consequence of this hydrogen bonding is to increase the basicity of the 4¢-amino group, its ability to act as a proton acceptor for the C2–H of the thiazo- lium ring being enhanced by several orders of magni- tude [11]. By deleting the 4¢-amino group altogether or replac- ing it with other functionalities such as a thiol, hydroxy or dimethylamino group, its important role in catalysis soon became apparent [9]. These analogues generally showed good binding capacity but were cata- lytically inactive, offering further proof of the proton acceptor role of the amino group. One exception is the 4¢-monomethylated amino group which has been shown to retain catalytic activity when bound to trans- ketolase with b-hydroxypyruvate as substrate [12]. However, the stability of the resulting enamine inter- mediate was reduced significantly. It was suggested that this is caused by impaired hydrogen bonding between the amino group and the dihydroxyethyl group attached to C-2. It has been shown that ThDP adopts a specific con- formation in the active site of the enzyme [13]. This so-called ‘V-conformation’ was first suggested when 6¢-methylated ThDP analogues were tested for enzymic activity by Schellenberger [9]. For PDC, it was reported that 6¢-methyl-ThDP was catalytically inac- tive, whereas 4-demethyl-6¢-methyl-ThDP was active. It was proposed that a 6¢-methyl group causes a steric clash with the 4-methyl group when the ThDP is in the V-conformation and the analogue therefore has to adopt an altered conformation. Interestingly, the 4-demethylated analogue, which retains its catalytic activity, is a relatively weak binder to the apoenzyme, suggesting the requirement of hydrophobic interactions between the 4-methyl group and the apoenzyme for good binding affinity. It was later reported that in transketolase the 6¢-methyl-ThDP does have $ 50% of the normal catalytic activity [14], whereas the 4-dem- ethyl-6¢-methyl-ThDP is inactive [15]. Crystal structures of three of these pyrimidine-modi- fied analogues, 1¢-pyridyl-, 3¢-pyridyl- and 6¢-methyl- ThDP, bound to transketolase have been obtained [16]. The pyrimidine ring is found in a very similar position to that of the normal ThDP complex in all three analogues, confirming that the lack of activity of the N 3 0 -pyridyl analogue is caused by the loss of hydrogen bonding and not by a change of conformation. Modifications to the thiazolium ring The positively charged thiazolium ring of ThDP has long been recognized as the catalytic centre of the coenzyme [4]. Synthetic modifications to the thiazolium moiety (Fig. 5) have been instrumental in providing insights into the catalytic mechanism of ThDP as well as providing information on how reaction intermedi- ates of the coenzyme are stabilized by the various ThDP-dependent enzymes [17,18]. Modifications at C-2 Substituents at the C-2 position of ThDP can lead to potent inhibitors of ThDP-dependent enzymes. Two such compounds are thiamin 2-thiazolone diphosphate (ThTDP), in which the C2–H has been replaced by C=O, and thiamin 2-thiothiazolone diphosphate (ThTTDP), in which the C2–H is replaced by C=S Fig. 3. Modifications to the aminopyrimidine ring of ThDP. Fig. 4. Amino acid residues surrounding ThDP in PDH E1 from Escherichia coli (from PDB entry 1L8A). The carbons of ThDP are in green and other carbons in grey. Conserved glutamate residue Glu571 hydrogen bonds to N 1 0 of ThDP. Figures were drawn using PYMOL [59]. K. Agyei-Owusu and F. J. Leeper Studies with analogues of thiamin diphosphate FEBS Journal 276 (2009) 2905–2916 ª 2009 The Authors Journal compilation ª 2009 FEBS 2907 [18]. Both are tight binding but reversible inhibitors of the E1 component of PDH of Escherichia coli. ThTDP, in particular, was found to have a K i value in the very low nm range, as measured by a number of methods. It can be seen that both ThTDP and ThTTDP mimic the neutral enamine intermediate com- mon to all ThDP-dependent enzymes and, as such, it is not surprising that they bind tightly to the enzyme. An interesting result obtained from studying these two analogues was the discovery of a new positive CD band at 330 nm when ThTTDP is added to the E1 component of PDH [18], which is absent when either ThDP or ThTDP is added to the subunit. This phe- nomenon is thought to be caused by the chiral confor- mation imposed on ThTTDP when it is bound in the active site. 2-Methyl-ThDP was also found to inhibit the enzyme by preventing formation of the ThDP com- plex [9]. Several structures of ThTDP bound to ThDP- dependent enzymes have been solved, including human branched-chain 2-keto acid dehydrogenase (Protein Database [PDB] entries 2BFC and 2BFF), benzoyl- formate decarboxylase (1YNO), oxalyl CoA decarbox- ylase (2C31) and