Development of Inhibitors of the 2C-Methyl-d ... - ACS Publications

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Development of Inhibitors of the 2C‑Methyl‑D‑erythritol 4‑Phosphate (MEP) Pathway Enzymes as Potential Anti-Infective Agents Tiziana Masini and Anna K. H. Hirsch* Stratingh Institute for Chemistry, University of Groningen, Nijenborgh 7, NL-9747 AG Groningen, The Netherlands

ABSTRACT: Important pathogens such as Mycobacterium tuberculosis and Plasmodium falciparum, the causative agents of tuberculosis and malaria, respectively, and plants, utilize the 2C-methyl-D-erythritol 4-phosphate (MEP, 5) pathway for the biosynthesis of isopentenyl diphosphate (1) and dimethylallyl diphosphate (2), the universal precursors of isoprenoids, while humans exclusively utilize the alternative mevalonate pathway for the synthesis of 1 and 2. This distinct distribution, together with the fact that the MEP pathway is essential in numerous organisms, makes the enzymes of the MEP pathway attractive drug targets for the development of anti-infective agents and herbicides. Herein, we review the inhibitors reported over the past 2 years, in the context of the most important older developments and with a particular focus on the results obtained against enzymes of pathogenic organisms. We will also discuss new discoveries in terms of structural and mechanistic features, which can help to guide a rational development of inhibitors.

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INTRODUCTION Isoprenoids constitute an extremely large class of natural products present in fungi, bacteria, plants, and mammals.1 More than 35 000 isoprenoids have been identified to date, with striking structural diversity and fulfilling very different functions of biological relevance such as protein degradation, apoptosis, regulation of transcription, and post-translational processes.2 In the life cycle of Mycobacterium tuberculosis and several other bacterial pathogens, for example, they play essential roles in the biosynthesis of the components of the bacterial cell wall.3 Despite their remarkable diversity, isoprenoids are biosynthesized in all organisms using two common building blocks, namely, isopentenyl diphosphate (1) and dimethylallyl diphosphate (2). Until the beginning of the 1990s, it was believed that the mevalonate pathway (named after the central intermediate of the pathway and utilizing acetyl-CoA as a unique building block) was the only metabolic pathway used by all organisms for the biosynthesis of 1 and 2.4,5 The work of Rohmer6,7 and Arigoni,8 independently, set the stage for the discovery of a completely distinct pathway for the synthesis of 1 and 2, employing pyruvate (3) and D-glyceraldehyde 3phosphate (4) as building blocks (Scheme 1).9,10 This biosynthetic pathway was initially named non-mevalonate pathway, although the more recent literature uses the term 2C-methyl-D-erythritol 4-phosphate (MEP, 5) pathway, named after the first committed precursor 5. After the discovery of the MEP pathway, the taxonomic distribution of the two pathways has been extensively studied,11 which demonstrated that whereas the mevalonate pathway is used by humans, animals, © XXXX American Chemical Society

archaebacteria, and fungi, the MEP pathway is the sole source of 1 and 2 for green algae and numerous pathogenic bacteria and apicomplexan protozoa, including important human pathogens such as M. tuberculosis and Plasmodium falciparum, causative agents of tuberculosis and malaria, respectively.12−14 Interestingly, higher plants use both pathways with a clear compartmentalization: the biosynthesis of sterols and triterpenes occurs in the cytoplasm via the mevalonate pathway, whereas the MEP pathway is used in plastids to biosynthesize carotenoids and phytol.15 This distinct distribution among organisms, together with the fact that the MEP pathway is essential in several organisms (including P. falciparum,16−18 M. tuberculosis,19 Toxoplasma gondii, Pseudomonas aeruginosa, and other infectious-disease-causing organisms),14,20 has encouraged scientists to target the enzymes of the MEP pathway on the way to the development of novel anti-infective agents as well as novel and more potent herbicides.21 In particular, there is an urgent need for drugs with a novel mechanism of action for malaria and tuberculosis, which are responsible nowadays for the death of approximately two million people per year.22−24 In fact, most of the currently available drugs are not effective anymore because of the emergence of multidrugresistant and extensively-drug-resistant (MDR and XDR, respectively) strains.25 The fact that most of the intermediates of the MEP pathway are phosphorylated means that the corresponding active sites of the enzymes are particularly polar, Received: July 20, 2014

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dx.doi.org/10.1021/jm5010978 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Scheme 1. Synthesis of the Isoprenoid Precursors Isopentenyl Diphosphate (1) and Dimethylallyl Diphosphate (2) via the 2CMethyl-D-erythritol 4-Phosphate (MEP) Pathway

rendering the rational drug design of inhibitors for these enzymes very challenging. Nevertheless, we have recently reported a systematic druggability assessment of the enzymes of the MEP pathway with the program DogSiteScorer, showing that all the substrate- and cofactor-binding pockets are overall druggable, although the apolar amino acid ratio (AAAr), as a single feature, is below the average for druggable pockets in all cases.26 Moreover, the problem of the polarity of the substrateand cofactor-binding pockets could be overcome by targeting allosteric pockets. Potent allosteric inhibitors for Arabidopsis thaliana IspD have been reported by Witschel et al.,27 and we identified other potentially allosteric pockets for DXS, IspD, IspF, and IspG during our druggability assessment of the enzymes of the MEP pathway,26 which could be worth exploring. Unfortunately, a structure-based design of allosteric

inhibitors for the enzymes of the MEP pathway appears to be more challenging. For A. thaliana IspD, for instance, these pockets are often formed only upon binding of a suitable ligand. As a result, high-throughput screening (HTS) approaches are better suited for the identification of an initial allosteric hit, which can subsequently be rationally optimized. A rapidly growing number of (co)crystal structures of the seven enzymes of the MEP pathway have been deposited in the RCSB Protein Data Bank (PDB) over the past 2 decades for the constituent enzymes of the MEP pathway, including several structures from pathogenic bacterial and plasmodial enzymes.9,28 No such structures have been reported so far for DXS, IspG, and IspH, which are, in fact, the least studied among the enzymes of the MEP pathway, especially in terms of inhibitors reported in the literature. Not only the structural information but also the B



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Chart 1. Inhibitors 9−19 of 1-Deoxy-D-xylulose-5-phosphate Synthase (DXS)

progress made in terms of assay development29 and sitedirected mutagenesis studies30 have enhanced our understanding of the structure and function of these enzymes, greatly facilitating the identification of potent inhibitors. Very little is known regarding the mechanism of regulation of the MEP pathway, although a sound understanding of this aspect is essential when targeting its constituent enzymes. A recent in silico study of the MEP pathway in P. falciparum using an elaborated kinetic model highlighted how two main strategies can be used to develop inhibitors blocking isoprenoid biosynthesis at the MEP-pathway level. The first strategy consists of reducing the flux through the pathway by inhibiting the two enzymes having the highest flux-control coefficients (DXS and IspC). The second strategy consists of inhibiting the enzymes having the lowest flux-control coefficients (IspG and IspH), thereby causing the accumulation of certain metabolites leading to toxicity in the cell.31 Both types of enzyme targets proved to be valuable drug targets in the in silico study. The fact that DXS might control the flux at the starting point of the pathway had already been proposed by Brown et al.32 Several studies have also focused on the potential regulatory role of the bifunctional enzyme IspDF.33 Very recently, a detailed analysis reported by Guggisberg et al. revealed a fundamental regulatory role of the MEP pathway: the loss of function of a cytosolic sugar phosphatase, which phosphorylates the intermediates of glycolysis, causes increased levels of the metabolites of the MEP pathway, particularly at the IspC level. It was shown how this loss of function can be used as a strategy to overcome the inhibition of the enzymes of the MEP pathway. In fact,

dramatic effects on the emergence of fosmidomycin resistance in P. falciparum have been observed.34 In this review, we will provide an overview of the recent developments of inhibitors for the enzymes of the MEP pathway since the review of Hale et al. in early 2012, with a particular focus on their in vitro activity against enzymes from pathogenic organisms. 1-Deoxy-D-xylulose-5-phosphate Synthase (DXS). DXS catalyzes the first and rate-limiting step of the MEP pathway, which consists of the formation of 1-deoxy-D-xylulose 5-phosphate (DXP, 6) in a head-to-tail condensation of pyruvate (3) and glyceraldehyde 3-phosphate (4) and concomitant thiamine diphosphate (TDP)- and Mg2+-dependent decarboxylation.35 As shown in Scheme 1, 6 is an intermediate not only in isoprenoid biosynthesis but also in vitamin B1 (7)36 and vitamin B6 (8)37 biosynthesis, rendering the DXS-catalyzed reaction a crucial branch point in pathogen metabolism and therefore DXS a very attractive drug target. It is therefore surprising that DXS is the least studied among all the enzymes of the MEP pathway, with just two crystal structures deposited in the PDB of Deinococcus radiodurans and Escherichia coli DXS in complex with its cofactor TDP and Mg2+ (PDB codes 2O1X and 2O1S, respectively)38 and very few inhibitors reported in the literature.39−43 TDP binds tightly to DXS (D. radiodurans DXS, Kd = 0.114 μM; M. tuberculosis DXS, Kd = 3.1 μM) in a relatively buried pocket of the protein, and we found that the TDP-binding pocket is druggable, although possessing a remarkably low lipophilic character.26 We have recently shown how the diphosphate moiety of TDP C



