Current Pharmaceutical Design, 2007, 13, 1161-1177 1161

Targeting the Methyl Erythritol Phosphate (MEP) Pathway for Novel

Antimalarial, Antibacterial and Herbicidal Drug Discovery: Inhibition of
1-Deoxy-D-Xylulose-5-Phosphate Reductoisomerase (DXR) Enzyme

Nidhi Singh1, Gweneal Chevé1, Mitchell A. Avery1,3,4 and Christopher R. McCurdy1,2,4,*

1
Department of Medicinal Chemistry and Laboratory for Applied Drug Design and Synthesis, School of Pharmacy
2
Department of Pharmacology, School of Pharmacy 3Department of Chemistry and Biochemistry 4National Center for
Natural Products Research, University of Mississippi, University, MS 38677-1848, USA

Abstract: The 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway for isoprenoid biosynthesis has come under in-
creased scrutiny as a target for novel antimalarial, antibacterial and herbicidal agents. 1-Deoxy-D-xylulose 5-phosphate
reductoisomerase (DXR) is a key enzyme of the pathway that catalyzes the rearrangement and nicotinamide adenine
dinucleotide phosphate (NADPH)-dependent reduction of 1-deoxy-D-xylulose 5-phosphate (DXP) to MEP. The unique
properties of DXR make it a remarkable and rational target for drug design. First, it is a vital enzyme for synthesis of iso-
prenoids in algae, plants, several eubacteria including the pathogenic bacteria like Bacillus anthracis, Helicobacter pylori,
Yersinia pestis, Mycobacterium tuberculosis and the malarial parasite, Plasmodium falciparum. Second, there are no func-
tional equivalents to DXR in humans, making it an attractive target for therapeutic intervention. Third, DXR appears to be
a valid target and the results from fosmidomycin (1), the only available DXR inhibitor under clinical trials, suggests syn-
ergistic effects with the lincosamide antibiotics, lincomycin and clindamycin. Despite drug design efforts in this area, no
successful drug specifically designed to inhibit DXR has emerged yet. This review summarizes the recent and promising
developments with respect to the current knowledge of the MEP pathway with emphasis on the understanding of the struc-
ture and the catalytic mechanism of the DXR enzyme and the global quest for therapeutically useful inhibitors of DXR.
Key Words: Isoprenoids, MEP pathway, mevalonate, fosmidomycin, FR900098, DXP reductoisomerase, DXR.

INTRODUCTION units, isopentenyl diphosphate (IPP) and its isomer dimethy-

lallyl diphosphate (DMAPP). Since the initial discovery of
Isoprenoids constitute the largest single class of natural
the Acetate/Mevalonate (MVA) pathway in the early 1950’s,
products, [1] with over 35,000 compounds identified to date it was assumed that the isoprenoids are biosynthesized
[2]. They represent a heterogeneous group of biologically
through this route in all organisms (Fig. 2) [14-16]. This
active molecules that constitute a major class of primary and
pathway includes condensation of two molecules of acetyl-
secondary metabolites. Compounds derived from the path-
CoA to form acetoacetyl-CoA, which is then converted to 3-
way carry out biological functions fundamental for the nor-
hydroxy-3-methylglutaryl CoA (HMG-CoA). HMG-CoA in
mal growth and developmental processes in all living organ-
the next step is reduced to MVA, the reaction being cata-
isms (Fig. 1) [3-5]. These include functioning as modulators lyzed by HMG-CoA reductase, a key regulatory enzyme of
of membrane properties (prenyllipids in archaebacteria [6]
this pathway that has been extensively studied [17]. MVA
and sterols in eubacteria and eukaryotes [7]), as carriers for
then undergoes two successive phosphorylation steps to give
electron transport (menaquinone, plastoquinone, phylloqui-
mevalonic-5-monophosphate (MVA-P) that is converted to
none and ubiquinone), [8] as light harvesting and photo-
mevalonic-5-diphosphate (MVA-PP). An adenosine triphos-
protecting pigments (carotenoids, phytol side chain of chlo-
phate (ATP)-dependent decarboxylation of MVA-PP results
rophyll), as regulators of growth and development (steroid in formation of IPP that is subsequently transformed to
hormones, gibberellins, cytokinins, abscisic acid) [9] and in
DMAPP by IPP isomerase. In due time, however, several
signal transduction (prenylated proteins) [10]. Isoprenoids
observations emerged in the literature that were not consis-
also serve as attractants for pollinators and seed dispersers
tent with this pathway. For example, [13C] acetate was not
[11] and as antibiotics, and herbivore repellents as well as
incorporated into ubiquinone of Escherichia coli [18] or into
toxins in plants [12, 13].
pentalenolactone produced by Streptomyces chromofuscus
Despite the diversity of structure and function, all isopre- [19]. It was also found that mevinolin, a potent and highly
noids originate from the common five-carbon (C5) building specific inhibitor of HMG-CoA reductase, that blocks sterol
biosynthesis in the cytosol, did not affect carotenoid biosyn-
thesis in plastids [20]. These results provided strong support
*Address correspondence to this author at the National Center for Natural for the existence of a different biosynthetic pathway for IPP
Products Research, University of Mississippi, University, MS 38677-1848, formation.
USA; Tel: +1-662-915-5882; Fax: +1-662-915-5638;
E-mail: cmccurdy@olemiss.edu

1381-6128/07 $50.00+.00 © 2007 Bentham Science Publishers Ltd.