PDH E1 (1RP7). In the last of these examples, the tighter binding of ThTDP than ThDP was ascribed to an increase in the number of hydrogen bonds between the protein and the ligand [19]. A further derivative of ThDP modified at C-2 is tetrahydroThDP 2, which is formed by reduction of ThDP with sodium borohydride. TetrahydroThDP is reported to inhibit yeast TK with a K i value of 0.4 lm [20], yeast PDC with a K i value of 6.5 lm [21] and PDH complex with an IC 50 of 0.046 lm [22]. Tetra- hydroThDP made by sodium borohydride reduction of ThDP consists of two racemic diastereoisomers. The diastereoisomers have been separated and it was found that the cis isomer was a much more potent inhibitor (K i = 0.02–0.15 lm) than the trans isomer (K i = 5–10 lm) [23]. Structures representing the pre-decarboxylation stage of the reaction of PDH E1 [24–25] and pyruvate oxi- dase [26] have been obtained by incubating the native enzyme with methyl acetylphosphonate. This reacts with the ThDP ylid to produce 2-phosphonolactyl- ThDP, an analogue of 2-lactyl-ThDP (Fig. 2) that has -PðOMeÞO À 2 in place of -CO À 2 . Similarly, reaction of benzaldehyde lyase with methyl benzoylphosphonate produces a phosphonate analogue of 2-mandelyl-ThDP [27]. Interestingly with benzoylformate decarboxylase, if benzoylphosphonate is used instead of its methyl ester, a phosphoryl group transfer occurs to an active- site serine residue [28]. Succinylphosphonate and its monoethyl esters, inactivate a-ketoglutarate dehydro- genase [29], so presumably this also forms phosphono analogues of the predecarboxylation intermediate, though in this case no crystal structure has been reported as yet. 3-Deazathiamin diphosphate and derivatives Among the most potent inhibitors of ThDP-dependent enzymes is 3-deazathiamin diphosphate (3-deazaThDP) 3, first synthesized by Hawksley et al. (Fig. 6) [30,31]. In this compound, a carbon atom replaces the N-3 Fig. 6. 3-DeazaThDP 3 and its derivatives 4 and 5, triazole ana- logues 6 and 7, and open-chain analogues 8 and 9. Fig. 5. Some modifications to the thiazolium moiety that have been studied. One enantiomer of the cis isomer of tetrahydro-ThDP 2 is shown. Studies with analogues of thiamin diphosphate K. Agyei-Owusu and F. J. Leeper 2908 FEBS Journal 276 (2009) 2905–2916 ª 2009 The Authors Journal compilation ª 2009 FEBS atom of ThDP resulting in a neutral thiophene ring in place of the thiazolium ring of the coenzyme. This analogue is of the same size and steric profile as ThDP and the only difference is the absence of a positive charge at the 3-position, which precludes the formation of the reactive ylid required for catalysis. Enzymatic studies with 3-deazaThDP carried out on PDC from Zymomonas mobilis (ZmPDC) showed it to be an essentially irreversible inhibitor, binding $ 25 000 times more tightly than ThDP [31]. Binding to a-keto- glutarate dehydrogenase (EC 1.2.4.2) E1 component by 3-deazaThDP was also found to be 500 times stron- ger than for ThDP [31]. In both enzymes studied, binding of the analogue occurred at a faster rate than the natural coenzyme. Similar results have been obtained with pyruvate dehydrogenase and branched- chain 2-keto acid dehydrogenase E1 components (see below) as well as with transketolase (K. M. Erixon & F. J. Leeper, unpublished results). It is initially surprising to find that 3-deazaThDP binds much tighter than the natural coenzyme. The key to rationalizing this result is to note that it is a good mimic of the ylid structure in which the five- membered ring is neutral overall. As a consequence of the charge being reduced from +1 to neutral, there is an increase in hydrophobic interactions between the enzyme’s active site residues and the analogue, which in turn leads to tighter binding. It has been shown that the enzyme stabilizes the neutral ylid and enamine intermediates in the catalytic cycle by providing an active site of low dielectric constant [32]. This would assist the formation of these neutral intermediates from their charged precursors. In studies on PDH E1, it was found that the protein displays half-of-sites reactivity towards proteolysis of the active-site loops when ThDP is bound but is pro- tected from cleavage at both active sites of the a 2 b 2 tetramer when 3-deazaThDP is bound. This observa- tion led to the ‘proton-wire’ hypothesis in which com- munication between the active sites is mediated by the shuttling of a proton down a solvent-filled tunnel that connects the two active sites [33]. 