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accounts for a good part of the binding energy.44 Targeting the TDP-binding pocket efficiently would require the use of efficient and druglike diphosphate mimics. In addition, the development of competitive inhibitors with respect to TDP has to take into account the fact that TDP is also the cofactor of several human enzymes (e.g., transketolase (TK)45 and pyruvate dehydrogenase (PDH)46) and that TDP-binding pockets are usually highly conserved.44 On the other hand, DXS has several features that distinguish it from other TDPdependent enzymes, such as its peculiar domain arrangement, with the TDP-binding site located within the same monomer,38 the particularly large active site,47 and the requirement for a ternary complex between TDP, 3, and 4 for the catalytic process to occur.48 In contrast, mammalian TDP-dependent enzymes bear the TDP-binding pocket at the dimer interface49 and follow classical ping-pong kinetics.50 These differences suggest that selective inhibition of DXS over mammalian TDPdependent enzymes should be possible, in particular when also targeting the substrate-binding pocket. Recently, it has been shown that DXS also has an important regulatory role for the whole MEP pathway.51 Interestingly, 1 and 2 were found to inhibit Populus trichocarpa DXS by competing with TDP (1, Ki = 65.4 μM; 2, Ki = 81 μM). Computational studies predict the binding modes of 1 and 2 to be similar to that of TDP, with the alkyl chain being essential for the appropriate orientation of their diphosphate groups. The fact that diphosphate alone does not show any inhibitory effect corroborates this assumption. Despite the absence of crystallographic information, the expression of pathogenic DXS from M. tuberculosis and Plasmodium vivax DXS has been reported.43,52 Although no structural information is available about the substrate-binding pocket, previously reported mutagenesis studies had identified which amino acid residues of DXS are involved in the substraterecognition process.38 Recently, the group of Freel Meyers reported the development of substrate-competitive inhibitors for DXS such as compounds 9 and 10 (Chart 1). Taking advantage of the previously discovered flexibility of DXS toward nonpolar acceptor substrates53 and considering that α-keto acids modified at the acyl position had been found to be poor alternative donor substrates for DXS,54 they designed a series of acetyl phosphonates as bisubstrate inhibitors, accommodating features from both 3 and unnatural acceptor substrates.42 These derivatives inhibit E. coli DXS in a competitive manner with respect to 3, with single-digit micromolar Ki values (9, Ki = 6.7 μM; 10, Ki = 5.6 μM), and as expected, increasing the length of the acyl groups results in a dramatic decrease of the inhibitory activity. Their mode of action presumably involves reaction with bound TDP to form a stable phosphonolactyl-TDP intermediate, which prevents the enzyme from catalyzing the decarboxylation step to yield 6. Interestingly, remarkable selectivity over mammalian TDP-dependent enzymes was also reported, in particular against porcine PDH and Saccharomyces cerevisiae TK. Butylacetyl phosphonate 10 (Chart 1) has singledigit micromolar inhibitory activity against Yersinia pestis (Ki = 7.5 μM), Salmonella enterica (Ki = 8.4 μM), and M. tuberculosis DXS (Ki = 4.0 μM) but exhibits weak antimicrobial activity against both Gram-negative and Gram-positive strains including E. coli and S. enterica, probably due to poor cellular uptake.43 The fact that the activity of DXS is rescued in the presence of 1deoxy-D-xylulose or thiamine, or under conditions of DXS overexpression, proves that the antimicrobial activity of 10 is a consequence of the specific inhibitory effect of 10 against DXS.

Despite its weak antimicrobial effects, 10 can act synergistically with established antimicrobial agents such as fosmidomycin (11, Figure 1) (reducing the flux of the MEP pathway at the

Figure 1. Major modifications on the chemical scaffolds of 11 and 20 to improve their potency against IspC in vitro and in vivo.

DXS and IspC level as well as inducing loss of IspF activity by rapidly depleting levels of 5) and ampicillin (influencing the ability of microbes to biosynthesize their own cell wall and therefore facilitating at the same time the intracellular uptake of 10). Drug synergism in the MEP pathway has already been reported55 and should be considered as a powerful strategy to prevent the emergence of drug resistance.56 Freel Meyers and co-workers have recently analyzed also the peculiar features of the DXS active site with respect to other TDP-dependent enzymes.47 They have shown that the particularly big size of the active site of DXS, which can accommodate also sterically demanding scaffolds, could be successfully exploited to design aromatic-substituted acetylphosphonates as selective inhibitors of DXS. Benzylacetylphosphonate (12) was shown to inhibit E. coli DXS with a Ki value of 10.4 μM and exhibits an increased selectivity (∼85-fold) with respect to 10 toward PDH. The herbicide ketoclomazone (13, Chart 1) is known to inhibit Chlamydomonas DXS with an IC50 value of 0.1 mM.40 A more recent study reports its ability to suppress the growth of Haemophilus inf luenzae with a minimum inhibitory concentration (MIC) of 12.5 μg/mL, demonstrating that 13 is able to inhibit H. inf luenzae DXS at low micromolar concentration (Ki = 23 μM) in a noncompetitive and mixed-type manner with respect to 3 and 4, respectively.57 Recently 14, a derivative of 13 produced by the hydrolysis of the latter, was reported to inhibit H. inf luenzae DXS with an IC50 value of 1.0 μM and shown to inhibit the growth of H. inf luenzae with a MIC value of 32 μg/mL.39 Addition of 1-deoxy-D-xylulose, used as exogenous nutrient of the MEP pathway and capable of rescuing the second step of the MEP pathway, led to a suppression of the inhibitory effect, showing that the antibacterial activity can be ascribed to inhibition of DXS. The absence of the hydroxyl group on the nitrogen atom, the dimethyl substitution, or the removal of the carbonyl group of the carboxylic acid of 13 resulted in a significant loss of the in vitro activity. Nevertheless, compound 15 (IC50 = 3.1 μM, H. inf luenzae DXS) showed a moderate MIC value (MIC = 64 μg/ mL), suggesting that cell permeation might be partially prevented by the negative charge present on 14. The fact that 13 exhibits stronger cell-based activity than 14 despite their comparable in vitro activity against H. inf luenzae DXS suggests that cell permeability might be an issue and that 13 might be a prodrug for the generation of 14 in situ. In 2013, BASF initiated an HTS-based program to identify additional inhibitors of the enzymes of the MEP pathway on the way to D



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Chart 2. Inhibitors 21−34 of 1-Deoxy-D-xylulose-5-phosphate Reductoisomerase (IspC) with Modified Hydroxamate Moieties

the development of new herbicides, using the plant enzymes from A. thaliana.58 In the HTS screening with A. thaliana DXS, two main clusters of compounds have been identified, corresponding to hydrazine derivatives and diphenyl oxazole scaffolds, with the best inhibitors being 16 and 17 for the two clusters, respectively (16, IC50 = 33 μM; 17, IC50 = 32 μM; Chart 1). The results were rather disappointing considering that 13 gave an IC50 of 80 nM in the same assay. Moreover, the absence of convincing structure−activity relationships (SARs) among the best hits did not allow for further rationalization of the results. In the context of a fragment-based design of inhibitors for DXS, we recently reported compound 18 to be a moderate inhibitor of D. radiodurans DXS (IC50 = 595 μM, Chart 1).44 The design of 18 was guided by an innovative NMR technique involving saturation-transfer difference (STD) NMR, transferNOE (trNOE), and INPHARMA, enabling validation of the binding mode of 18 in solution. 18 is about as potent as deazathiamine (19), which also lacks the diphosphate group of TDP. 18 has the potential to be grown toward the substratebinding pocket of DXS, where selectivity over human TDPdependent enzymes can be gained.

In our druggability assessment of DXS, we identified three other druggable pockets lying at the interface of two monomers of DXS. Despite being smaller than the TDP-binding pocket and particularly solvent-exposed, these three pockets might be worthwhile exploring for the development of potentially allosteric inhibitors, given their higher lipophilic character with respect to the TDP-binding pocket.26 1-Deoxy-D-xylulose-5-phosphate Reductoisomerase (IspC). IspC (Dxr) is the most studied among the enzymes of the MEP pathway,59 and it catalyzes the second (and first committed) step in the pathway, consisting of an intramolecular isomerization and reduction of the substrate 6 to afford 5 (Scheme 1).59,60 IspC uses NADPH as a hydride donor, and the presence of a divalent cation (Mg2+, Co2+, or Mn2+) is essential for its catalytic activity. IspC was shown to be essential for the survival of several pathogens like M. tuberculosis.19 Over 30 (co)crystal structures of IspC from several organisms have been reported, including those of important pathogens such as M. tuberculosis (e.g., PDB code 3ZHX)61 and P. falciparum (e.g., PDB codes 4KP762 and 3AU963). The overall structure of IspC consists of a homodimer, each monomer being divided into three domains. The N-terminal E



