Antimalarial and structural studies of pyridine-containing inhibitors of 1-deoxyxylulose-5-phosphate reductoisomerase

Article abstract of DOI:10.1021/ml300419r

1-Deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) in the nonmevalonate isoprene biosynthesis pathway is a target for developing antimalarial drugs. Fosmidomycin, a potent DXR inhibitor, showed safety as well as efficacy against Plasmodium falciparum malaria in clinical trials. On the basis of our previous quantitative structure-activity relationship (QSAR) and crystallographic studies, several novel pyridine-containing fosmidomycin derivatives were designed, synthesized, and found to be highly potent inhibitors of P. falciparum DXR (PfDXR) having K_i values of 1.9-13 nM, with the best one being ～11× more active than fosmidomycin. These compounds also potently block the proliferation of multidrug resistant P. falciparum with EC ₅₀ values as low as 170 nM. A 2.3 A? crystal structure of PfDXR in complex with one of the inhibitors is reported, showing that the flexible loop of the protein undergoes conformational changes upon ligand binding and a hydrogen bond and favorable hydrophobic interactions between the pyridine group and the PfDXR account for the enhanced activity.

Full text of DOI:10.1021/ml300419r

Letter
pubs.acs.org/acsmedchemlett
Antimalarial and Structural Studies of Pyridine-Containing Inhibitors
of 1‑Deoxyxylulose-5-phosphate Reductoisomerase
Jian Xue,† Jiasheng Diao,† Guobin Cai,†,‡ Lisheng Deng, Baisong Zheng, Yuan Yao, and Yongcheng Song*
Department of Pharmacology, Baylor College of Medicine, 1 Baylor Plaza, Houston, Texas 77030, United States
S
* Supporting Information
ABSTRACT: 1-Deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) in
the nonmevalonate isoprene biosynthesis pathway is a target for developing
antimalarial drugs. Fosmidomycin, a potent DXR inhibitor, showed safety as
well as efficacy against Plasmodium falciparum malaria in clinical trials. On the
basis of our previous quantitative structure−activity relationship (QSAR) and
crystallographic studies, several novel pyridine-containing fosmidomycin
derivatives were designed, synthesized, and found to be highly potent
inhibitors of P. falciparum DXR (Pf DXR) having Ki values of 1.9−13 nM, with
the best one being ∼11× more active than fosmidomycin. These compounds
also potently block the proliferation of multidrug resistant P. falciparum with EC50 values as low as 170 nM. A 2.3 Å crystal
structure of Pf DXR in complex with one of the inhibitors is reported, showing that the flexible loop of the protein undergoes
conformational changes upon ligand binding and a hydrogen bond and favorable hydrophobic interactions between the pyridine
group and the PfDXR account for the enhanced activity.
KEYWORDS: 1-deoxy-D-xylulose-5-phosphate reductoisomerase, antimalarial activity, X-ray protein crystallography, inhibitor design
ukaryotic parasite Plasmodium spp. are the causative agents
Eof malaria, among which Plasmodium falciparum produces
the most severe form of human malaria and is responsible for
the vast majority of deaths of malaria patients (∼1 million/
year). In addition, this dreadful number could be rising because
of the increased drug resistance of P. falciparum to inexpensive
drugs such as chloroquine. Since 2001, the World Health
Organization (WHO) has strongly recommended the use of
artemisinin-based combination therapies to treat malaria.1
However, Plasmodium parasites are known to be able to
develop drug resistance. After 10 years, P. falciparum strains
that are resistant to these new drug combinations have been
observed.1 There is therefore a pressing need to develop new
antimalarial drugs.