3-DeazaThDP has also been used to obtain crystal structures of the ThDP-dependent enzymes with their substrates bound. In most cases, equivalent structures with ThDP bound could not be obtained because the reaction occurs too quickly. Thus, the structure of a complex of oxalyl CoA decarboxylase with 3-dea- zaThDP and oxalyl CoA was solved and this revealed both the CoA binding site and the ordering of the C-terminus of the protein to form a lid over the CoA chain (PDB entry 2JI6) [34]. In the case of phenylpyru- vate decarboxylase, crystallizing the enzymes with 3-deazaThDP bound allowed a structure of the enzyme to be obtained which had substrate molecules in both the active site and the regulatory site, thus unveiling the mechanism of allosteric substrate activation [35]. As well as being a very potent inhibitor, 3-deaza- ThDP has the advantage over neutral analogues such as ThTDP in that it can be functionalized at the C-2 position in order to make very close mimics of reaction intermediates in the catalytic cycle of various ThDP- dependent enzymes. In so doing, very selective and potent inhibitors of the E1 components (E1p and E1b) of pyruvate dehydrogenase complex (PDHc) and the related branched-chain keto acid dehydrogenase com- plex (EC 1.2.4.4) have been produced (M. D. H. Wood and F. J. Leeper, unpublished results). PDHc and branched-chain 2-keto acid dehydrogenase complex belong to the highly complex 2-oxoacid dehydrogenase family of enzymes. Enzymes in this family have a molecular mass of between 4 and 10 · 10 6 Da. PDHc comprises three different enzymic components (E1, E2 and E3) that work in tandem to catalyse the oxidative decarboxylation of pyruvate to give acetyl CoA, CO 2 and NADH as products (Fig. 7). This is carried out with the aid of no fewer than five coenzymes, namely ThDP, CoA, lipoic acid, NAD + and FAD. The first reaction catalysed by PDHc is the ThDP-dependent decarboxylation of pyruvate by the E1 component, an a 2 b 2 heterotetramer with two active sites located at the interfaces between the a- and b-subunits [33]. 3-DeazaThDP proved to be a potent inhibitor of both E1p and E1b with K i values of 0.14 and 0.48 nm, respectively. For comparison, ThDP has a K D for both E1 components of $ 3 lm. The time-course of the inactivation of the holoenzyme is relatively slow, because of the slow rate of unbinding of ThDP. It was observed that there is a faster initial rate of inactiva- tion followed by a much slower rate. This was observed for both enzymes although it is more pro- nounced for E1b. This profile may be evidence for the already mentioned half-of-sites reactivity. The two active sites of the E1 component are in alternate Fig. 7. Overall reaction catalysed by the pyruvate dehydrogenase complex. K. Agyei-Owusu and F. J. Leeper Studies with analogues of thiamin diphosphate FEBS Journal 276 (2009) 2905–2916 ª 2009 The Authors Journal compilation ª 2009 FEBS 2909 conformational states, with one being ‘open’ and the other ‘closed’. The more rapid phase of the inactiva- tion may be caused by binding of the inhibitor after ThDP has unbound from the ‘open’ conformation, whereas the slow phase may be caused by binding of the second molecule of inhibitor in the second active site, which has to change from a ‘closed’ to an ‘open’ conformation to allow its ThDP to unbind. Analogues 4 and 5 (Fig. 6) synthesized readily from 3-deazathiamin by Friedel–Crafts acylation at the C-2 position, followed by reduction of the ketone and pyrophosphorylation, proved to be even better inhibi- tors for their corresponding E1 components than deazaThDP (Fig. 5). 4 closely mimics the enamine reaction intermediate of the E1p reaction, whereas 5 mimics the enamine intermediate of E1b. For the E1p component, analogue 4 showed excellent affinity with a K i value of 0.1 nm. Analogue 5, by contrast, showed no detectable inhibition, thus confirming that the E1 component of PDHc shows a high degree of selectivity for its natural substrate pyruvate over branched-chain keto acid substrates. The opposite is true for the E1b subunit in which analogue 5 binds with a higher affin- ity (K i = 0.17 nm) than 4 (K i = 0.44 nm). The fact that 4 binds with less affinity can be attributed to its shorter methyl side chain which would have fewer hydrophobic interactions with the enzyme compared with the longer branched side chain. The selectivity of both E1 components for the R and S enantiomers of 4 and 5 was also probed. Interest- ingly neither enzyme shows any preference for either enantiomer of 4 but E1b shows a three- to four-fold preference for binding the R enantiomer of 5. Crystal structures have been obtained of 4 bound in the ThDP-binding sites of phenylpyruvate decarboxy- lase [35] and the branched-chain keto acid decarboxy- lase from Lactococcus lactis [36]. These structures have helped define the conformations that the enzymes would adopt when the reaction mechanisms have reached the enamine intermediates. Using 3-dea- zaThDP