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moiety is fundamental for chelating the divalent cation present in the active site of IspC.74 The reverse hydroxamate analogues 21 and 22 (Chart 2) also show comparable inhibitory activity with respect to 11 and 20 against E. coli IspC.75 On the other hand, most hydroxamates may be rapidly hydrolyzed in vivo or degraded by glucuronidation or sulfation and they may suffer from poor pharmacokinetic and toxicological profiles.76 Furthermore, they have significant binding affinities for many metal cations such as Zn2+, Cu2+, and Ni2+.77 To overcome the limitations associated with the presence of the hydroxamate moiety, several modifications have been explored, where the hydroxamic group has been replaced, for example, by a hydroxyurea, a benzoxazolone, a benzoxazolethione, or a catechol moiety.78,79,82 Ortmann80 and Link81 reported interesting derivatives of hydroxamic acid, leading to compounds with double-digit nanomolar activity against P. falciparum IspC. Replacing the hydroxamate moiety with bulky heteroaryl moieties, aimed at both increasing the overall hydrophobicity and providing a proper chelating group, does not result in the expected strong inhibitory potency, probably because of the particularly narrow geometry of the metal-binding subpocket of the active site of IspC.82 Recently, a series of amide derivatives with the generic formula 23 were synthesized (Chart 2), featuring an aromatic ring bearing ortho-substituents, which are expected to participate in the chelation of the metal cation.83 An analogue bearing a methylsulfonyl group at the ortho-position of the phenyl ring was also synthesized, which should lead to deprotonation of the acidic N-H hydrogen atom at physiological pH, improving the interaction of the binder with the active-site metal ion. None of the compounds showed any inhibitory activity against E. coli and M. tuberculosis IspC (IC50 > 100 μM) or against P. falciparum K1 in human erythrocytes (IC50 > 64 μM). Despite clearly improving the hydrophobic character of the original scaffold 21, the poor flexibility around the amide bond might be responsible for the lack of binding and therefore inhibitory activity. It had already been shown that a cis geometry of the two oxygen atoms of the hydroxamate moiety is required for metal chelation to occur.63 When catechol derivatives 24 and 25 were tested against E. coli IspC, 25 (IC50 = 4.5 μM, Chart 2) was found to be a more potent inhibitor than 24 (IC50 = 24.8 μM), showing how important the position of the chelating atom is for the inhibitory activity.84 25 was also shown to have double-digit micromolar activity against M. tuberculosis IspC (IC50 = 41 μM), although it did not show any activity against M. tuberculosis.82 The group of Rohmer has recently explored hydrazides (26), dithiocarbamates (27, 28), and O-methylated hydroxamate moieties (29) as bidentate chelators and mimics of the hydroxamate group of varying chain length (Chart 2).85 Unfortunately, none of the derivatives synthesized were found to possess strong inhibitory activity against E. coli IspC. As for the hydrazides, the fact that the group is protonated at the pH of the enzymatic assay (pH 7.5) probably explains why they do not chelate Mg2+ cations. Dithiocarbamates are well-known metal-complexing compounds,86 but the soft-base character of the sulfur atoms might not allow efficient coordination of hard cations such as Mg2+. 29 is the only compound that resulted in weak inhibitory potency against E. coli IspC after preincubation, but its IC50 of 930 μM is still negligible with respect to that of 11 in the same assay (IC50 = 0.032 μM). In some recent studies

domain binds NADPH, while the central domain hosts the majority of the residues lining the active site. The C-terminal domain is believed to have mainly a structural role.64 A flexible loop is responsible for a substantial conformational change upon binding of 6 or other strong binders like 11. This rearrangement shields the active site from bulk solvent and was also observed for other isomerases, where a domain or a loop movement is required to protect a reactive intermediate from being solvent-exposed.60 Despite the abundance of crystallographic information about IspC from several organisms, the dramatic conformational change of IspC upon ligand binding renders the rational design of new inhibitors very challenging. Also, the rational optimization of the most successful and therefore most studied IspC inhibitor, 11, is a challenge, given its nature as a slowbinding inhibitor, which leads to strong conformational differences in IspC depending on the stage of the binding event.65 Owing to the extremely flexible active site of IspC, some derivatives of 11 were proposed to bind in a “reversed” fashion by docking studies, with the phosphonate moiety binding to the Mg2+ cation and the hydroxamate located in the phosphonate-binding site, rendering their rational optimization challenging.66 Our druggability assessment of IspC revealed the presence of a particularly druggable subpocket, consisting of the active site excluding the ADP-binding pocket, which could be exploited in order to enhance hydrophobic features of inhibitors of IspC.26 We also found that the adenosine-binding pocket of NADPH is druggable, although its small size and its low lipophilicity suggest that a bisubstrate inhibitor targeting this pocket and the substrate-binding pocket might result in a more efficient inhibition of IspC. Most of the IspC inhibitors developed so far are analogues of 11 or of its acetyl derivative FR-900098 (20) (Figure 1).59 Both 11 and 20 are natural products isolated from Streptomyces lavendulae and S. rubellomurinus, respectively.67,68 The use of 11 as a single-drug treatment for malaria suffers from several drawbacks including low bioavailability, rapid clearance from the parasite, a short serum half-life, and recrudescent infections.69 In fact, 11 is currently under clinical investigation in conjunction with piperaquine;70 the clinical trials of the fosmidomycin−clindamycin combination have been discontinued because of lack of efficacy in Mozambican children less than 3 years old.71 Moreover, 11 is a potent inhibitor of M. tuberculosis IspC in vitro but does not have any inhibitory activity in mycobacteria, presumably because of its inability to cross the mycobacterial cell wall. It was shown that M. tuberculosis lacks the glycerol 3-phosphate transporter GlpT, which is responsible for the mechanism of transport of polar compounds in other bacteria like E. coli and P. aeruginosa.72 Despite its particularly polar character, 11 is actively transported inside the cell in P. falciparum; the mechanism of transport, however, remains to be elucidated.73 The most recent efforts in terms of development of inhibitors for IspC are focused both on improving the bioavailability of 11 and 20 and on enhancing their lipophilic character to obtain whole-cell antimycobacterial activity (Figure 1). Over the past 3 years, numerous attempts to replace the hydroxamic moiety of 11 and 20 with more lipophilic hydroxamate bioisosteres possessing improved bioavailability have been reported.59 On the one hand, the ability of hydroxamates to chelate metal cations as bidentate ligands makes them very strong inhibitors of metalloenzymes. It was shown for 11 that its hydroxamate F



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Chart 3. Inhibitors 35−48 of IspC with Modified C3 Linker

tetrahedral alcohol functionality of 6 and would allow for further functionalization, which could lead to potential five- or six-membered chelating rings. Furthermore, the possibility to tune the hardness and softness parameters of the heteroatoms would allow for the identification of the best chelating group for the IspC cation. Nevertheless, the activity of this class of compounds against IspC has yet to be determined. Overall, identifying a suitable replacement for the hydroxamic moiety of 11 appears to be challenging because of difficulties in finding an efficient chelating group for the IspC cation. Among the recently reported scaffolds lacking a hydroxamate moiety, the catechol derivatives look the most promising (Chart 2). A very interesting scaffold (33), completely unrelated to that of 11, had been found by the group of Deng in 2009, displaying 1.4 μM activity against E. coli IspC.84 34, which also possesses a relatively new chemical scaffold, was found to have submicromolar activity against E. coli IspC (IC50 = 0.84 μM).66 These findings might suggest that exploiting chemical diversity could be an alternative strategy for the development of efficient inhibitors of IspC.

by the group of Kaye, the effects of the carboxamide Nsubstituents and the length of the carbon linker have been explored.87 In general, the N-arylcarboxamide derivatives bearing phosphonic acid salts showed better inhibition of E. coli and P. falciparum IspC than the corresponding phosphonate esters, whereas the opposite was observed in the case of N-heteroarylcarboxamide. The most successful inhibitors (e.g., 30, 45% inhibition at 250 μM, Chart 2) still show much weaker inhibitory potency than 11 (99% inhibition at 300 μM) for P. falciparum IspC. 31, despite displaying a disappointing 11% inhibition at 250 μM P. falciparum IspC, was found to inhibit the growth of the P. falciparum 3D7 strain (chloroquine sensitive but sulfadoxine resistant) and to be nontoxic against the human cell line Hst578T. Moreover, the SARs show that increasing the number of methylene groups in the spacer (particularly three and four methylene groups) results in a dramatic loss of inhibition, presumably due to steric constraints in the active site. Substituted phosphinic acids (e.g., 32, Chart 2)88 have also been proposed as replacement of the hydroxamic moiety of 11 and 20.89 The presence of a phosphinic acid would mimic the G



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Modifications at the level of the carbon chain of 11 might involve modification of the chain length as well as introduction of substituents at different positions on the linker (Chart 3). The ideal length of the carbon linker of 11 and 20 has been recently confirmed to be a C3 moiety. In fact, the inhibitory potency of M. tuberculosis IspC of derivatives of 11 and 20 bearing C2, C4, and C5 linkers drops dramatically.90 The presence of one double bond in the C3 linker (compound 35, IC50 = 1.07 μM) resulted in an improved inhibitory activity against M. tuberculosis IspC with respect to its parent compound 21 (IC50 = 2.39 μM). The C3 spacer of 11 and 20−22 is particularly suitable for derivatization. Attempts have been made to increase the inhibitory activity by introducing substituents at the α-carbon atom of the phoshonate moiety such as alkyl, hydroxymethyl, or bismethyl substituents with none of them showing improved inhibition of IspC.91 The incorporation of an aromatic substituent in the C3 spacer resulted in loss of activity.92 The introduction of other substituents resulting in α-halogenated derivatives,93 or α-arylmethyl-substituted derivatives, afforded submicromolar activity against various strains of P. falciparum.94 In particular, several studies have shown that the introduction of phenyl residues with different substitution patterns at the α position of the reverse derivatives 21 or 22 can significantly increase their inhibitory potency of P. falciparum IspC. For example, 36 was found to possess single-digit nanomolar inhibition of P. falciparum IspC (IC50 = 3.1 nM), compared to the parent compound 20 displaying an IC50 of 15 nM.95 Similar derivatives also afforded single-digit nanomolar inhibition of P. falciparum IspC (e.g., 37, IC50 = 3.0 nM) as well as potent inhibitory activity against the MDR K1 strain of P. falciparum, about 10-fold higher than the one of 11 and no cytotoxicity on human MRC-5 cells.96 On the other hand, the dichloro derivative 38, although possessing submicromolar activity against M. tuberculosis IspC, does not show any activity on M. tuberculosis whole cells.97 It has also been shown that the replacement of the βmethylene group of 11 or 20 by oxygen results in inhibitors that are as potent as 21 or 22 against P. falciparum IspC (e.g., 39) but have superior activity against the P. falciparum 3D7 strain compared to 11 or 20.98 In a recent study, the group of Kurz attempted the combination of the above-mentioned structural modifications, but the resulting derivatives were found to be less potent against P. falciparum IspC in vitro than 36 and 37 while possessing potent in vitro antiplasmodial activity both against a chloroquinone-resistant strain and against a chloroquinone-sensitive strain of P. falciparum.99 To elucidate the effect of a given atom at the β-position, reverse thia analogues of 11 and 20 have been synthesized and tested (e.g., 40), which show increased inhibitory activity with respect to the carba and the oxa analogues (e.g., 41, 38) of M. tuberculosis IspC but decreased inhibition of P. falciparum IspC.62 Nevertheless, some of the thia analogues display double-digit micromolar activity against various strains of P. falciparum. The involvement of the sulfur atom in a favorable hydrophobic interaction with a strictly conserved methionine residue is probably the reason why, in general, the thia analogues are more potent than the oxa analogues, where the oxygen atoms suffer from repulsive interactions with the sulfur atom of the methionine residue. Crystallization studies show that P. falciparum IspC selectively binds the S-enantiomer of these derivatives (Figure 2). The apparent IC50 values for the (+)- and (−)-enantiomers of 42 are 9.4 nM and 12 μM,