1162 Current Pharmaceutical Design, 2007, Vol. 13, No. 11
Fig. (1). A schematic representation showing branching of various isoprenic compounds at different steps of the pathway in diverse systems.
More recently, however, 13C-incorporation into various
isoprenic compounds in bacteria, algae and plants proved
useful for the elucidation of a novel pathway to IPP in these
organisms [21-26]. With mutants of Escherichia coli defi-
cient in the enzymes of the triose phosphate pathway, incor-
poration of 13C-labeled glycerol or pyruvate into the prenyl
chain of ubiquinone Q8 revealed that the isoprenic units
were formed in the new metabolic route from pyruvate and
glyceraldehyde-3-phosphate [21]. To distinguish this alterna-
tive pathway for the biosynthesis of the isoprenoid building
units from the MVA pathway, it was named as the non-
mevalonate route. This pathway is also known as the 1-
deoxy-D-xylulose-5-phosphate (DXP) pathway, where DXP
is the first intermediate; or 2C-methyl-D-erythritol-4-phos-
phate (MEP) pathway since MEP is considered to be the first
committed precursor [27, 28].
The MEP pathway is comprised of seven enzymatic steps
(Fig. 2). The first step is the condensation of glyceraldehyde-
3-phosphate and pyruvate to produce DXP [29] in a transke-
tolase-type reaction catalyzed by DXP synthase. DXP is a
precursor not only for IPP, the common metabolic precursor
of all isoprenoids, but also for thiamine (vitamin B1) [29-32]
and pyridoxal (vitamin B6), two important cofactors for
many metabolic enzymes [29, 30, 33]. The next step is the
conversion of DXP into MEP [34] in a reaction mediated by
the DXR enzyme in a single step by intramolecular rear-
rangement and reduction, the exact mechanism of which is
not yet clear. The transformation of MEP to 4-diphospho-
cytidyl-2C-methyl-D-erythritol (CDP-ME) [35] is catalyzed
in a cytidine triphosphate (CTP)-dependent reaction by
CDP-ME synthase. The adenosine triphosphate (ATP)-
dependent CDP-ME kinase then phosphorylates CDP-ME at
the 2- hydroxy group, forming 4-diphosphocytidyl-2-C-
methyl-D-erythritol-2-phosphate (CDP-ME2P) and adeno-
sine diphosphate (ADP) [36]. This CDP-ME2P, in the next
Singh et al.
step, is converted into 2-C-methyl-D-erythritol-2,4-cyclodi-
phosphate (MECP) and cytidine monophosphate (CMP) in a
reaction catalyzed by MECP synthase [37]. 1-Hydroxy-2-
methyl-2-(E)-butenyl-4-diphosphate synthase (HMBPP syn-
thase) catalyzes the conversion of MECP into HMBPP [38,
39]. Finally, HMBPP is converted into a mixture of IPP and
DMAPP (usually in 5:1/3:1 ratio), [40] the reaction being
catalyzed by HMBPP reductase (also called as IPP/DMAPP
synthase), seemingly replicating IPP isomerase activity [41].
By contrast, IPP isomerase favors an equilibrium ratio of 3:7
between IPP and DMAPP [42].
The MEP pathway is found operative in most eubacteria,
[43] algae, [26, 44-46] cyanobacteria and diatoms [47] but is
absent in animals, fungi, archaebacteria and certain bacteria
like streptococci, staphylococci and lactobacilli [48]. Al-
though higher plants possess both the MEP pathway as well
as the MVA pathway for isoprenoid synthesis, [46, 49, 50]
distinct compartmentalization occurs; [51-57] the MVA
pathway occurs in the cytosol and mitochondria (e.g. for
sterols and ubiquinone) while the MEP pathway, for synthe-
sis of both IPP and DMAPP, occurs in plastids (e.g. for iso-
prene, plastoquinone-9, carotenoids and phytol) [58, 59].
Data from labeling and inhibitory studies from several labo-
ratories indicated the existence of substantial cross talk be-
tween both cellular pathways [60-64]. Even though the
transportation of isoprenoid building units, synthesized in
plastids through the MEP pathway, into cytosol has been
reported, the inverse cooperation is known to occur to a rela-
tively lesser extent depending on the physiological state and
the developmental stage of the plant cells. In spite of the
evidence suggesting the existence of some cooperation be-
tween both IPP producing pathways, lack of one can never
be fully compensated by the other [65]. Nonetheless, the
existence of two different pathways for IPP biosynthesis in
plants is fascinating and perhaps, is of evolutionary signifi-
MEP Pathway for Novel Antimalarial, Antibacterial and Herbicidal Drug Discovery Current Pharmaceutical Design, 2007, Vol. 13, No. 11 1163

MVA PATHWAY (CYTOSOL) MEP PATHWAY (PLASTID)

O O O
O
Acetyl CoA Glyceraldehyde-3-P
SCoA COO- H O P O-
+
Pyruvate OH O-
Acetyl CoA Acetyl transferase
CO2 DXP synthase (dxs)

O O OH
O
1-Deoxy-D-xylulose-5-P (DXP)
SCoA O P O-
Acetoacetyl-CoA O OH O-

Fosmidomycin NADPH
HSCoA
NADP+
O HMG-CoA synthase
DXP reductoisomerase (dxr/ispC)
OH O
SCoA
HO O P O- 2-C-methyl-D-erythritol-4-P (MEP)
Hydroxymethylglutaryl-CoA(HMG-CoA) OH OH O-
COOH COSCoA CTP
PPi
HMG-CoA reductase NH2
NADPH CDP-ME synthase (ispD/ygbP)
CoA
NADP+ OH N
Mevanolin O O
O P O P O N O
HO O
OH OH O- O-
Mevalonic acid (MVA)
COOH CH2OH CDP-methyl-D-erythritol(CDP-ME) HO OH
ATP
ATP
Mevalonate kinase ADP
ADP CDP-ME kinase (ispE/ychB) NH2
O
N
HO O- P O O O
O- O- O
N O
Mevalonic-5-monophosphate (MVA-P) O P O P O
COOH CH2O P O- O- O-
OH OH
O
ATP CDP-methyl-D-erythritol-2-P(CDP-ME-2P) HO OH
Mevalonate 5-phosphate
ADP kinase CMP
MECP synthase (ispF/ygbB)
HO
O-
O- O- O O
P O
COOH CH2O P O P O- O P
O-
O O O

Mevalonic-5-diphosphate (MVA-PP) 2-C-methyl-D-erythritol-2,4-diphosphate (MECP)

OH OH

Mevalonate 5-diphosphate HMBPP synthase (ispG/gcpE)

decarboxylase

O- O-
ATP CO2 O P O P O-
ADP + Pi OH O O
NADH
HMBPP reductase (ispH/lytB)
1-Hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate (HMBPP)

Isopentenyl diphosphate O- O- O- O-
(IPP) O P O P O- O P O P O- Dimethylallyl diphosphate
Isopentenyl diphosphate
O O O O (DMAPP)
isomerase

Fig. (2). Biosynthesis of IPP via the MVA and the MEP pathway.
1164 Current Pharmaceutical Design, 2007, Vol. 13, No. 11 Singh et al.