and terpenoids, including important substances such as C15−
C55 isoprenyl (e.g., farnesyl and undecaprenyl) diphosphates
for anchoring proteins to the cell membrane. DXR is essential
for the growth of all of these species including P. falciparum. In
addition, humans and animals use the mevalonate pathway to
synthesize IPP and DMAPP, making DXR an attractive target
for discovering novel antimalarial drugs.3,4
Fosmidomycin (1, Chart 1) and its close analogue FR900098
(2) were found to be potent DXR inhibitors5 and possess
antibacterial and -malarial activities.6 Particularly, several recent
Chart 1. Structures of DXR Inhibitors
1-Deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) is
the second enzyme in the nonmevalonate isoprene biosynthesis
pathway, catalyzing the reductive isomerization of 1-deoxy-D-
xylulose-5-phosphate (DXP) to 2-methyl-D-erythritol-4-phos-
phate (MEP) using Mg2+ (or Mn2+) and NADPH as the
cofactors, as schematically illustrated in Figure 1.2 This is used
by most bacteria, as well as apicomplexan parasites such as
Plasmodium spp., to make isopentenyl diphosphate and
dimethylallyl diphosphate. These two diphosphate compounds
are the only precursors for the biosynthesis of all isoprenoids
Received: November 25, 2012
Accepted: December 11, 2012
Published: December 11, 2012
Figure 1. Reaction catalyzed by DXR.
© 2012 American Chemical Society
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clinical trials showed that in combination with clindamycin, 1 is
safe for human use and effective against P. falciparum malaria.7,8
However, because of a short half-life in plasma (∼1 h) as well as
poor oral availability of 1, considerable interest has therefore
been generated to develop potent, lipophilic DXR inhibitors
with the hope that these compounds could have broad anti-
infective activities as well as improved pharmacokinetic
properties.9−20
substituted 6 and 7 were prepared similarly, starting from
benzaldehyde.
The enzyme inhibitory activities of the newly synthesized
compounds 4−7 were first tested against recombinant EcDXR.
As shown in Table 1, compounds 4p and 5m were found to
Table 1. Activity of DXR Inhibitors 1−7
In our early work,19,20 a series of lipophilic, pyridine- or
quinoline-containing phosphonate compounds, such as com-
pound 3 in Chart 1, were found to be a new class of DXR
inhibitors. Our structure−activity relationship (SAR) and
quantitative SAR (QSAR) studies all showed the importance
of the presence of an electron-deficient aromatic ring, such as a
pyridine, at the α-position of the phosphonate group, as shown
in Figure S1a in the Supporting Information. In addition,
superposition of the crystal structures of Escherichia coli DXR
(EcDXR) in complex with 1 and 3 (Figure S1b in the
Supporting Information) suggests fosmidomycin derivatives
with an α-pyridine substituent, such as compounds 4m, 5m, 4p,
and 5p (m stands for meta-substituted pyridinyl compound and
p stands for para-substituted pyridinyl) shown in Chart 1, could
be particularly active, since these compounds likely have all
favorable interactions with the protein as found for both 1 and
3. Compounds 6 and 7 (Chart 1) with a more electron-rich
phenyl ring were included in the study as a comparison.
The general method for synthesizing compounds 4−7 is
shown in Scheme 1. Pyridine-3-carboxaldehyde was reduced
IC50 against P. falciparum
enzyme Ki (μM)
EcDXR PfDXR
proliferation (μM)
3D7 strain
Dd2 strain
1
0.034
0.087
0.035
0.042
0.082
1.5
0.021
0.013
0.0089
0.0019
0.013
0.48
1.17
0.34
0.18
0.44
0.63
0.44
0.18
0.17
0.31
0.46
4m
4p
5m
5p
6
a
a
NT
NT
7
0.93
0.085
NT
3.5
1.7
CQ
NT
0.021
0.12
a
NT, not tested.
exhibit potent activity with Ki values of 35 and 42 nM,
respectively, being as active as 1 (Ki = 34 nM). Compounds 4m
and 5p (Ki = 87 and 82 nM) have only slightly reduced
inhibition against EcDXR. However, compounds 6 and 7
containing a phenyl substituent were found to be markedly less
active with Ki values of 1.5 and 0.93 μM, respectively, showing
an average of ∼20-fold activity reduction as compared to
pyridine-containing compounds 4 and 5.