Figure 2. Cartoon representation of Plasmodium falciparum IspC (key amino acid residues shown as sticks) in complex with S-(+)-42 and NADPH (PDB code 4KP7).62 Color code is as follows. Protein skeleton: C, gray. Ligand S-(+)-42 skeleton: C, cyan; NADPH skeleton, pink; O, red; N, blue; S, yellow; P, orange. Mn2+ cation is shown as a yellow sphere. Water molecules involved in hydrogen bonds with S-(+)-42 are shown as red spheres. Hydrogen bonds below 3.1 Å are shown as dashed lines. Figures were generated using the software PyMOL.100

respectively, where the active enantiomer can be unequivocally assigned as S-(+)-42, given the results of the crystallographic studies. 62 These results suggest that development of enantiopure inhibitors for IspC might lead to dramatically enchanced inhibitory potency. Inspired by the above-mentioned results with inhibitors of IspC containing pyridine moieties (e.g., 34),66 the group of Song synthesized and tested several derivatives of 11 and 20, bearing pyridines at the α-position of the phosphonate group, which were found to exhibit up to 11-fold enhanced inhibitory activity against P. falciparum IspC with respect to 11 (e.g., 43, Ki = 1.9 nM, Chart 3).101 Previously reported results had shown, in fact, the importance of the presence of an electrondeficient aromatic ring at the α-position of the phosphonate group.102 Expectedly, pyridine derivatives such as 43 showed considerably improved antimalarial activity with respect to 11 against the 3D7 strain (sensitive to chloroquine) and the Dd2 strain (resistant to chloroquine, pyrimethamine, and mefloquine) of P. falciparum and were found not to be cytotoxic. Cocrystallization experiments revealed the binding mode of these scaffolds to be similar to that of 11. The pyridine moiety is hosted in a rather hydrophobic cavity formed by the flexible loop, where the pyridine nitrogen atom is also favorably engaged in a hydrogen bond with the protein. When the pyridine ring is replaced with other nitrogenbearing substituents such as azides (44), amides, or substituted H



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overcome inhibition of the constituents enzymes),34 the development of antimalarials, which are able to cross the cell wall without need for a transporter, would overcome the resistance problem.109 49 is able to inhibit the growth of Mycobacterium smegmatis (MIC = 250−500 μM), although less efficiently than the antituberculotic drug isoniazid (MIC = 10−20 μM) (Chart 4).

triazoles (e.g., 45) at the same position (Chart 3), the inhibitory activity against E. coli IspC decreases. Given that no information is available about the in vitro activity against P. falciparum IspC, a close comparison with the pyridine derivatives discussed above is not possible; their single-digit micromolar inhibition against P. falciparum K1 in human erythrocytes is, however, very encouraging. Lately, a new approach has been exploited, aimed at targeting both the substrate- and the cofactor-binding sites of IspC.61 One of the best compounds of the series, 46 (Chart 3), has an IC50 of 0.32 μM against M. tuberculosis IspC, which is 2-fold higher than that of the corresponding compound with no substitution at the hydroxamate position (47, IC50 of 0.15 μM). Therefore, the substituent at the hydroxamic position apparently does not contribute to the inhibitory activity. None of the derivatives reported in this study were able to inhibit the growth of the M. tuberculosis strain H37Ry, but they did show excellent inhibitory activity of P. falciparum growth with IC50 value in the double-digit nanomolar range; in particular, 46 has a much higher inhibitory potency (IC50 = 0.04 μM) than 11, 20, and 47. Other bisubstrate inhibitors for IspC have been designed by the group of Dowd, who have introduced both aromatic and aliphatic rings either at the amide or at the hydroxamate oxygen atom, to test them as mimics of the nicotinamide ring of NADPH.103 The best result was obtained for 48 (Chart 3), which inhibits M. tuberculosis IspC with an IC50 of 17.8 μM in a competitive manner with respect to DXP but in a noncompetitive manner with respect to NADPH, in disagreement with its modeled binding mode. Although the in vitro activity of 48 with respect to 11 and 20 decreases, the cell-based activity against the M. tuberculosis strain H37Rv of its diethyl ester is comparable to that of the diethyl ester of 11, probably owing to its increased lipophilic character. Other attempts to address the NADPH-binding pocket with acyl-substituted analogues of 11 bearing aliphatic or (hetero)aromatic groups did not result in an improved in vitro activity against M. tuberculosis IspC with respect to 11.61 Modifications at the phosphonate moiety of 11 and 20−22 have followed two main approaches. On the one hand, the replacement of the phosphonate group of 21 and 22 with isosteric groups such as a carboxylate or a sulfonate resulted in a drastically decreased inhibitory activity against E. coli IspC.104 The phosphate derivatives of 11 and 20 have higher inhibitory potency against Synechocystis IspC than 11 but, being prone to hydrolysis, do not show any activity in vivo.105 On the other hand, in order to develop analogues of 11 with potent mycobacterial activity, the strategy of masking the polar phosphonate moiety with hydrophobic groups has recently been exploited. These prodrugs would be able to cross the very apolar mycobacterial cell wall before being hydrolyzed by an endogenous esterase, releasing a hemiketal, which would spontaneously break down into formaldehyde and the active form of the inhibitor.106 In addition, there is also the need to increase the bioavailability of 11 and 20 as antimalarials. Considering that the ionization of their phosphonate groups is believed to contribute to their low bioavailability, the use of their lipophilic esters could help to overcome this problem.107,108 Given that a glpT mutation is one of the few reported paths toward resistance against 11 (together with the recently discovered loss of function of a cytosolic sugar phosphatase, which is able to increase the levels of the metabolites of the MEP pathway to

Chart 4. Inhibitors 49−52 of IspC with Modified Phosphate Groups

Surprisingly, no effect was observed when testing the corresponding non-N-methylated prodrug.110 Also, the corresponding phosphonate esters of 11 and 20 do not show any inhibitory activity. 50 and 51 showed improved antituberculotic activities with MIC values of 50−100 and 25−100 μg/mL, respectively (Chart 4).111 Interestingly, the corresponding derivatives of 50 and 51 bearing a secondary ester functionality do not show any activity. This fact could be explained by a lack of substrate recognition in the case of branched esters by the esterases responsible for their intracellular hydrolysis. More functionalized lipophilic ester prodrugs (e.g., 52, Chart 4) have been recently reported by Kurz and co-workers displaying significantly better IC50 values than 11 in P. falciparum growth assays including a drug-resistant strain of P. falciparum (52, IC50 = 0.022 μM; 11, IC50 = 0.81 μM against P. falciparum Dd2).99 The antimalarial activity of these reversed derivatives is also better compared to the previously reported pivaloyloxymethyl ester analogues of 11 and 20.112,113 Further derivatization at the hydroxamic nitrogen atom of 52 does not lead to an improved inhibitory potency. 4-Diphosphocytidyl-2C-methyl-D-erythritol Synthase (IspD). The third enzyme of the MEP pathway, IspD, catalyzes the formation of 4-diphosphocytidyl-2C-methyl-D-erythritol (53) by transferring a diphosphocytidyl unit from cytidine triphosphate (CTP) to 5 (Scheme 1). The catalytic process requires Mg2+, which has been shown by kinetic studies with E. coli IspD, to coordinate to the enzyme first, followed by CTP and 5.30 Several (co)crystal structures of bacterial IspD have been reported,114 including some from mycobacteria. In I



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Chart 5. Inhibitors 54−60 of 4-Diphosphocytidyl-2C-methyl-D-erythritol Synthase (IspD)

particular, the apo structure of M. tuberculosis IspD (PDB code 3OKR)115 and crystal structures of M. tuberculosis IspD in complex with CTP and Mg2+ (PDB code 2XWN)116 as well as with 53 (PDB code 3Q80)117 have been deposited in the PDB. This is particularly interesting given that the IspD-encoding gene, ispD, was shown to be essential for M. tuberculosis growth.118 The structure of IspD consists of a homodimer, the formation of which is promoted by a long β-arm, extending from the main globular domain having an α/β fold. The active site is highly conserved among several species (bacteria and plants), and it is mainly formed by amino acid residues belonging to the main domain, although some residues of the β-arm are also involved. As a result, the active site is located at the dimer interface, making it rather solvent-exposed. Moreover, some degree of flexibility was observed in the cytosinebinding site of IspD when comparing the structures of the apoand the CTP-bound form of M. tuberculosis IspD. P. falciparum IspD shows remarkable differences in the amino acid sequence with respect to other bacterial IspD, being more than 3 times larger than its corresponding bacterial homologues.116 A region of the amino acid sequence of P. falciparum IspD, close to the C-terminus and similar in length to other IspD orthologues, shows 19% and 16% sequence identity to E. coli IspD and to M. smegmatis and M. tuberculosis IspD, respectively. Nevertheless, out of the 22 amino acid residues that are strictly conserved in most bacterial species,119 only 17 are conserved in P. falciparum IspD. The β-arm of P. falciparum IspD is extended by nine amino acids, but other

motifs of the sequence defining conformational features of the protein are mostly conserved. The presence of a particularly polar region lying in the middle of the sequence could suggest that P. falciparum IspD is formed by two domains connected by this polar linker. We showed that IspD possess the least lipophilic active site among all the enzymes of the MEP pathway.26 This feature, together with the fact that the CTP-binding pocket is rather solvent-exposed, renders the development of substratecompetitive inhibitors for IspD very challenging and suggests that targeting allosteric sites would lead to more druglike, potent inhibitors for IspD. On the one hand, 54 (Chart 5), the only substrate-competitive inhibitor for IspD reported so far, bears a phosphate moiety and displays very weak inhibition of E. coli IspD (IC50 = 1.36 mM).120 On the other hand, potent allosteric inhibitors have been discovered with a HTS approach as novel herbicides by Witschel et al. with inhibitory activity in the nanomolar range against A. thaliana IspD (e.g., 55 IC50 = 140 nM and 56 IC50 = 35 nM against A. thaliana IspD, Chart 5) and very potent herbicidal activity.27 Recently, by screening a library of 100 000 compounds using a photometrically monitored assay, Kunfermann et al. discovered halogenated marine alkaloids of the pseudilin-type having potent inhibitory activity against A. thaliana IspD (e.g., 57, Chart 5).121 Interestingly, the presence of a divalent metal cation, in particular Cd2+, resulted in a 7-fold increase of the activity. Other divalent metal cations such as Cu2+ and Zn2+ also enhance the inhibitory potency. The fundamental role of a J