Table 1. Diseases and their Causative Organisms in Plants and in Humans that may be Alleviated by Inhibition of DXR.a

Causative Organism Infection/Disease Type of Pathogen

Human Pathogen

Helicobacter pylori Gastritis, peptic ulcer, gastric adenocarcinoma, gastric Bacteria

mucosa-associated lymphoid tissue lymphoma

Plasmodium falciparum Malaria Protozoa

Mycobacterim tuberculosis Tuberculosis Bacteria

Bacillus anthracis Anthrax Bacteria

Bordetella purtussis Whooping cough Bacteria

Neisseria meningitidis Meningitis Bacteria

Fusobacterium nucleatum Periodontitis Bacteria

Porphyromonas gingivalis Chronic adult periodontitis and progressive periodontitis Bacteria

Chlamydia trachomatis Chlamydia, Ttachoma Bacteria

Neisseria gonorrhoeae Gonorrhea Bacteria

Treponema pallidum Syphilis Bacteria

Campylobacter jejuni Gastroenteritis Bacteria

Clostridium botulinum Food poisoning Bacteria

Clostridium tetani Tetanus Bacteria

Corynebacterium diphtheriae Diptheria Bacteria

Escherichia coli Enteric bacteria Bacteria

Francisella tularensis Tularemia Bacteria

Haemophilus influenzae Bacteremia and acute bacterial meningitis Bacteria

Pseudomonas aeruginosa Bacteremia Bacteria

Klebsiella pneumoniae Pneumonia Bacteria

Mycobacterium leprae Leprosy Bacteria

Vibrio cholerae Cholera Bacteria

Yersinia pestis Plague Bacteria

Bacillus anthracis Anthrax Bacteria

Toxoplasma gondii Toxoplasmosis Protozoa

Plasmodium vivax Malaria Protozoa

Shigella dysenteriae Shigellosis Bacteria

Salmonella enteritidis Gastroenteritis Bacteria

Chlamydia psittaci Psittacosis Bacteria

Clostridium difficile Antibiotic induced diarrhea, pseudomembranous colitis Bacteria

Enterococcus faecalis Urinary tract infection Bacteria

Salmonella typhi Typhoid fever Bacteria

Salmonella typhimurium Gastroenteritis Bacteria

Propionibacterium acnes Acne vulgaris Bacteria

MEP Pathway for Novel Antimalarial, Antibacterial and Herbicidal Drug Discovery Current Pharmaceutical Design, 2007, Vol. 13, No. 11 1165

(Table 1) contd….

Causative Organism Infection/Disease Type of Pathogen

Plant Pathogen

Agrobacterium tumefaciens Crown gall disease Bacteria

Ralstonia solanacearum Southern wilt Bacteria

a
This information is obtained from the SWISSPROT protein sequence data bank (http://us.expasy.org/sprot/; DXR was used as the keyword).

cance to plants providing them with better adaptability to vivo, Mg2+ has been suggested to be the relevant divalent
environmental changes [66]. cation in vivo [73, 74, 76]. The substrate for the reaction
catalyzed by the enzyme is DXP. The first evidence for DXP
Interestingly, it has been found that isoprenoids are syn-
as an isoprenoid precursor was provided by the studies of
thesized exclusively via this pathway in various pathogenic
Arigoni et al. and Broers [45]. Since 1-deoxy-D-xylulose
bacteria like Escherichia coli, Mycobacterium tuberculosis,
(DX) was reported to be used by a wild-type Escherichia
Mycobacterium leprae, Helicobacter pylori, Vibrio cholerae
and Bacillus anthracis and in the apicoplast of the apicom- coli strain in place of DXP, [46] the possibility of using DX
to produce MEP via a DXR catalyzed reaction was also in-
plexan protozoan, P. falciparum; refer to Table 1 [34, 53,
vestigated. No oxidation of NADPH was observed under
67]. In addition, knockouts of DXR in E. coli [34, 41] and
such conditions that led to the conclusion that DXP and not
Bacillus subtilis [68] are lethal. However, it has not yet been
proven, if within the infected host, the DXP/MEP pathway is
the sole provider of de novo synthesized isoprenoids in the
pathogen or that the products and/or the intermediates may
be utilized from the alternative pathway operating in the
host. Sole dependence on the DXP/MEP pathway within the
diseased host for IPP/DMAP will increase the significance of
the target since the theoretical potential of this target to cure
disease hinges on the importance of the target in the infected
host cell. Since the amino acid sequences of the constituent
enzymes of the DXP/MEP pathway are highly conserved
(except HMBPP synthase that contains a large plant-specific
internal domain of unknown function) [69] and have no
known human counterparts, [57] the constituent enzymes of
the DXP/ MEP pathway offer the potential for the develop-
ment of novel antibacterial, antimalarial and herbicidal com-
pounds perhaps, without toxicity to humans.

DXR ENZYME: MECHANISTIC STUDIES

The DXR is the second enzyme in the cascade of the
MEP pathway that has been characterized at the enzymatic
and molecular level in Escherichia coli [34, 69] and its
orthologues from the cyanobacteria, Synechocystis, [70] the
plants Arabidiopsis [71] and Mentha piperata [72] and the
malarial parasite, P. falciparum [67] The structural and bio-
chemical studies conducted on this enzyme show that the
DXRs exist as homodimers (Fig. 3) with molecular weights
between 42-45 kDa. The enzyme catalyzes the rearrange-
ment and NADPH-dependent reduction of DXP to MEP.
Studies on Escherichia coli and Mycobacterium tuberculosis
enzymes [73, 74, 89] have afforded direct evidence for the
reversibility of the reaction catalyzed by both enzymes, with
Fig. (3). The ribbon representation of the physiological homodimer
a reaction equilibrium that is essentially in favor of MEP
of DXR. The upper monomer is colored blue (N-terminal domain;
production.
residues 1–150), pink (connective domain; residues 160-270), and
DXR requires a divalent metal ion for catalysis; Co2+, green (C-terminal domain; residues 312-398). The catalytic loop
Mn and Mg2+ have been shown to activate the enzyme in
2+
(residues 186–216) surrounding the inhibitor-binding site is indi-
an optimum concentration of 1mM, [73] albeit with varying cated in cyan. The lower monomer is colored grey with NADPH (in
kinetic parameters [34]. With other divalent metal ions such stick representation and colored yellow) and fosmidomycin (repre-
as Ca2+, Ni2+, Zn2+, minimal or no activity was observed [34, sented as ball and stick and colored by atom type) cocrystallized
73, 75, 76]. However, due to the abundance of its levels in [82].
1166 Current Pharmaceutical Design, 2007, Vol. 13, No. 11 Singh et al.