and its substituents, as well as the phosphono analogues mentioned above, crystal structures can now be obtained that represent ‘snapshots’ of ThDP-depen- dent enzymes at almost every stage of the reaction. Other synthetic analogues The synthesis of 3-deazaThDP is a 12-step procedure which, although producing good yields, is difficult and time-consuming. Two inhibitors with marginally less potency than 3-deazaThDP but which can be obtained much more easily in four synthetic steps are the triaz- ole analogues 6 (K i =20pm) and 7 (K i =30pm against ZmPDC) (Fig. 6) [37]. A disadvantage of these triazole compounds is that they cannot be functional- ized at the 2-position to produce analogues of reaction intermediates. They do, however, provide a readily accessed scaffold for synthesizing mimics of the diphosphate moiety and this is discussed in the follow- ing section. Having established that neutral analogues of the thiazolium moiety such as 3-deazaThDP bind with a much higher affinity than the natural coenzyme and knowing that the diphosphate group is the most important group for binding strength, we wondered if ‘open-chain’ analogues such as 8 and 9 would show any potency as inhibitors (Fig. 6). The ‘open-chain’ analogues have the advantage of being much more readily synthesized than their thiophene-containing counterparts. Initial results from affinity studies with apo-ZmPDC suggest K i values in the nm range (K. Agyei-Owusu & F. J. Leeper, unpublished results). These analogues are not expected to have the potency of 3-deazaThDP because of their higher degree of con- formational freedom, but they are readily synthesized and easily functionalized into mimics of reaction inter- mediates and, with potential K i values in the nm range, show much promise as selective ThDP-dependent enzyme inhibitors. ThDP analogues formed during crystallization or X-ray irradiation In several cases, the crystal structures of ThDP-depen- dent enzymes, once solved, have revealed that the thia- zolium ring of the ThDP has degraded, presumably by hydrolysis. Examples include Zm PDC (PDB entry 1ZPD) and carboxyethylarginine synthase (PDB entry 2IHU), where C-2 has been lost altogether, and both yeast and Arabidopsis thaliana acetohydroxyacid syn- thases in complex with herbicides such as metsulfuron methyl (e.g. PDB entries 1YHY and 1T9D), in which the ethyl pyrophosphate side chain appears to have become detached from the pyrimidine ring with at most fragments of the thiazolium ring remaining. It is worth noting that hydrolysis of the thiazolium ring of ThDP generates open-chain neutral species similar to 8 and 9. In view of the fact that the enzymes bind neu- tral species more tightly than positively charged ones, it is reasonable to assume that the enzyme causes the hydrolysis reaction to be more thermodynamically favourable. In Klebsiella pneumoniae acetolactate synthase, the structure obtained after soaking the crystals with pyru- vate appeared to show a 2-(1-hydroxyethyl)ThDP which had cyclized from the 4¢-amino group to C-2 to Studies with analogues of thiamin diphosphate K. Agyei-Owusu and F. J. Leeper 2910 FEBS Journal 276 (2009) 2905–2916 ª 2009 The Authors Journal compilation ª 2009 FEBS give a dihydrothiachrome derivative (PDB entry 1OZG). Pyruvate ferredoxin oxidoreductase is an anaerobic enzyme that is inactivated by exposure to both pyruvate and air. The structure of this inactive complex shows electron density for a noncovalently bound pyruvate molecule and an additional atom apparently attached to the 4¢-NH 2 (PDB entry 2C3U). It was suggested that hydroxylation of this amino group has occurred to give the hydroxylamine [38]. Diphosphate group mimics The diphosphate group of ThDP forms the most important binding interactions with the enzyme by coordinating to Mg 2+ [9]. It has been shown that inor- ganic diphosphate on its own will compete with the coenzyme for its active site. ThDP binds at the inter- face of two subunits of the enzyme, with the diphos- phate group bound to one subunit and the aminopyrimidine moiety bound in a cavity between the two subunits. In order to synthesize potential drug molecules with good pharmacokinetic profiles based on ThDP, it would be essential to make mimics of the diphosphate group that are not as highly charged but which retain good affinity to the enzyme. Because of its high charge, the diphosphate group would make it difficult for an analogue to penetrate the lipid mem- branes of cells with consequent poor uptake and bio- availability. With this in mind, a number of analogues of ThDP have been synthesized with isosteres of the diphosphate group (Fig. 8) [37]. As mentioned above, the triazole analogues of ThDP can be readily accessed and show