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homologues cannot be extrapolated for plasmodial homologues. Another class of compounds was identified in the HTS-based program initiated by BASF toward new herbicidals, consisting of acetylated aminobenzothiazoles such as 58, displaying an IC50 of 1.8 μM against A. thaliana IspD (Chart 5). Nevertheless, the binding mode of these scaffolds was not further investigated, and so far, their activity has not been optimized.58 A series of virtual hits, designed so as to occupy the active site of M. tuberculosis IspD, has been reported by Varikoti et al. bearing a central aminopyrimidine scaffold decorated with different substituents at the 2 and 6 positions of the pyrimidine ring and at the amino group (e.g., 59, Chart 5). The compounds were selected based on their ADMET properties and synthetic accessibility using different software, but their in vitro activity against M. tuberculosis IspD has yet to be tested.123 Domiphen bromide (DMB, 60) was identified by a HTS approach and inhibits M. smegmatis IspD with an IC50 of 33 μg/ mL (Chart 5).124 Besides its activity against IspD, DMB was found to inhibit the growth of M. smegmatis also by affecting IspD-independent pathways and in addition, it displays comparable MIC values with respect to isoniazide and rifampicin against MDR and XDR strains of tuberculosis. Further studies are necessary to elucidate the mechanism of action of DMB. 4-Diphosphocytidyl-2C-methyl-D-erythritol Kinase (IspE). IspE is an ATP- and Mg2+-dependent kinase that catalyzes the transfer of the γ-phosphoryl group from ATP to the 2-hydroxyl group of 53, affording 4-diphosphocytidyl-2Cmethyl-D-erythritol 2-phosphate (61, Scheme 1). To date, 18 (co)crystal structures of IspE have been deposited in the PBD, including some structures of M. tuberculosis IspE such as its apo form (PDB code 3PYD) and cocrystal structures of the enzyme with 53 (PDB code 3PYE), the nonhydrolyzable ATP analogue AMP-PNP (PDB code 3PYF) and ADP (PDB code: 3PYG).125 On the one hand, IspE was shown to be essential for M. smegmatis, and its absence in mammalian cells makes it an attractive drug target.126 On the other hand, IspE belongs to the galactose/homoserine/mevalonate/phosphomevalonate (GHMP) kinase superfamily, named after several mammalian proteins (galactose kinase, homoserine kinase, mevalonate kinase, and phosphomevalonate kinase), and exhibits a high degree of similarity (in terms of sequence alignment and structural features) with these ATP-dependent proteins,127 which could cause selectivity problems when targeting IspE.128 Nevertheless, significant differences between the active site of IspE and that of the kinases of the GHMP superfamily exist, suggesting that IspE could be targeted selectively.129,130 There is a high level of sequence identity among different IspE orthologues (30−38% identity between M. tuberculosis IspE and Thermus thermophilus, E. coli and Aquifex aeolicus IspE) as well as high overall structural similarity. IspE was characterized as a monomer in solution,130 and it displays the characteristic twodomain fold of the GHMP kinase superfamily, consisting of an ATP- and a substrate-binding domain.131 The active site is enclosed in a deep cleft between two domains. The N-terminal domain is highly conserved and folds into a phosphate-binding loop that accommodates the nucleotide in an unusual syn conformation. The substrate is bound to the C-terminal domain, with the cytosine moiety of 53 deeply buried in the cavity. A comparison of the apo-structure of M. tuberculosis IspE with the substrate-bound M. tuberculosis IspE shows that the

divalent cation for the inhibitory activity was confirmed when solving the cocrystal structure of 57 with A. thaliana IspD (PDB code 4NAK, Figure 3).121 57 binds in the same allosteric

Figure 3. Cartoon representation of Arabidopsis thaliana IspD (key amino acid residues shown as sticks) in complex with the allosteric inhibitor 57 (PDB code 4NAK).121 Color code is as follows. Ligand 57 skeleton: C, yellow; O, red; N, blue; Br, cyan. Cd2+ cation is represented as an orange sphere. A water molecule coordinating Cd2+ is represented as a red sphere. Hydrogen bonds below 3.3 Å are shown as dashed lines. The halogen bond between a bromine atom of 57 and the protein is shown as a blue dashed line.

pocket as the previously described azolopyrimidine derivatives;27 the pocket is located in proximity of the active site within only one monomer, and Cd 2+ is tetrahedrally coordinated by the phenolic hydroxyl group, the pyrrole nitrogen atom of 57, a water molecule, and the Gln238 side chain of A. thaliana IspD. The remarkable plasticity of this pocket, formed just upon binding of small molecules and not present in the apo form of IspD, allows for accommodation of very different scaffolds, like 55 and 57, and has dramatic consequences on the conformation of the active site of IspD, locking the catalytic active site and therefore preventing the binding of CTP. The halogenated pseudilin derivatives were also tested against P. vivax IspD and were found to be slightly less potent (e.g., 57 IC50 = 48 μM). The fact that 56 was found to be highly cytotoxic against mammalian cells together with its ability to inhibit the P. falciparum blood stages with EC50 value of 1.3 μM suggests that targets other than IspD are most probably involved in the inhibitory activity of 57. Pseudilin-type inhibitors are, in fact, known to have other possible biological targets.122 Also, no information about the binding mode of 57 within P. vivax IspD is known. The authors consider the possibility that the allosteric pocket observed for A. thaliana IspD might not be present or might be less flexible in other IspD orthologues. This could justify the lower inhibitory potency of 57 against P. vivax IspD, considering also the abovementioned sequence dissimilarities between P. falciparum IspD and its bacterial orthologues. Plasmodial IspD as an antimalarial target would therefore need a dedicated approach, given that inhibitory or crystallographic results obtained on other IspD K



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Chart 6. Inhibitors 62−78 of 4-Diphosphocytidyl-2C-methyl-D-erythritol Kinase (IspE)

number of first-generation inhibitors of E. coli IspE successfully targeting just the substrate-binding pocket and exploiting a lipophilic pocket lying in proximity of the cytidine diphosphate (CDP) binding site (the hydrophobic cleft is lined by Val57, Val60, Leu66, Ile67, Lys96, Met100, and Phe185 in E. coli IspE).132,133 The best inhibitor, 62 (Chart 6), displays a Ki in the upper nanomolar range against E. coli IspE, with the cyclopropyl ring of 62 efficiently filling the above-mentioned lipophilic pocket. The lipophilic pocket identified by Diederich and co-workers in E. coli IspE is highly conserved also in M.

active site is stabilized by a network of water-mediated interactions upon substrate binding, while the loop present in this part of the pocket is more flexible in apo-IspE. Our druggability assessment of M. tuberculosis IspE in complex with 53 confirmed the possibility of successfully targeting the ATP- and the ME-binding pockets of 53, although their low lipophilic character and solvent exposure might render it challenging.26 Moreover, targeting the ATP-binding pocket could lead to selectivity issues over human kinases. To overcome this problem, Diederich and co-workers reported a L