DX is the substrate for the reaction [69] and that DX needs
to be converted to DXP before it can be used as a substrate
[34]. Also, the conversion of DXP into MEP by DXR re-
quires the presence of NADPH as a cofactor, in preference to
NADH [34, 77]. The results from recent studies on the My-
cobacterium tuberculosis DXR suggest that although NADH
can be utilized as a cofactor in vitro, NADPH is the relevant
cofactor in vivo because of its higher kcat/Km values [73].
The reaction catalyzed by the enzyme includes both the
rearrangement and the reduction of DXP. The presence of a
rearrangement in the MEP pathway was demonstrated by
studies on the incorporation of [4,5-13C2] glucose into ho-
panoids and ubiquinones from Methylbacterium fujisa-
waense [43] which was in agreement with another similar
study in Zymomonas mobilis [23]. The experiments on the
biosynthesis of diterpenoids of Ginkgo biloba embryos led to
similar conclusions [63]. In the first step, the C3-C4 bond in
DXP is broken with concomitant formation of a new bond
between C2 and C4 of the substrate to produce a putative
aldehyde intermediate, 2-C-methyl-D-erythrose 4-phosphate,
which in the next step is reduced by NADPH (Fig. 4).
The stereochemistry of the reaction catalyzed by DXR
with the recombinant enzymes from Synechocystis [73], E.
coli [70], and Mycobacterium tuberculosis, [78] shows that
the C-1 proS hydrogen of MEP is derived from H-3 of DXP
and the hydride transferred from C-4 of NADPH is delivered
to the Re face of the intermediate aldehyde (Fig. 5) [79].
Based on these results, the enzyme has been classified as a
class B dehydrogenase [70, 78]. Investigation of the stereo-
chemical course of the reduction step in Liriodendro tulipif-
era confirmed these findings [80]. The analysis of the crystal
structures of DXR from E. coli [81, 82] provides additional
support to the above statement.
A similar reaction has already been detailed for acetohy-
droxy acid isomeroreductase [83-87]. Although the reaction
catalyzed by DXR and acetohydroxy acid isomeroreductase
are related, their overall amino acid sequences are different.
Moreover, even though both enzymes have a comparable
induced fit mechanism, their X-ray crystal structures differ
extensively [86, 88]. Like acetohydroxy acid isomeroreduc-
tase, the catalysis by the E. coli enzyme has been shown to
obey an ordered mechanism in which NADPH and a divalent
cation binds first resulting in a minor conformational change
before substrate binding that causes more pronounced con-
formational change prior to the enzymatic reaction [76, 89].
Notably, the results of product inhibition studies and isotope
effect experiments on the Mycobacterium tuberculosis DXR
suggested a steady-state random mechanism [73]. However,
a preferred order of substrate addition, where NADPH pre-
cedes DXP, could not be ruled out.
Nevertheless, due to the similarities in the overall reac-
tions catalyzed by DXR and the acetohydroxy acid iso-
meroreductase, it was generally accepted that their mecha-
nism was also analogues. However, several other studies
suggested that the aldehyde intermediate could be formed by
a retro-aldol/aldol mechanism [90]. The proposed mecha-
nism for both the reactions is shown in Fig. (6).

OH 1 OH
OH
5 Mg2+ 5 NADPH
2 4 O 4 HO
1 OPO32-
3 OPO32- 2 OPO32-
3
O OH OH OH

DXP 2-C-Methyl-D-erythrose-4-phosphate MEP

Fig. (4). The reaction catalyzed by DXR.

HA HA
N N N HA
HB HB
O O O
H2N H2N H2N

O CH3 HO HO
CH3 CH3
O
OH HO OH
HO H3 H
H3
OH H3
OPO32- OPO32-
Re
OPO32-

DXP MEP

Fig. (5). Outline of the stereochemical features of the DXR reaction. (Copyright obtained from Ref. [79]).
MEP Pathway for Novel Antimalarial, Antibacterial and Herbicidal Drug Discovery Current Pharmaceutical Design, 2007, Vol. 13, No. 11 1167

A B

H H
O O O O
O P O- O P O-
O O
O- O-
OH O
DXP DXP H

-ketol
Retro-aldolization
rearrangement

H
O O O O
HO P O- - O P O-
O O
O- Aldolization O-
OH O

H H
O O NADPH O O
O P O- HO P O-
O O
O- O-
OH OH
MEP

Fig. (6). The stereochemistry of the proposed mechanism for the rearrangement of DXP to MEP; (A)
-ketol rearrangement, (B) retro-
aldol/aldol reaction. The shaded circle represents a Mg2+ ion.