high affinity to the enzyme PDC. Several analogues of the triazole diphosphate 7 have been prepared and provide more evidence for the critical role of diphosphate in binding. For example, there is a difference in binding to ZmPDC between the diphosphate 7 and its alcohol precursor of the order of 1 · 10 7 . The general trend observed in binding studies with the diphosphate mim- ics was a decrease in affinity with decreasing anionic charge. The methylene diphosphonates 10 and 11, which are trianionic, show good binding affinity rela- tive to the other diphosphate mimics studied, with K i values only $ 30–40 times greater than that of 7. There is a successive decrease in affinity going from 10 and 11 (trianionic) to the phosphoramidic analogue 12 (dianionic), to carbamate 13 and malonate 14 (mono- anionic), to the iminodiacetate analogue 15, for which no binding was observed. Pyrithiamin, riboswitches and thiamin pyrophosphokinase Pyrithiamin (Fig. 9) was first synthesized in 1941 by Tracy and Elderfield [39], the culmination of efforts by a number of groups to produce the pyridinium isostere of the thiazolium moiety in ThDP. A 0.5 mg dose of this analogue was found to be lethal when adminis- tered to a newly weaned mouse, whereas a 0.17 mg dose retarded growth in the mouse [40]. Over the years, it has been used to produce symptoms of thia- min deficiency in mice [41] and has been shown to be toxic to species of fungi, algae and bacteria [42–44]. In enzymatic studies, pyrithiamin diphosphate was found to be only a modest inhibitor of apo-TK (K i = 110 lm) [20] and apo-PDC (K i =78lm) [21], which suggested that its antithiamin effects were not the result of competition with ThDP for binding to the enzymes. Moreover, a number of organisms that bio- synthesize thiamin and would be expected to upregu- late its production in the presence of pyrithiamin were found to be unable to do so [43]. As far back as 1976, Fig. 8. Diphosphate mimics based on the triazole analogue of ThDP. Fig. 9. Structures of pyrithiamin and amprolium. K. Agyei-Owusu and F. J. Leeper Studies with analogues of thiamin diphosphate FEBS Journal 276 (2009) 2905–2916 ª 2009 The Authors Journal compilation ª 2009 FEBS 2911 pyrithiamin was shown to disrupt the regulatory mech- anisms of thiamin biosynthesis, causing a decrease in thiamin production, although the underlying mecha- nism for this phenomenon remained unknown. The effects of pyrithiamin on microorganisms at the molecular level became clearer with the discovery of ThDP riboswitches [7]. Riboswitches (Fig. 10) are ele- ments of mRNA that form receptors for a specific ligand which, when bound, controls expression of the gene(s) encoded in the mRNA. A number of different classes of riboswitches have been discovered, each class named after its specific ligand. ThDP riboswitches are generally sited upstream of the genes coding for the enzymes involved in thiamin biosynthesis, transport and the salvaging of precursors to the thiazole and pyrimidine moieties of ThDP. Depending on the organ- ism, they can, when bound to their ligand, act either as terminators of transcription (e.g. in Bacillus subtilis)or as inhibitors of translation (by masking the ribosome binding site, e.g. in E. coli) or to cause mis-splicing of the RNA (in eukaryotes) resulting in premature termi- nation of translation or instability of the mRNA [45]. It has been shown that pyrithiamin is taken up into cells by thiamin transporters and pyrophosphorylated by the enzyme TPK before binding to the ThDP ribo- switch. Pyrophosphorylation of pyrithiamin by TPK has also been carried out in vitro [46]. On binding of pyrithiamin diphosphate to the riboswitch, the conse- quent reduction in the levels of thiamin biosynthetic enzymes leads to the depletion of intracellular thiamin levels, preventing cell growth. Pyrithiamin-resistant mutants of the bacteria B. subtilis and E. coli [43], the fungus Aspergillus oryzae [47] and the alga Chlamydo- monas reinhardtii [44] have all been characterized and in each case it turns out that the mutation is in a ThDP riboswitch. This causes the corresponding biosynthetic gene(s) to be constitutively expressed, no longer under the control of the riboswitch. Three separate groups have obtained crystal struc- tures of ThDP riboswitches, two using the ThiM ribo- switch from E. coli [48,49] (Fig. 10) and one a riboswitch from Arabidopsis thaliana [50]. Two sepa- rate domains (P2 ⁄ P3 and P4 ⁄ P5) recognize the amino- pyrimidine and diphosphate moieties of ThDP, respectively. Binding of ThDP brings these two domains together and causes the helix P1 to form, which is what ultimately controls expression of the gene(s). Interestingly, it appears that there is little binding