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the problem of the flexibility and address the phosphatebinding region of ATP in a more direct and preorganized manner, several benzimidazole derivatives lacking the methylene linker were also synthesized and tested, which do not show an improved inhibitory potency with respect to the imidazole derivative 68. Efforts to develop bisubstrate inhibitors targeting both the substrate- and the ATP-binding pockets of IspE have been reported in 2010 by Börner and co-workers, where 69 (Chart 6) showed an IC50 of 8.7 μM against E. coli IspE when tested with a tailor-made poly(ethylene oxide)−peptide carrier.137 Because of its particularly rigid structure and its pronounced hydrophobicity, 69 is, in fact, not water-soluble and the presence of the carrier (which was shown not to affect the biological activity of 69) allowed its solubilization in water. On the way to get structural diversity with respect to the cytidine-derived inhibitors and to the cytosine scaffolds discussed above, Tang et al. tested existing small-molecule inhibitors of GHMP kinases,138 which were also shown to cross-inhibit E. coli IspE.139 Two novel druglike scaffolds were identified (70 and 71, Chart 6), with double- and single-digit micromolar inhibitory potency against E. coli IspE. Their binding mode was predicted to be in the binding pocket of 53 within E. coli IspE, where their extensive aromaticity allows for several π−π interactions with the cytidine-binding subpocket, although the hydrophobic subpocket is just partially filled. Remarkably, no significant inhibition of human galactokinase 1 (GALK1) was observed, pointing out that selectivity among GHMP kinase inhibitors can be fulfilled. Interestingly, 70 and 71 were also tested against the orthologue Y. pestis IspE and showed comparable or improved inhibitory activity. 70 was also shown to weakly inhibit E. coli bacterial growth in culture. In the same study, computational HTS of over two million druglike compounds directed against the active site of IspE yielded an additional novel scaffold, consisting of the tetrahydro-1,3,5-triazine. In particular, compound 72 (Chart 6) was able to inhibit 80% of the activity of E. coli IspE at 20 μM. Several derivatives of 70 and 71 were also tested, but the SARs were not consistent with the predicted binding mode and cocrystallization studies would be necessary to confirm the binding mode and further optimize the hits rationally. Non-substrate-like inhibitors with completely new chemical scaffolds were also found by Tidten-Luksch et al. through a combination of in silico and in vitro screening against A. thaliana IspE targeting the substrate- and both the substrateand the cofactor-binding sites of IspE, respectively.140 The best three compounds (73, 74, and 75) are shown in Chart 6 and possess inhibitory activity from three- to singledigit micromolar against E. coli IspE. 73 displays good druglike properties and a very good ligand efficiency (0.50 kcal/mol), and it is a very promising scaffold for a fragment-to-lead project. A few derivatives of 73, 74, and 75 were also tested, but they do not show any substantial improvement of the inhibitory potency. Nevertheless, SARs are consistent with the putative binding mode, where the molecules bind in the cytidinebinding pocket of IspE, forming favorable π−π stacking interactions with two tyrosine residues. Further structural diversity was accomplished in the context of the HTS program of BASF in search of new herbicides.58 Isoindoline 76 (Chart 6) displays single-digit micromolar inhibition of tomato IspE (IC50 = 7.2 μM), and its binding mode was postulated to mimic the cytosine moiety of 53. 76

tuberculosis and P. falciparum IspE, suggesting that these scaffolds could have high inhibitory potency also against plasmodial and mycobacterial enzymes. Nevertheless, the replacement of a phenylalanine residue in E. coli IspE (Phe185) with a tyrosine residue in both M. tuberculosis IspE (Tyr185) and P. falciparum IspE (Tyr257) decreases the hydrophobic character of the pocket. The propargylic sulfonamide moiety provides an excellent vector for favorably addressing the tyrosine residue while interacting with the highly conserved, catalytically essential residues Lys9 and Asp130 (A. aeolicus IspE). In fact, several derivatives (e.g., 63, Chart 6) were recently synthesized where the cyclopropyl moiety was replaced by more polar substituents that should interact with the hydroxyl group of tyrosine by hydrogen-bonding.134 The inhibitory activity of 63 was tested against A. aeolicus IspE (where the phenylalanine of the hydrophobic pocket is also replaced by a tyrosine (Tyr175) like in the pathogenic bacterial enzymes), but surprisingly, no activity was detected below 500 μM, probably because of the very energetically demanding replacement of a water cluster. To overcome this problem, sugar derivatives such as 64 (Chart 6) were synthesized, which were expected to replace a large number of the water molecules of the cluster and form a complete network of hydrogenbonding interactions with the protein. Unfortunately, 64 showed weak or no activity against E. coli and A. aeolicus IspE, respectively. Also, removing the propargyl linker (compound 65, Chart 6) resulted in a dramatic drop of the inhibitory potency with respect to the initial scaffold 62. Schütz et al. synthesized several derivatives of 62 with different substituents at the N1 position of the cytosine ring so as to improve its water solubility and, at the same time, optimize the interactions with the ribose subpocket of the binding site of 53, to improve the inhibitory potency.135 Unfortunately, while there was a clear improvement in the water solubility that could also help the cocrystallization experiments, the inhibitory activity of all the synthesized compounds does not improve with respect to 62. The fact that 66 (Chart 6) gives a comparable IC50 with respect to 62 suggests that the local dipoles induced by the oxygen or sulfur atoms are responsible for the binding affinity rather than an S···π interaction. Other derivatives were prepared, where the cytosine ring of 62 was replaced by a 2-aminopyridine ring (e.g., 67, Chart 6), bearing aryl moieties to fill the ribose subpocket. In all cases, the inhibitory potency dropped dramatically (67, IC50 = 242 μM against E. coli IspE) suggesting that the ribose subpocket of E. coli IspE is best filled by cycloalkyl substituents. The scaffold of 62, the best inhibitor among all the inhibitors of IspE discussed so far, was used as a starting point to explore the phosphate-binding region of the ATP-binding site of IspE. This neutral glycine-rich loop was addressed using imidazole and benzimidazole moieties featuring different exit vectors.136 The best compound of the series was found to be 68 (Chart 6), where the cyclohexyl ring fits the small hydrophobic pocket of E. coli IspE mentioned above, while the trifluoromethyl substituent, used as phosphate surrogate, can undergo favorable orthogonal dipolar interactions. The fact that 68 still does not show an improved inhibitory potency with respect to 62 could be justified by the flexibility of the carbon linker, which might allow the phosphate surrogate to be oriented toward bulk water rather than binding to the loop. Other derivatives bearing different phosphate isosteres at this position, such as triazole, tetrazole, and nitrile, are all less potent than 68. To overcome M



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Chart 7. Inhibitors 80−83 of 2C-Methyl-D-erythritol 2,4-Cyclodiphosphate Synthase (IspF)

as well as the binary complex of P. falciparum IspF with CDP (PDB codes 4C81 and 4C82, respectively).151 IspF forms bellshaped homotrimers, where the active sites are located at the interface of two monomers. In particular, the pocket involved in binding the phosphate moiety of the substrate is capped with a flexible loop that becomes completely ordered when the reaction product is bound. However, the structure of the rest of the substrate-binding pocket hardly changes upon substratebinding.144 A hydrophobic cavity is found at the core of the IspF trimer.152 Crystallographic studies145 and mass spectrometry experiments153 revealed the presence of downstream isoprenoid products such as 1, 2, geranyl diphosphate (GDP), and farnesyl dihposphate (FDP) in this pocket, suggesting that this cavity in IspF might be involved in feedback regulation of the MEP pathway. Nevertheless, as we will discuss below, no direct inhibition of E. coli IspF by 1, 2, GDP, or FDP has been observed.154 During our druggability assessment of IspF with DoGSiteScorer, we found this pocket to be druggable,26 and given its presumed role in regulating the MEP pathway, it could be particularly interesting to explore for the development of inhibitors for IspF. There is a high degree of sequence and structural similarity among most IspF orthologues. The key residues in the active site, which coordinate Zn2+, are strictly conserved, as well as the key interactions and structural features involved in the substrate specificity and reaction mechanism. This is particularly interesting given that most attempts of crystallization of small-molecule ligands in complex with IspF from pathogenic organisms such as P. falciparum have failed so far151 and suggests that any structural model of IspF might be suitable to guide a structure-based design or optimization project. From our previously reported druggability assessment of the enzymes of the MEP pathway, the active site of IspF emerged as the

also showed moderate bleaching herbicidal properties in the Lemna minor test system. When tested in a P. falciparum cellbased assay, most of the isoindolines identified in the context of the HTS program showed excellent activity, with 77 being the best inhibitor of P. falciparum proliferation in red blood cells (IC50 = 58 nM against P. falciparum NF54, Chart 6).141 The synthesis of a variety of derivatives allowed for a remarkable improvement of the antiplasmodial activity of 77, with the best compound 78 (Chart 6) having an IC50 of 18 nM against P. falciparum NF54. However, none of the tested derivatives showed any inhibitory activity against P. falciparum IspE in vitro below 100 μM, suggesting that the biochemical target associated with the potent antimalarial activity in the cellbased assay might not be related to the inhibition of IspE. The fact that some of the active compounds in the cell-based P. falciparum assay also display strong growth inhibition of rat skeletal myoblast (L-6) cells suggests that there might be a correlation between these effects. 2C-Methyl-D-erythritol 2,4-Cyclodiphosphate Synthase (IspF). The conversion of 61 into the cyclic product 2C-methyl-D-erythritol 2,4-cyclodiphosphate (79) is catalyzed by IspF and occurs through nucleophilic attack of the 2phosphate of 61 on the β-phosphate of the same substrate, leading to intramolecular cyclization and release of cytidine monophosphate (CMP) (Scheme 1).142,143 Mn2+ and Zn2+ cations are required for catalysis, promoting the proper intramolecular positioning and stabilization of the developing charge in the pentavalent transition state.144,145 More than 50 (co)crystal structures of IspF from different organisms have been deposited in the PDB, most of them from strains of E. coli,144,145 but also from H. inf luenzae,146 Salmonella typhimurium,147 and T. thermophilus,148 among others, and IspF was shown to be essential in pathogenic organisms.149 The binary complex of M. smegmatis IspF with CDP has been reported (PDB code 2UZH)150 and, very recently, also the apo-structure N



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most druggable among the enzymes of the MEP pathway, with a particularly high AAAr. Despite extensive studies of the structural and mechanistic features of IspF, few inhibitors for this enzyme have been reported in the literature so far, with the most successful ones being the non-cytidine-like thiazolopyrimidines derivatives reported by Diederich and co-workers in 2010,155 having single-digit micromolar activity against both P. falciparum and M. tuberculosis IspF (e.g., 80, IC50 = 9.6 μM against P. falciparum IspF; IC50 = 6.1 μM against M. tuberculosis IspF; Chart 7). No information about their binding mode is available, and their optimization has not been reported to date. A series of multiple ligand-bound structures of Burkolderia pseudomallei IspF were reported in 2011 by Begley et al.156 The fragment-like hits were identified in a fragment-based screening with B. pseudomallei IspF using NMR spectroscopy, and their binding modes were validated by X-ray crystallography, showing that different fragments could occupy the cytosinebinding pocket, the Zn2+-binding site, and a previously unknown binding surface, outside the active site. In a more recent study, the fragment-like hits have been chemically linked to develop “fusion” ligands targeting at the same time the cytidine- and the Zn2+-binding pockets of IspF, and 81 was found to possess binding affinity equal to that of cytidine diphosphate (81, Kd = 70 μM; cytidine diphosphate, Kd = 75 μM; Chart 7), despite lacking the metal-coordinating diphosphate group. The cocrystal structure of 81 with B. pseudomallei IspF (PDB code 3Q8H, Figure 4) shows that there