As of today, no conclusive evidence that rules out one of
these pathways has been found, but it is certain that the in-
termediate(s) remain tightly bound to DXR prior to the
NADPH mediated reduction. After reduction, it is suggested
that NADP+ is released before the release of MEP [91].
STRUCTURAL STUDIES
To date, several crystal structures of DXR from E. coli
[81, 82, 92-94] and one from Zymomonas mobilis [95] have
been reported by different research groups. The first crystal
structure of the E. coli DXR published, was an apo-enzyme,
which revealed the arrangement of the three domains: an N-
terminal domain that binds NADPH; a “connective” domain
that contains the active site; and a C-terminal domain also
involved in cofactor binding, to form an overall V-shaped
molecule with an apparent flexibility observed in the loop
region. The unoccupied site is filled with water molecules
[81]. The second enzyme structure, in complex with NADPH
and a sulfate ion, was also obtained from E. coli. This struc-
ture provided insight into the cofactor binding that stabilized
the protein molecule and exhibited a more ordered flexible
loop region. The sulfate ion was found positioned within the
putative phosphate-binding pocket in the DXR protein [82].
The first DXR-ligand complex structure with Mn2+ and fos-
midomycin bound was reported along with two other struc-
tures of DXR: the apo-enzyme and with Mn2+. Unfortu-
nately, the NADPH binding site was blocked by the C-
terminus of the neighboring monomer in the crystal. Conse-
quently, the catalytic loop region was only partially ordered
in this structure. No structural rearrangement within the en-
zyme was observed assuming that complete ordering of the
loop region requires substrate as well as cofactor binding
within the binding pocket. The active site consists of phos-
phate and divalent metal binding motifs separated by a hy-
drophobic region. The phosphonate moiety of fosmidomycin
binds to the site defined by Ser186, Gly187, Ser222,
Met225, Asn227, Lys228 and Ile250 (Fig. 7). The N-formyl
hydroxylamine moiety in fosmidomycin engages the metal
by a bidentate chelation. This structure is believed to mimic
substrate binding of the C2 carbonyl oxygen and the C3 hy-
droxyl group in DXP. These two functionalities along with
several acidic residues (Asp150, Glu152 and Glu231) hold
the metal ion in place forming an octahedral coordination
sphere [92].
The structure of Zymomonas mobilis DXR was solved in
the apo-form and as a binary complex with NADPH. Com-
parison with the E. coli DXR crystal structures revealed that
except for differences in the residues interacting with the
adenine ring of NADPH, the two enzymes shared significant
structural homology and highly conserved active site archi-
tecture [95]. Crystallographic structures of two biphospho-
nate inhibitors complexed to DXR were published but nei-
ther NADPH nor a metal ion is present in either structures.
Instead, a sulfate ion is observed which fits to the substrate
phosphate pocket in a similar fashion as seen in one of the
previous crystal structures [93]. However, phosphonate
1168 Current Pharmaceutical Design, 2007, Vol. 13, No. 11
Fig. (7). A view of the binding mode of fosmidomycin within the active site of E. coli DXR: Fosmidomycin is represented as a ball-and-stick
model colored by atom-type while NADPH as a stick model is indicated in yellow [94].
moieties of these compounds bind to the metal ion instead of
the phosphate-binding site that is utilized by fosmidomycin.
Besides, the loop region forms a different conformation
when it is located over the bisphosphonate inhibitors rather
than that when it is over fosmidomycin [91]. Recently, the
ternary structures of the DXR-NADPH-fosmidomycin, the
DXR-NADPH-substrate and the selenomethionine-labeled
DXR-NADPH-fosmidomycin complex in a tight binding
closed enzyme conformation were solved (Fig. 8) [94]. The
DXR-NADPH-fosmidomycin complex revealed a large con-
formational change in the “connective” domain to give a
relatively hydrophobic binding pocket that tightly binds the
inhibitor. The narrow size of the active site in the DXR-
fosmidomycin-NADPH complex explains why larger ana-
logues of DXP or fosmidomycin have little or no inhibitory
activity against the enzyme [94]. The absence of an expected
metal ion from the structure was attributed to the acidic crys-
tallization conditions (pH = 5) used at which the affinity of
DXR for metal is low. Furthermore, this structure had a fully
ordered loop conformation that closed upon the active site
making interactions with the ligand.
Site-directed mutagenesis studies of the recombinant E.
coli protein revealed the importance of conserved histidine
and glutamate residues for activity [69]. Glu231 was found
to be the most important residue for catalysis, with a sug-
gested role in formation of an octahedral complex with the
metal ion along with other strictly conserved residues. Muta-
tion of this residue to a Lys resulted in an enzyme with sig-
Singh et al.
nificantly reduced kcat but with minor changes in Km for both
DXP and NADPH. Mutations of three conserved His resi-
dues to Gln, recognized by comparing DXRs from E. coli
with those from other bacteria and plants, to identify the
residue crucial for catalytic activity of the enzyme were also
made. The His153Gln mutation resulted in a 10-fold de-
crease in kcat while demonstrating a 3.5-fold increase in
KmDXP, with no effect on the KmNADPH. Although surrounded
by conserved residues, this residue is not in close proximity
of the substrate. However, it has been implicated to have a
structural role [82]. The mutation of either His257 or His209
to a Gln, resulted in increased Km for both DXP and
NADPH but drastically reduced kcat, suggesting that these
residues have no enzymatic role in vivo. Recently, site di-
rected mutagenesis of highly conserved Trp204 in the
Synechocystis DXR (Trp212 in E. coli DXR) revealed the
important role of this residue in substrate/inhibitor binding.
The authors have suggested that it may also have a role in
discriminating substrates for DXR [96]. These findings have
been well supported by those available from published DXR
crystal structures.
INHIBITORS OF DXR: THE STATE-OF-THE-ART
Fosmidomycin (also known as FR-31564 or 3-(N-formyl-
N-hydroxyamino) propylphosphonic acid; Fig. 9) [28, 50]
was identified as a natural antibiotic from Streptomyces lav-
endulae in the late 1970s [97]. This compound was also
found to exert herbicidal activity [98, 99]. Studies on the
MEP Pathway for Novel Antimalarial, Antibacterial and Herbicidal Drug Discovery
Fig. (8). A representation of the E. coli DXR in a tight binding conformation with the inhibitor (PDB ID: 1Q0L). The NADPH molecule
(stick representation) and fosmidomycin (depicted in ball and stick) has been shown. Helices and sheets are represented as red cylinders and
yellow arrows, respectively [94].
inhibition of the biosynthesis of menaquinone and caro-
tenoids in Micrococcus luteus attributed this effect to the
inhibition of isoprenoid biosynthesis [100]. Kuzuyama et al.
performed a database search to identify specific inhibitors of
the non-mevalonate pathway [28]. This was accomplished by
identifying compounds that were active against E. coli and
Bacillus subtilis, which possess the non-mevalonate path-
way, but was inactive against Staphylococcus aureus, which
has the classical acetate/mevalonate pathway [101]. This
search led to the identification of fosmidomycin as a putative
pathway-specific inhibitor that was active against most
Gram-negative and some Gram-positive bacteria.
Further studies confirmed that fosmidomycin was a re-
markably non-toxic, potent, specific inhibitor of DXR [50,
94]. In an in vitro assay with the purified recombinant E. coli
DXR, it was shown that fosmidomycin inhibited the DXR
activity in a dose-dependent manner with an IC50 value of
8.2 nM. Also, in an E. coli growth assay, the inhibitory effect
of fosmidomycin was overcome by the addition of 0.025%
of 2-C-methylerythritol, the free alcohol of MEP, to the me-
dium. Moreover, in the presence of the above supplementing
agent, fosmidomycin had no effect on the growth of an E.
coli DXR knockout mutant. These results led to the conclu-
sion that fosmidomycin inhibited DXR activity [38, 50]. A
similar conclusion was reached from studies of isoprenoid
biosynthesis in barley (Hordeum vulgare L.), tomato (Ly-
copersicon esculentum L.), and duckweed (Lemna sp.) [102]
The crystal structure of E. coli DXR-metal-fosmidomy-
cin complex revealed a substrate-like binding of the inhibitor
Current Pharmaceutical Design, 2007, Vol. 13, No. 11 1169
[92]. Recent studies have defined fosmidomycin as a mixed-
type (both competitive and non-competitive) inhibitor of the
recombinant enzyme of E. coli (Ki = 38 nM) [38]. It has also
been reported to be a slow, tight-binding competitive inhibi-
tor of DXR [94]. Its activity as a competitive inhibitor of the
Zymomonas mobilis DXR was tested to a Ki of 0.6 μM [74].
The antibiotic also inhibits the enzyme from higher plants
[50, 103]. Fosmidomycin and its acetyl derivative, FR-
900098 (2, Fig. 9) inhibit the recombinant DXR from P. fal-
ciparum in a dose-dependent manner [67]. Both compounds
are effective antimalarial agents, since mice infected with
Plasmodium vinckei became free of parasites upon treatment
[50, 103]. Fosmidomycin also cured uncomplicated P. falci-
parum malaria in humans [104, 105]. However, a high rate
of recrudescence was observed with treatment regimens of
less than 4 days precluding the use of fosmidomycin as a
monotherapeutic agent. This led to a combination of fosmi-
domycin with clindamycin that has been shown to be well
tolerated and highly efficacious in the treatment of uncom-
plicated malaria and such therapies would seem to be more
promising [106]. Clinical studies to assess the efficacy and
OH O OH O
H N P O- N P O-
O- O-
O O
2
1
Fig. (9). Inhibitors of the DXR enzyme, Fosmidomycin (1) and
FR900098 (2).
1170 Current Pharmaceutical Design, 2007, Vol. 13, No. 11 Singh et al.