of the thiazolium moiety of ThDP and this provides scope for the development of analogues of the coenzyme that can interact with the riboswitch. In the case of the E. coli riboswitch, crystal structures were obtained with ThDP analogues thiamin mono- phosphate, benfotiamine and pyrithiamin bound [49], whereas for the A. thaliana riboswitch structures were solved with oxythiamin diphosphate and pyrithiamin diphosphate as ligands [50]. It is known that several types of antibiotics act by binding to ribosomal RNA [51]. Riboswitches likewise have well-defined and conserved 3D structures and so pose attractive targets for the design of drug mole- cules, particularly as they do not, as far as is known, occur in animals [45]. ThDP-dependent enzymes as drug targets Despite the essential nature of ThDP-dependent enzymes and the existence of several enzymes that A B Fig. 10. (A) Secondary structure of the ThiM riboswitch from Escherichia coli. Conserved nucleotides are shown in red and conserved secondary structure in blue. Helices are labelled P1 to P5. (B) Structure of the ThDP-binding site (from PDB entry 2HOJ). Studies with analogues of thiamin diphosphate K. Agyei-Owusu and F. J. Leeper 2912 FEBS Journal 276 (2009) 2905–2916 ª 2009 The Authors Journal compilation ª 2009 FEBS occur in microorganisms but not in animals, inhibitors of ThDP-dependent enzymes have not yet found use as antibacterials. However, it has been found that three separate classes of herbicides (including metsulfu- ron-methyl; Fig. 11), which had been in use for many years, all act by inhibiting acetohydroxyacid synthase, the first enzyme in branched-chain amino acid biosyn- thesis. Crystal structures of these herbicides bound to acetohydroxyacid synthase have been obtained and show that they do not bind in the ThDP-binding site but instead bind in the mouth of the active site, block- ing access to the ThDP [52]. Clomazone is a commer- cially available herbicide that acts by interfering with terpene biosynthesis. Only relatively recently has it been shown to act by inhibiting deoxyxylulose 5-phos- phate synthase, the first enzyme of the non-mevalonate pathway to terpenes [53]. However the inhibitor is not clomazone itself but a metabolite 5-ketoclomazone. Amprolium (Fig. 9) is a relative of pyrithiamin and is widely used for the treatment or prevention of coc- cidiosis in cats, dogs, poultry and cattle. It acts by blocking thiamin uptake into cells much more effec- tively in the parasite than in the host animal [54]. The antibiotic metronidazole, used to treat infections by anaerobic bacteria and protozoa, presents an interest- ing example of a novel thiamin analogue. It turns out that metronidazole can be a substrate for thiaminase (which catalyses the displacement of the thiazole unit of thiamin by various nucleophiles) to form a close analogue of thiamin (Fig. 12) [55]. This compound was found to inhibit thiamin pyrophosphokinase and it is possible that the side-effects of prolonged use of metronidazole are caused by the resulting ThDP deficiency. One ThDP-dependent enzyme that has gained con- siderable attention in the last decade has been TK. This is because it has been identified as playing an important role in cell proliferation in a number of tumour lines. TK is involved in the nonoxidative pen- tose 5-phosphate pathway that leads to formation of the ribose required for the synthesis of nucleotides and, therefore, the expression of TK and of other enzymes of the pathway is upregulated in cancer cells. It has been shown both in vitro and in vivo that target- ing TK with drugs based on ThDP leads to a decrease in tumour growth [56]. In tests on mice, oxythiamin 16 (Fig. 13) was found to inhibit Ehrlich ascites tumour cell proliferation by 84% at a dose of 500 mgÆkg )1 . The effect is enhanced when oxythiamin is adminis- tered alongside dehydroepiandrosterone, an inhibitor of the oxidative pentose 5-phosphate pathway, suggest- ing that the oxidative pathway is also important. Fig. 11. Structures of herbicides metsulfuron-methyl and cloma- zone. Fig. 12. Reaction of metronidazole with thiamin catalysed by thiaminase. Fig. 13. Structures of oxythiamin, N-3¢-pyridyl-thiamin and its disul- fide prodrug 18. K. Agyei-Owusu and F. J. Leeper Studies with analogues of thiamin diphosphate FEBS Journal 276 (2009) 2905–2916 ª 2009 The Authors Journal compilation ª 2009 FEBS 2913 A large number of thiamin analogues have been syn- thesized and tested for inhibition of TK in vitro, with and without pyrophosphorylation by TPK, and in vivo, as well as for their effect on tumour growth. These analogues included both uncharged compounds like 3-deazathiamin 3 [5] and analogues with a positively charged thiazolium ring [57]. The