A better understanding of the mechanism of regulation of the MEP pathway could have important implications for the efficient development of potent inhibitors for the enzymes of the MEP pathway. However, so far, little is known about its regulation.158 Interestingly, IspF was recently shown to play a crucial role in the regulation of the MEP pathway.154 The activity of E. coli IspF was shown to be particularly enhanced and sustained by 5, the first committed intermediate of the MEP pathway, formed by the IspC-catalyzed reaction. In particular, the methylerythritol (ME) scaffold itself, unique to this pathway, drives the activation and stabilization of active IspF. The feed-forward mechanism to maximize the production of 79 confirms that sustained levels of 79 are required for the synthesis of other essential metabolites, besides isoprenoids.159 The fact that 5 may act as a regulator of the levels of 79 in pathogenesis underlines that inhibitor combinations, effectively inhibiting both the upstream enzyme IspC and the downstream IspF, could result in a potent anti-infective action. Feedback inhibition of the IspF−5 complex by the downstream isoprenoid FDP was also observed, while FDP does not inhibit IspF itself, suggesting that the IspF−5 complex may be the physiologically relevant form of the enzyme. Further attempts to design substrate analogues as inhibitors of IspF failed.160 On the one hand, 82 (Chart 7), the closest structural analogue to the substrate of IspF 61, bearing a druglike, stable bisphosphonate group unable to undergo cyclization to release CMP, does not show any inhibition of E. coli IspF but on the contrary, weak, time-dependent inhibition (37% inhibition at 500 μM after 30 min of incubation) of the E. coli IspF−5 complex. On the other hand, 83 (Chart 7), lacking the cytidyl moiety but retaining the ME component (which is also recognized by IspF) and bearing a druglike bisphosphonate group were found to enhance and sustain E. coli IspF activity. Notably, 83 did not have any modulating effect on the IspF−5 complex. These results provide further evidence that IspF and its complex with 5 respond differently to the presence of potential modulators. An enhanced understanding of the mechanism and physiological relevance of the activation of IspF by 5 is fundamental for the development of effective inhibitors of IspF; 5 might, in fact, induce a conformational change of IspF that alters its susceptibility to inhibitors. It has already been shown, for example, that the inhibitory activity of CDP against IspF diminishes in the presence of 5.154 2C-Methyl-D-erythritol 2,4-Cyclodiphosphate Reductase (IspG) and 1-Hydroxy-2-methyl-2-(E)-butenyl-4diphosphate Reductase (IspH). The last two steps of the MEP pathway consist of the reductive ring opening of 79 to afford (E)-4-hydroxy-3-methylbut-2-enyl diphosphate (84), catalyzed by IspG, followed by the IspH-catalyzed dehydroxylation of 84 to afford a mixture of 1 and 2 (Scheme 1). The catalytic mechanism of both enzymes had not been fully elucidated for many years, explaining why IspG and IspH are among the less studied enzymes of the MEP pathway. Nevertheless, over the past few years, a lot of progress has been made in unraveling structural, functional, and mechanistic aspects of IspG and IspH, which has guided the development of potent inhibitors of both targets.161 Both proteins contain unusual [4Fe−4S] clusters, coordinated to three cysteine residues, with a unique fourth iron atom that is not in contact with any amino acid residue. The amino acids constituting the coordination motif surrounding the [4Fe−4S] cluster are highly conserved among different orthologues.162 The physiological

Figure 4. Cartoon representation of Burkholderia pseudomallei IspF (key amino acid residues shown as sticks) in complex with inhibitor 81 (PDB code 3Q8H).157 Color code is as follows. Ligand 81 skeleton: C, green. Zn2+ cation is represented as a cyan sphere. A water molecule bridging 81 and Zn2+ is shown as a red sphere. Hydrogen bonds below 2.9 Å are shown as dashed lines.

is no direct interaction of 81 with the catalytic Zn2+ cation, but the presence of the bicyclic aromatic ring, engaged in hydrophobic interactions, probably compensates for this lack, resulting in a strong affinity of 81 or B. pseudomallei IspF. Unfortunately, when tested for antibacterial activity against Burkholderia thailandensis and antimalarial activity against several strains of P. falciparum, 81 does not show any activity, probably owing to its particularly hydrophilic character.157 O



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Chart 8. Inhibitors 85−93 of 2C-Methyl-D-erythritol 2,4-Cyclodiphosphate Reductase (IspG) and 1-Hydroxy-2-methyl-2-(E)butenyl-4-diphosphate Reductase (IspH)

IspG. Very few crystal structures of IspG have been deposited in the PDB, and none of them correspond to the enzyme from pathogenic organisms. Ligand-free and 79-bound T. thermophilus IspG (PDB codes 2Y0F169 and 4G9P,170 respectively) and A. aeolicus IspG in complex with the [4Fe− 4S] cluster (PDB code 3NOY)171 have been reported. IspG is a two-domain (AB) protein, and it is catalytically active as a dimer, where the two active sites (the substrate-recognition site and the pocket hosting the [4Fe−4S] cluster) present in separate domains are able to interact with each other because of the head-to-tail arrangement of the protein.170 A certain degree of flexibility is associated with the catalytic process, where the binding of 79 to the N-terminal domain (A) induces a 60° rotation of the Fe-S-containing C-terminal domain (B), rendering it more accessible for the substrate. Despite its remarkably polar character, we found the active site of IspG to be druggable.26 Moreover, we identified two other pockets during our druggability assessment with DoGSiteScorer, with much higher lipophilic character, which could be exploited for the design of potentially allosteric inhibitors. Very few inhibitors for IspG have been reported so far, the best one being the substrate-analogue propargyl diphosphate (85, Chart 8), showing an IC50 of 750 nM against E. coli IspG.172,173 Several other substrate analogues have been tested against bacterial and plant IspG, and not surprisingly, the alkyne diphosphates proved to be the best inhibitors of IspG. This result is consistent with the expected inhibitory mechanism of 85, which is predicted to bind in proximity to the [4Fe−4S] cluster forming a π-complex with the unique iron of the [4Fe−4S] cluster, as evidenced by EPR/HYSCORE spectra, in a similar manner to the alkene substrate 84.172 The same results have been obtained for 86 in a more recent study (Chart 8).174 Evidence for the formation of organometallic species between synthetic [4Fe−4S] clusters and alkynes had been reported previously.175 Recently, 87 and 88 were found to inhibit IspG with singledigit micromolar IC50 values for different IspG ortologues (Chart 8).174 Interestingly, whereas the EPR spectra of 88 bound to IspG are indicative of π-bonding, with the thiol group not directly bound to Fe, the EPR spectra of 87 bound to IspG suggest that no π-interaction is involved.

electron donor for the reduction of the [4Fe−4S] cluster is either a ferredoxin or a flavodoxin, depending on the organism.163 Different catalytic mechanisms have been proposed for IspG and IspH, which have been extensively discussed by Oldfield and Wang in 2014.161 Recent spectroscopic findings (electron paramagnetic resonance (EPR) and Mössbauer spectroscopy, DFT calculations) suggest that the catalytic mechanism for both enzymes might involve bioorganometallic intermediates, involving direct iron−carbon interactions during catalysis. In particular, a ferraoxetane intermediate containing both Fe−C and Fe−O bonds is proposed to be involved in the IspGcatalyzed reaction, while a weak π-complex and an allyl η3complex are supposed to be involved in the IspH-catalyzed reaction. Mechanistic studies on IspG and IspH have been often supported by X-ray crystallography: Span et al., for example, report a rotation of the hydroxymethyl group of the substrate of IspH (84). Such detailed understanding might be fundamental when designing and optimizing inhibitors for IspH.164 Most of the inhibitors developed so far for IspG and IspH target the [4Fe−4S] cluster, in particular its unique fourth iron site. On the one hand, metalloproteins containing [4Fe−4S] clusters carry out a broad series of reactions in all organisms,165 including mammals, and targeting these clusters could lead to selectivity issues. On the other hand, the possibility of targeting both IspG and IspH at the same time looks very appealing for the development of more efficient anti-infective agents, potentially able to overcome drug resistance. Also, targeting the two enzymes at the same time might reduce the proinflammatory effects of 84 in acute infections. In fact, the inhibition of only IspH would lead to accumulation of 84, which would activate γδ T cells leading to inflammation-related damage.166 In addition to its role as a 2H+/2e−reductase, IspH in its oxidized form was recently found to be able to hydrate acetylenes to aldehydes and ketones.167 Other [4Fe−4S] cluster-containing proteins, which could be exploited as antiinfective or herbicidal targets, are also acetylene hydratases, such as fumarase A in malaria parasites.168 These findings suggest that a more efficient multiple-level inhibition could be possible when targeting IspH. P