safety of the combination of fosmidomycin with other anti-
malarial drugs have already been initiated in humans [107-
109]. Consequently, both fosmidomycin and FR900098 af-
forded valuable leads for further development towards clini-
cal use.
Additionally, these compounds have low toxicity and
their half-life in serum is very short [110]. Moreover, both
the compounds have poor oral bioavailability, with a resorp-
tion rate in the range of 20-40% [111] which may be attrib-
uted to low lipophilicity resulting from ionization of polar
phosphonate group at physiological pH values that restricts
their usage as single therapeutic agent. Since FR900098 pos-
sessed an approximately 2-fold greater antimalarial activity,
attempts were made to improve its efficacy upon oral ad-
ministration by synthesizing prodrugs [112-114]. These pro-
drugs were obtained by transforming the charged phospho-
nate moiety to yield phosphodiaryl (3), [112] acyloxyalkyl
esters (4) [113] and alkoxycarbonyloxyethyl (5) [114], with
increased oral antimalarial efficacy in mice infected with the
rodent malaria parasite, Plasmodium vinckei (Fig. 10).
Structural modifications directed towards the synthesis of
hydroxyurea [115] and carboxylic acid [116] analogues of
fosmidomycin and FR900098 have also been reported, how-
ever no enzyme inhibition studies of these compounds have
been reported to date. Fosmidomycin analogues containing a
benzoxazolone (6), benzoxazolothione (7) or oxazolopyridi-
none (8) ring have also been synthesized although their bio-
logical evaluation was reported in a model plant cell, Ca-
tharanthus roseus, in terms of its cell growth and the ability
to accumulate a monoterpenoid indole alkaloid, ajmalicine,
Fig. (9) [117]. It was found that fosmidomycin, as a control,
resulted in no effect on Catharanthus roseus cell growth but
inhibited production of ajmalicine, which has been proven to
originate from the MEP pathway [118], in a dose-dependent
manner with an IC50 of 10
M [119]. Of the twelve phos-
phonic esters and acids studied, none affected Catharanthus
roseus cell growth, but four of them (including 6, 7 and 8)
showed a significant inhibition (45-85% at 125
M) of ajma-
licine accumulation.
Recently, the synthesis of substituted 3-[2-(diethoxy-
phosphoryl) propyl] oxazolo [4, 5 - b] pyridine - 2(3H) - ones by
functionalization at 6-position with various substituents like
aryl, vinyl, carbonyl chains as fosmidomycin analogues have
been reported by the same group [120]. Some of the reported
derivatives (including 9; Fig. 10) were found to be more
powerful inhibitors of ajmalicine production than fosmido-
mycin. The inhibitory effect of these analogues and those
synthesized previously, against DXR has not been investi-
gated which is necessary to confirm that these compounds
are, in fact, DXR inhibitors. It has been suggested that inhib-
iting the MEP pathway of plants could be useful in the
search of novel antibiotics, antimalarials and herbicides
[121]. If positive results are obtained after further testing of

OH O 3 R= O

N P OR
O
OR
O 4 R=
O
O
5 R=
O O

Cl Cl Br

N
O O O
O N P OC2H5 O N P OC2H5 O N P OC2H5
OC2H5 OC2H5 OC2H5
O S O
6 7 8

N OH O OH O
O N P OH N P O-
O O
O N P OC2H5 OH O-
O O
OC2H5
O
9 10 11

Fig. (10). Structural analogues of fosmidomycin as DXR inhibitors.