compound selected for further development was the N-3¢ pyridyl analogue 17 (Fig. 13). This has poor pharmacokinetic properties leading to rapid clearance, however, and is better administered as its prodrug 18 [58]. (Thiamin itself has poor oral absorption because of the low capacity of its transporter in the gut and is often taken in similar pro- drug form in order to treat or prevent symptoms of thiamin deficiency.) The disulfide bond of prodrug 18 is reduced in vivo and cyclizes spontaneously to produce 17. Both in vitro and in vivo tests on the HCT-116 cell line showed encouraging EC 50 values for the prodrug, which were better than those of its parent thiazolium salt. Concluding remarks Enzymatic studies involving analogues of ThDP have revealed much information on the mechanistic intrica- cies of various ThDP-dependent enzymes. For exam- ple, the role of hydrophobic interactions between the enzyme and coenzyme in stabilizing reaction intermedi- ates has become clearer, and the features of the coen- zyme essential for binding and catalysis have been elucidated. New and intriguing features of some enzymes, for example, half-of-sites reactivity in the E1 component of PDH, have also been discovered. Crys- tal structures of enzymes with analogues of reaction intermediates bound have helped to define the roles of the various amino acid side chains at different stages of the reaction. The study of thiamin and its analogues has entered a new phase with the discovery of riboswitches and their regulatory role in the expression of genes of the thiamin biosynthetic pathway. This has, in turn, led to a new set of therapeutic targets in the fight against infections caused by pathogenic bacteria and fungi. Analogues targeting riboswitches in these species may be less susceptible to resistance by the target organisms because in most cases, more than one thiamin ribo- switch exists to regulate thiamin biosynthesis, so multi- ple mutations would be required to achieve resistance. Another promising area of research is the inhibition of the enzyme TK which has been implicated in a number of cancers. Potent inhibitors of TK continue to be developed and this will hopefully add to the arsenal of chemotherapeutic drugs already available. Acknowledgements We thank the EPSRC for a studentship to K.A O. References 1 Kluger R (1987) Thiamin diphosphate: a mechanistic update on enzymic and nonenzymic catalysis of decar- boxylation. Chem Rev 87, 863–876. 2 Lindqvist Y, Schneider G, Ermler U & Sundstro ¨ mM (1992) Three-dimensional structure of transketolase, a thiamine diphosphate dependent enzyme, at 2.5 A ˚ reso- lution. EMBO J 11, 2373–2379. 3 Reed L (1974) Multienzyme complexes. Acc Chem Res 7, 40–46. 4 Breslow R (1957) Rapid deuterium exchange in thiazolium salts. J Am Chem Soc 79, 1762–1763. 5 Thomas AA, De Meese J, Le Huerou Y, Boyd SA, Romoff TT, Gonzales SS, Gunawardana I, Kaplan T, Sullivan F, Condroski K et al. (2008) Non-charged thiamine analogs as inhibitors of enzyme transketolase. Bioorg Med Chem Lett 18, 509–512. 6 Eschbach M-L, Muller IB, Gilberger T-W, Walter RD & Wrenger C (2006) The human malaria parasite Plas- modium falciparum expresses an atypical N-terminally extended pyrophosphokinase with specificity for thia- mine. Biol Chem 387, 1583–1591. 7 Rodionov D, Vitreschak A, Mironov A & Gelfand M (2002) Comparative genomics of thiamine biosynthesis in procaryotes. New genes and regulatory mechanisms. J Biol Chem 277, 48949–48959. 8 Golbik R, Neef H, Hubner G, Konig S, Seliger B, Meshalkina L, Kochetov GA & Schellenberger A (1991) Function of the aminopyrimidine part in thiamin pyrophosphate enzymes. Bioorg Chem 19, 10–17. 9 Schellenberger A (1967) The structure and mechanism of the active centre of yeast pyruvate decarboxylase. Angew Chem Int Ed 6, 1024–1035. 10 Wikner C, Meshalkina L, Nilsson U, Nikkola M, Lindqvist Y, Sundstrom M & Schneider G (1994) Analysis of an invariant cofactor–protein interaction in thiamin diphosphate-dependent enzymes by site-directed mutagenesis – glutamic-acid-418 in transketolase is essential for catalysis. J Biol Chem 269, 32144–32150. 11 Kern D, Kern G, Neef H, Tittmann K, Killenberg Jabs M, Wikner C, Schneider G & Hubner G (1997) How thiamine diphosphate is activated in enzymes. Science 275, 67–70. 12 Meshalkina LE, Kochetov GA, Brauer J, Hu ¨ bner G, Tittmann K & Golbik R (2008) New evidence for cofactor’s amino group function in thiamin catalysis by transketolase. Biochem Biophys Res Commun 366, 692– 697. 13 Nemeria N, Chakraborty S, Baykal A, Korotchkina LG, Patel MS & Jordan F (2007) The 1¢,4¢-iminopyrimidine Studies with analogues of thiamin diphosphate K. Agyei-Owusu and F. J. Leeper 2914 FEBS Journal 276 (2009) 2905–2916 ª 2009 The Authors Journal compilation ª 2009 FEBS [...]