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In the context of a cell-based screening for inhibitors of the MEP pathway, maculosin (89, Chart 8) was found to possess weak antimicrobial activity against E. coli and, in particular, IspG was found to be a 89-binding protein.176 Nevertheless, its inhibitory activity for IspG in vitro is not reported. 89 might be worthwhile exploring as a potentially allosteric inhibitor for IspG. IspH. Several (co)crystal structures of IspH have been reported, but as for IspG, no structure of IspH from pathogenic organisms has been deposited to date. The structure of E. coli IspG in complex with 2 (PDB code 3KEF)177 confirms the presence of the [4Fe−4S] cluster, which had not been observed in previously reported crystal structures,178,162probably because of the loss of one Fe atom from the cluster during crystallization. IspH is a monomeric protein, with the [4Fe− 4S] cluster lying at the center of three α/β domains, and the conformation of the unbound form is different from the substrate-bound form, the latter being more closed. As for IspG, this is caused by the necessity of bringing the catalytic [4Fe−4S] cluster in proximity to the substrate that is bound in a central cavity completely shielded from bulk solvent. Although the AAAr is close to that of undruggable pockets, our druggability assessment of IspH suggests that the active site of IspH can be targeted. The presence of adjacent pockets to the active site suggests that this pocket can accommodate inhibitors bigger in size than the substrate itself.26 Two kinds of substrate analogues have been studied so far as inhibitors for IspH. The first class includes compounds in which the diphosphate group of 84 has been replaced by another moiety like a carbamate (e.g., 90) or an aminosulfonyl carbamate (e.g., 91), but their inhibitory activity against IspH (as well as against IspG) was found to be rather low (∼60% inhibition of A. aeolicus IspH at 1 mM) (Chart 8).179 Moreover, it was found that substitution of the diphosphate moiety of 84 abolishes the ability of these compounds to activate human Vγ2Vδ2 cells.180 The second class of compounds retains the diphosphate moiety while bearing other groups instead of the terminal hydroxyl group, therefore preventing the binding of the substrate to the unique fourth iron site of the [4Fe−4S] cluster. 87 and 88 were found to be potent inhibitors of IspH, with IC50 values against E. coli IspH of 210 and 150 nM, respectively.181,182 Both 87 and 88 are competitive reversible inhibitors of IspH with Ki values of 20 and 54 nM, respectively, with 87 being a slow-binding inhibitor, probably due to the necessity for deprotonation of the terminal amino group present in its protonated form at physiological pH. The binding mode of 87 and 88 in complex with E. coli IspH was confirmed by crystallographic studies,183 but it is still not clear whether the inhibitory action of 87 and 88 is due to their activity against the oxidized cluster, the reduced cluster, or both. A recent EPR investigation of the binding mode of 87 and 88 with IspG and IspH highlighted a remarkable difference in their binding behavior with the two enzymes. This unexpected diversity should be considered especially when targeting both IspG and IspH at the same time.174 Replacement of the terminal hydroxyl group of 84 with a pyridine moiety, which can interact with the [4Fe−4S] cluster via a Lewis acid−base mechanism, was less successful, with the best compound, 92, showing just single-digit micromolar inhibition against A. aeolicus IspH (Chart 8).184 Very recently, crystallographic studies validated the binding mode of pyridine analogues such as 93 bound to E. coli IspH (PDB code 4MUX, Figure 5).185

Figure 5. Cartoon representation of Escherichia coli IspH (key amino acid residues shown as sticks) in complex with inhibitor 93 and the [4Fe−4S] cluster (PDB code 4MUX).185 Color code is as follows. Ligand 93 skeleton: C, pink. [4Fe−4S] cluster: Fe, cyan; S, yellow. A water molecule bridging 93 with Glu126 is represented as a red sphere. Hydrogen bonds are shown as dashed lines below 2.6 Å.

Considering the very close proximity of two pyridine ring atoms to the fourth iron atom of the cluster and the continuous electron density between the fourth iron and the pyridine nitrogen atom, the authors propose a η2 side-on metal−ligand coordination of 93, as shown in Figure 5. Considering the inhibitory potency of compounds such as 87, 88, 92, and 93, it becomes evident how the ability of a certain ligand to interact with the fourth iron atom of the [4Fe−4S] cluster constitutes a key feature for enhancing its inhibitory potency with respect to IspH. Interestingly, when a fluorine atom replaces the terminal hydroxyl group of 84, the molecule can react with IspH forming the same intermediate as observed with the natural substrate.186 As for IspG, a series of substrate analogues bearing alkyne moieties, which can interact with the [4Fe−4S] cluster, have also been designed and tested against IspH,187 with the best one, 86, resulting in strong inhibition of A. aeolicus IspH (IC50 = 450 nM).188

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CONCLUSIONS The enzymes of the MEP pathway are attractive drug targets for the development of new anti-infective agents and herbicides but present several challenges. First, the fact that their active sites are remarkably polar makes the development of druglike inhibitors particularly demanding. Second, selectivity issues can arise when targeting DXS, IspE, IspG, and IspH, given their structural or functional resemblance to human enzymes. The literature data reported in the past decade and reviewed by us and others28,189 demonstrate, however, that these challenges can be overcome. The mechanisms of regulation of the MEP pathway including its branch points with other metabolic pathways have not been sufficiently taken into account to date, especially when trying to develop inhibitors that might be less prone to the emergence of resistance in pathogenic organisms. In this context, DXS and IspF are particularly interesting targets, given the involvement of DXS in the biosynthesis of both vitamin B1 and vitamin B6 and the crucial role of IspF in the regulation of the MEP pathway. Nevertheless, both targets Q



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are underexplored both in terms of structural studies and in terms of development of inhibitors. IspC, in constrast, is the most widely studied among the enzymes of the MEP pathway. The number of derivatives of fosmidomycin (11) and FR900098 (20), potent inhibitors of IspC and antimalarial agents, reported in the literature so far is enormous, and substantial progress has recently been made especially aimed at improved bioavailability for the use as antituberculotic drugs. M. tuberculosis and P. falciparum are, in fact, very different organisms from several points of view, and this aspect should be taken into account from the early stages of the drugdevelopment process. The fact that 5, the product of the IspC-catalyzed reaction, has supposedly a regulatory effect on the levels of 79 in pathogenesis would suggest that drug combinations of IspC and IspF inhibitors could result in potent anti-infective activity. This is particularly important also in light of the recent discovery of a new mechanism of regulation of the MEP pathway, involving a sugar phosphatase whose loss of function is able to increase the level of the metabolites in the MEP pathway, particularly at the IspC level. It has been shown that overproduction of the substrate of IspC (6) can overcome competitive inhibition of P. falciparum IspC by 11, rendering the emergence of resistance to 11 a real problem. The strategy of targeting multiple MEP pathway enzymes at the same time could be applied for IspG and IspH as well, both containing a [4Fe−4S] cluster. In fact, whereas inhibition of only IspH would lead to accumulation of 84, leading to acute infections, targeting both enzymes simultaneously would reduce the proinflammatory effects of 84. The fact that optimization of the activity of the cytosine derivative 62 against E. coli IspE has not succeeded so far might suggest that a bottleneck has been reached for this scaffold and that chemical diversity needs to be explored. Moreover, there are no data reported regarding the activity of 62 and its derivatives for enzymes of pathogenic organisms. Focusing on the development of allosteric inhibitors seems to be the solution for enzymes such as IspD, which possess a particularly hydrophilic active site. Accordingly, the allosteric inhibitors with nanomolar activity against P. vivax IspD should be further investigated. Over the past decade, substantial progress has been made in the development of inhibitors of the MEP pathway enzymes. The development of potent and selective anti-infective drugs and herbicides, however, necessitates an enhanced understanding of the regulation and branch points of the pathway.

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of rational design and synthesis of pharmacologically interesting compounds as inhibitors of the first enzyme of the non-mevalonate pathway, 1-deoxy-D-xylulose-5-phosphate synthase (DXS). Anna K. H. Hirsch read Natural Sciences at the University of Cambridge, U.K., developing the double conjugate addition of dithiols to propargylic carbonyl systems under the supervision of Prof. S. V. Ley. She obtained her Ph.D. from the ETH Zurich in 2008, having worked on the design and synthesis of inhibitors of the kinase IspE from the methylerythritol phosphate pathway under the supervision of Prof. F. Diederich. After a postdoc with Prof. J.-M. Lehn focusing on the design and synthesis of dynamic polymers inspired by proteins, she took up her current position as Assistant Professor at the University of Groningen, The Netherlands, in 2010, working on rational approaches to drug design in combination with dynamic combinatorial chemistry on multiple targets, including DXS from the methylerythritol phosphate pathway.

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ACKNOWLEDGMENTS A.K.H.H. received funding from The Netherlands Organisation for Scientific Research (NWO-CW, VENI grant) and from the Ministry of Education, Culture and Science (Gravitation Program 024.001.035).

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ABBREVIATIONS USED MEP, 2C-methyl-D-erythritol 4-phosphate; DXS, 1-deoxy-Dxylulose-5-phosphate synthase; IspC, 1-deoxy-D-xylulose-5phosphate reductoisomerase; IspD, 4-diphosphocytidyl-2Cmethyl-D-erythritol synthase; IspE, 4-diphosphocytidyl-2Cmethyl-D-erythritol kinase; IspF, 2C-methyl-D-erythritol 2,4cyclodiphosphate synthase; IspG, 2C-methyl-D-erythritol 2,4cyclodiphosphate reductase; IspH, 1-hydroxy-2-methyl-2-(E)butenyl-4-diphosphate reductase; MDR, multidrug resistant; XDR, extensively drug resistant; AAAr, apolar amino acid ratio; TDP, thiamine diphosphate; TK, transketolase; PDH, pyruvate dehydrogenase; STD, saturation-transfer difference; trNOE, transfer NOE; CTP, cytidine triphosphate; CDP, cytidine diphosphate; GHMP, galactose/homoserine/mevalonate/ phosphomevalonate; GALK1, human galactokinase 1; GDP, geranyl diphosphate; FDP, farnesyl diphosphate

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*E-mail: [email protected]. Phone: +31 (0)50 363 4275. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest. Biographies Tiziana Masini obtained her Master’s degree at the University of Pisa, Italy, and developed a new synthetic route to isoflavanones via a palladium-catalyzed direct arylation of 4-chromanones and structural analogues for her Master’s research project. She is currently a Ph.D. student in the group of Dr. A. K. H. Hirsch at the University of Groningen, The Netherlands, and her research interest lies in the field R



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