MEP Pathway for Novel Antimalarial, Antibacterial and Herbicidal Drug Discovery
these compounds for their specific activity against DXR, it
will only corroborate the above statement and may provide
interesting starting points for the development of antibacte-
rial as well as antimalarial agents.
In a study by Woo et al., several analogues of fosmido-
mycin were synthesized and evaluated against recombinant
DXR from Synechocystis sp. PCC6803 [122]. In this study
the polar phosphonate moiety was modified to a phosphate,
carboxylate and sulfamate while the hydroxamic moiety was
substituted with N-formyl, N-methyl and N-acetyl, N-methyl
or converted to the reversed hydroxamate. It was found that
although the phosphate analogue of FR900098 (10) was de-
termined to be more potent mixed inhibitor of Synechocystis
DXR (Ki = 2nM) than fosmidomycin, due to the phosphate
moiety in its structure, it was prone to cleavage by phopha-
tases that inactivated this compound. The other two changes
led to large increase in the Ki values. Similarly, the hydrox-
amic group was suggested to be highly sensitive to changes
with even a minor modification leading to decreased inhibi-
tion.
In another study by Mercklé et al., a fragment-based ap-
proach was employed in an attempt to understand the inhibi-
tion of DXR by fosmidomycin using a kinetic assay based on
consumption of NADPH and fosmidomycin [125]. A series
of fosmidomycin fragments and cyclic analogues (11) were
synthesized and examined. While the fragments showed
weak inhibition mostly in the mM range the cyclic analogues
showed high-
M binding at best.
Modification of the propylene spacer between the hy-
droxamic moiety and the phophonate moiety has been
scarcely reported in the literature. A previous attempt to
shorten the three-carbon spacer by Hemmi et al. only com-
promised antibacterial activity, which shows that the length
of this carbon chain is critical for optimal activity [123].
More recently, Haemers et al. reported the synthesis of -
aryl-substituted fosmidomycin analogues [124]. In this
study, the influence of aromatic substituents in the -position
of the phosphonate moiety was investigated. The activity of
these analogues was evaluated against E. coli DXR and two
OH O OH
H N P OH R N
OH
O O
12 Cl
Cl
Compound 12
E. coli 0.059
IC50 (μ M) P. falciparum Dd2 0.028
P. falciparum 3D7 0.090
Fig. (11). Conformationally restricted fosmidomycin analogues.
Current Pharmaceutical Design, 2007, Vol. 13, No. 11 1171
P. falciparum strains. Of the several active analogues that
were reported, compound 12 (Fig. 11) was found to be 3
times more potent than FR900098 in inhibiting the growth of
P. falciparum. Moreover, the authors have suggested that the
presence of the aromatic ring would improve the ability of
these compounds to cross the parasitic cell membrane.
The synthesis of conformationally restricted fosmidomy-
cin analogues has also been reported by incorporating the C-
1 and C-2 atoms in a three-membered ring [126]. The (1R,
2S)-trans-analogue 13 showed an important inhibitory activ-
ity toward E. coli DXR, whereas 14 appeared to be as effec-
tive as fosmidomycin in growth inhibition of Dd2 and 3D7
strains of P. falciparum. Conversely, synthesis of its -
phenyl-substituted analogue led only to the cis-isomer 15,
which appeared to have no significant activity toward E. coli
DXR.
In addition to the aforementioned inhibitors, several
structural analogues of DXP have shown inhibitory activity
against DXR. Two DXP analogues, each lacking a hydroxyl
group at C-3 (16) and C-4 (17) respectively, were designed
and synthesized as substrates for DXR. However, they were
found to be reversible, mixed-type inhibitors of the E. coli
DXR with Ki values of 800
M and 120
M respectively
(Fig. 12) [127]. This study established the importance of
hydroxyl groups in enzyme catalysis. A similar conclusion
was reached in another study, in which the corresponding 3-
and 4-fluoro analogues (18, 19) of DXP acted as noncom-
petitive inhibitors with Ki values of 444
M and 733
M
respectively [128].
Phaosiri and Proteau reported the synthesis of six DXP
analogues and their evaluation against recombinant Synecho-
cystis PCC6803 DXP reductoisomerase [129]. Five of these
acted as relatively weak competitive inhibitors when com-
pared to fosmidomycin. In accordance with the previous
report, the absence of the C3 hydroxyl (16, Ki = 150
M)
and C4 hydroxyl (17, Ki = 30
M), the Km DXP for both of
which was found to be approximately six-fold lower than Ki,
supports the idea that the hydroxyl at these positions are not
critical for binding but essential for catalysis. Moreover, the
H O OH H
P OH N
OH
H O HO P O
OH
13 R=H
15
14 R=CH3
13 14 15
0.313 0.050 > 30
2.0 0.48 --
2.1 0.32 --
1172 Current Pharmaceutical Design, 2007, Vol. 13, No. 11 Singh et al.

Ki for compound 20 and the Km for DXP were found to be
similar, indicating the importance of the stereochemistry of
both hydroxyl moieties for catalysis. The inhibitory activity
of compound 21 was explained to be due to either the incor-
rect alignment of the compound within the active site or due
to a steric interaction upon rearrangement. In fact, a recently
reported mutation study, using this compound, confirmed
that it was steric interaction with an amino acid side chain
that prevents DXR from using the compound as a substrate
[96]. It was suggested that the lack of turnover of compound
22 (Fig. 12) by the enzyme was perhaps, due to lower elec-
trophilicity at the carbonyl moiety when compared to the
substrate DXP.
Synthesis and evaluation of fluoro analogues of DXP
[130] (23, 24) as modest inhibitors with IC50 values of 2.0
and 3.4 mM respectively, and 6.2 mM for compound 21, also
revealed the importance of steric interactions within the ac-
tive site substantiating the findings of Phaosiri et al.
More recently, the same group reported the synthesis of
six DXP analogues of which four compounds (22; Fig. 12
and 25-27; Fig. 12) inhibited the E. coli DXR with IC50 val-
ues ranging from 0.25 to 1.0 mM. Their activity was attrib-
uted to their size that approximated that of the substrate,
DXP, and their ability to coordinate with the active site diva-
lent metal ion [131]. Although none of the DXP analogues
were as potent as fosmidomycin, inhibition by 22 and 25-27
suggested that negatively charged or neutral donor atoms are
accepted as chelators of the active site Mg2+ ion. Inhibition
by analogue 27 demonstrates that a carbonyl group is not
required for inhibition. Interestingly, amine 28 (Fig. 13) did
not inhibit the enzyme. Since Mg2+ forms complexes with
amines, the lack of inhibition may reflect that the amino
group in 28 is protonated. Also, the hydroxylamine analogue
29 (Fig. 13) did not possess inhibitory activity that was at-
tributed to the extremely confined volume of the active site,
which is observed after the enzyme binds its substrates. The
chain of analogue 29 was one atom longer than that of the
potent hydroxamic acid isostere of fosmidomycin, stressing
the importance of the three-carbon chain length separating
the hydroxamic and the phophonate group.
The synthesis of two novel related inhibitors of DXR, the
4-(Hydroxyamino)-4-oxobutylphosphonic acid, 30 (Fig. 14)
and its methylated analogue, 31 (Fig. 14) has been reported
by Kuntz et al. [132]. The two inhibitors were characterized
by the same features, a negatively charged phosphonate moi-
ety and a chelating hydroxamate group linked by a chain of
the same length as that found between the functional groups
of fosmidomycin, but in a different arrangement. Like fos-
midomycin, the hydroxamic acids 30 and 31 were also com-
petitive slow-binding inhibitors with the Ki values of both
compounds against DXP as 169 nM for 30 and 54 nM for 31.
In addition to targeting the enzyme active site, ap-
proaches focused on developing inhibitors to block the
NADPH binding site of the enzyme have also been investi-
gated. Several novel adenosine derivatives that displayed
moderate but significant activity against the P. falciparum