... Heinrich P, Janser P, Wiss O & Steffen H (1972) Studies on the reconstitution of apotransketolase with thiamin pyrophosphate and analogues of the coenzyme Eur J Biochem 30, 533–541 Wittorf J & Gubler C (1971) Coenzyme binding in yeast pyruvate decarboxylase Kinetic studies with thiamin diphosphate analogues Eur J Biochem 22, 544–550 Strumilo S, Czygier M & Markiewicz J (1995) Different extent of inhibition... Phosphonate analogues of a-ketoglutarate inhibit the activity of the a-ketoglutarate dehydrogenase complex isolated from brain and in cultured cells Biochemistry 44, 10552–10561 Hawksley D, Griffin DA & Leeper FJ (2001) Synthesis of 3-deazathiamin J Chem Soc Perkin Trans 1, 144– 148 Mann S, Melero CP, Hawksley D & Leeper FJ (2004) Inhibition of thiamin diphosphate enzymes by 3-deazathiamin diphosphate. .. Structure 14, 217–224 Tracy A & Elderfield R (1941) Studies in the pyridine series II Synthesis of 2-methyl-3-hydroxyethylpyridine and of the pyridine analogue of thiamine (vitamin B1) J Org Chem 6, 54–62 Wooley DW (1950) Pyrithiamine and neopyrithiamine J Am Chem Soc 72, 5763–5765 Todd K & Butterworth R (1999) Mechanisms of selective neuronal cell death due to thiamine deficiency Ann NY Acad Sci 893, 404–411... reaction Biochem Int 10, 479–486 Schellenberger A (1982) The amino group and steric factors in thiamin catalysis Ann NY Acad Sci 378, 51–62 Konig S, Schellenberger A, Neef H & Schneider G (1994) Specificity of coenzyme binding in thiamin diphosphate- dependent enzymes – crystal-structures of yeast transketolase in complex with analogs of thiamin diphosphate J Biol Chem 269, 10879–10882 Bearne SL, Gish G,... Leeper F (2007) Inhibition of pyruvate decarboxylase from Z mobilis by novel analogues of thiamin pyrophosphate: investigating pyrophosphate mimics Chem Commum 9, 960–962 Cavazza C, Contreras-Martel C, Pieulle L, Chabriere E, Hatchikian EC & Fontecilla-Camps JC (2006) Flexibility of thiamine diphosphate revealed by kinetic crystallographic studies of the reaction of pyruvate–ferredoxin oxidoreductase... discussion of antithiamin compounds and thiamin antagonists Ann NY Acad Sci 378, 157–160 55 Alston TA & Abeles RH (1987) Enzymatic conversion of the antibiotic metronidazole to an analog of thiamine Arch Biochem Biophys 257, 357–362 56 Rais B, Comin B, Puigjaner J, Brandes JL, Creppy E, Saboureau D, Ennamany R, Lee WNP, Boros LG & Cascante M (1999) Oxythiamine and dehydroepiandrosterone induce a G1... 2-thiazolone and thiamin 2-thiothiazolone diphosphates – evidence for reversible tight-binding inhibition J Biol Chem 276, 45969–45978 Arjunan P, Chandrasekhar K, Sax M, Brunskill A, Nemeria N, Jordan F & Furey W (2004) Structural determinants of enzyme binding affinity: the E1 component of pyruvate dehydrogenase from Escherichia coli in complex with the inhibitor thiamin thiazolone diphosphate Biochemistry... pyrophosphorylation of the primary hydroxyl group in (hydroxyethyl )thiamin by modified phosphoric acid-cresol solutions and evaluation of extension of the method to nucleosides Bioorg Chem 17, 224–230 Nemeria N, Yan Y, Zhang Z, Brown AM, Arjunan P, Furey W, Guest JR & Jordan F (2001) Inhibition of the Escherichia coli pyruvate dehydrogenase complex E1 subunit and its tyrosine 177 variants by thiamin 2-thiazolone and thiamin. .. Sax M, Brunskill A, Chandrasekhar K, Nemeria N, Zhang S, Jordan F & Furey W (2006) A thiamin- bound, pre-decarboxylation reaction intermedi- Studies with analogues of thiamin diphosphate 25 26 27 28 29 30 31 32 33 34 35 ate analogue in the pyruvate dehydrogenase E1 subunit induces large scale disorder-to-order transformations in the enzyme and reveals novel structural features in the covalently bound... snapshots of oxalyl-CoA decarboxylase give insights into catalysis by nonoxidative ThDP-dependent decarboxylases Structure 15, 853–861 Versees W, Spaepen S, Wood MDH, Leeper FJ, Vanderleyden J & Steyaert J (2007) Molecular mechanism of allosteric substrate activation in a thiamine FEBS Journal 276 (2009) 2905–2916 ª 2009 The Authors Journal compilation ª 2009 FEBS 2915 Studies with analogues of thiamin diphosphate . MINIREVIEW Thiamin diphosphate in biological chemistry: analogues of thiamin diphosphate in studies of enzymes and riboswitches Kwasi Agyei-Owusu and Finian J. Leeper Department of Chemistry,. understanding of ThDP-dependent enzymes, as well as studies of thiamin analogues as drug molecules and in the field of riboswitches. Modifications to the aminopyrimidine moiety Analogues of ThDP. Studies in the pyridine series. II. Synthesis of 2-methyl-3-hydroxyethylpyridine and of the pyridine analogue of thiamine (vitamin B1). J Org Chem 6, 54–62. 40 Wooley DW (1950) Pyrithiamine and