O OH O F O
P O- P O- P O-
O O O
O- O- O-
O OH O O OH

16 17 18

OH O OH O OH O
P O- P O- P O-
O O O
O- O- O-
O F O OH O OH

19 20 21

OH O OH O OH O
H2N P O- F3C P O- HF2C P O-
O O
O- O- O-
O OH O OH O OH

22 23 24

Compound 16 17 18 19 20 21 22 23 24

E. coli 800 120 -- -- -- 6200 253 2000 3400

IC50 (μ M)
Synechocystis PCC6803 150 30 444 733 180 630 90 -- --

Fig. (12). Analogues of substrate, DXP as DXR inhibitors.

MEP Pathway for Novel Antimalarial, Antibacterial and Herbicidal Drug Discovery
OH O OH
HO P O-
O O
O-
O OH
25
NH2 OH
O O
O OH
28
Compound 25
IC50 (μM) - E. coli 551
Fig. (13). Substrate analogues as DXR inhibitors.
strain Dd2 in the low-micromolar range were reported for
DXR inhibition by Herforth et al. However, the binding to
the NADPH binding site was not confirmed in the study
[133]. Also, the fact that the E. coli DXR was utilized for
DXR inhibition studies when the in vivo target would be the
P. falciparum DXR further complicates the issue. Addition-
ally, application of a high-throughput screen to DXR, which
is based on the ability of a compound to compete with a pep-
tide “surrogate ligand”, at either or both DXP and NADPH
binding sites of the E. coli DXR enzyme, has also been ex-
ploited. A library of 32,000 compounds was screened from
which 89 potent and specific inhibitors were registered as
hits. However, the structure-activity data for these com-
pounds have not been reported [134].
OH O OH
HN P O- N P
O O
O-
O O
30
31
Fig. (14). Inhibitors of DXR.
Molecular modeling techniques have been employed to
establish a detailed and extensive structure-activity relation-
ship for forty-three fosmidomycin analogues, tested as in-
hibitors of E. coli DXR in order to predict affinity data for
new inhibitors [135]. The adaptations of fields for molecular
comparison (AFMoC) approach, a 3D-QSAR technique that
incorporated information about the interaction properties of
the surrounding protein available from several published
DXR X-ray structures, was implemented. This technique
was shown to overcome the drawbacks of field-based meth-
ods that relies solely on ligand-based information and makes
no use of available information about the interaction proper-
ties of the surrounding protein from the existing crystal
structure. The binding affinities of the inhibitors spanned
over 4 logarithmic units. The training set consisted of 27
Current Pharmaceutical Design, 2007, Vol. 13, No. 11 1173
O OH OH O
O P O- P O-
O
O- O-
O OH O OH
26 27
O OH OH O
P O- HN P O-
O- O-
O OH
29
26 27 28 29
1024 310 > 5000 > 5000
compounds while the test set consisted of 16 compounds. A
statistically significant model with the regression coefficient,
r2 = 0.86 and predictive power, q2 = 0.46 was obtained.
Recently, a 3D model of DXR enzyme from P. falcipa-
rum has also been determined by means of comparative
modeling by Singh et al. [136]. In this study, a structure of
the enzyme-inhibitor complex was also modeled by docking
fosmidomycin into the putative active site. The proposed
model helped to identify several critical ligand-receptor con-
tact points in the binding site that might serve as the key mo-
lecular determinants of ligand recognition by the DXR en-
zyme. The authors suggest that the model may be used as a
significant tool to enhance the understanding of interactions
of DXR inhibitors with the protein at the atomic level. Such
modeling studies may prove useful in the early design and
O development of inhibitors by either de novo drug design or
O- virtual screening of large chemical databases leading to de-
velopment of new agents against the enzyme.
O-
CONCLUSION
Despite substantial progress, the quest for therapeutically
significant inhibitors of DXR has not yet led to the develop-
ment of drugs that are clinically useful. Several inhibitors of
DXR have been identified, with fosmidomycin and its acetyl
analogue being the most potent to date. Although these com-
pounds exhibit poor pharmacological properties and proba-
bly do not have the potential to become drugs, they provide a
reference for the evaluation of DXR inhibition in cell lines.
Studies on analogues of fosmidomycin suggest that it will be
difficult to improve upon the inhibition qualities of fosmi-
domycin. Although several X-ray crystal structures of DXR
have been published, these structures still leave the active
fosmidomycin conformation and the detailed reaction
mechanism undetermined. The evaluation of DXR inhibition
by the fosmidomycin analogues should aid in defining the
structural requirements for the design of potent DXR inhibi-
tors in conjunction with the X-ray structural data and avail-
able SAR studies. Efforts towards understanding and eluci-
1174 Current Pharmaceutical Design, 2007, Vol. 13, No. 11 Singh et al.

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