E. coli DXR has been used in the vast majority of previous
studies, although EcDXR inhibition may not be relevant and
predictive in the context of developing antimalarial drugs. To
this end, we next tested the inhibitory activities of these
compounds against recombinant Plasmodium falciparum DXR
(Pf DXR) enzyme. In our previous work,21 1 was determined
also to be a potent inhibitor of Pf DXR with a Ki value of 21
nM. We were pleased to find that pyridine-containing
compounds 4m,p and 5m,p possess considerably higher
inhibitory activity against PfDXR, as can be seen in Table 1
and representatively shown in Figure S2 in the Supporting
Information. The Ki values of these compounds range from 1.9
to 13 nM, with the best compound 5m (Ki = 1.9 nM) having an
acetyl and a pyridin-3-yl group being ∼11× more active than 1.
The phenyl-containing compounds 6 and 7 again exhibited
significantly lowered inhibitory potencies with Ki values of 480
and 85 nM, showing that the pyridine groups in 4m,p and 5m,p
are able to provide an average of ∼30-fold enhanced activity
against PfDXR.
a
Scheme 1. General Synthesis for Compounds 4−7
a
Reagents and conditions: (i) NaBH4, MeOH. (ii) SOCl2. (iii)
HP(O)(OEt)2, NaH, THF, 43% for three steps. (iv) n-BuLi, then allyl
bromide, −78 − 0 °C. (v) O3, −78 °C, then Me2S. (vi) NH2OBn. (vii)
NaBH3CN, pH 3, MeOH, 40% from 8. (viii) For R = H, HCOOH/
Ac2O; for R = Me, Ac2O. (ix) TMSBr. (x) H2, Pd/C, 76% from 10.
With these highly potent inhibitors of P. falciparum DXR in
hand, we next investigated the antimalarial activities of these
compounds against the proliferation of erythrocyte-stage P.
falciparum. The 3D7 strain of P. falciparum is sensitive to
current antimalarial drugs, while the Dd2 strain is multidrug
resistant, including chloroquine (CQ), pyrimethamine, and
mefloquine. Chloroquine and 1 were included in our studies as
positive controls. As also shown in Table 1, 1 was found to have
good activity against the growth of P. falciparum strains 3D7
and Dd2 with EC50 values of 1.2 and 0.44 μM, respectively,
while chloroquine exhibits more activity against the 3D7 strain
(EC50 = 21 nM) than it does against the drug-resistant Dd2
strain (EC50 = 120 nM). The antimalarial activities of 1 and
chloroquine are similar to those reported previously.6 As
compared to 1, the novel pyridine-containing DXR inhibitors
and converted to 3-picolinyl chloride, which was reacted with
sodium salt of diethylphosphite to give 8. It was alkylated with
allyl bromide, followed by ozonization to give aldehyde 9.
Upon reductive amination with O-benzyl-hydroxylamine and
NaBH3CN, the resulting hydroxylamine 10 was formylated or
acetylated, followed by hydrolysis using bromotrimethylsilane
(TMSBr) and hydrogenation, to give 4m or 5m. Compounds
4p and 5p were similarly prepared, starting from pyridine-4-
carboxaldehyde. However, analogous compounds with an ortho-
substituted pyridinyl cannot be synthesized from pyridine-2-
carboxaldehyde, due to instability of the corresponding
aldehyde 9 (or its O-benzyl-hydroxylamine oxime). Phenyl-
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Figure 2. (A) Overall structure of PfDXR in complex with Mn2+ (brown sphere), 5p, and NADPH. (B) Close-up view of the active site of
PfDXR:5p. (C) Active sites of the aligned structures of PfDXR:5p (in green) and PfDXR:1 (in orange) complexes. For clarity, only the flexible
loops containing residues 290−299 of the two structures are shown as curved lines. (D) Aligned structures of PfDXR:5p (in green) and EcDXR:3
(in purple) complexes. The colored curved lines represent the flexible loops of the proteins.
were found to have enhanced antimalarial activities. Formyl
compounds 4m and 4p exhibit potent antimalarial activity
(EC50 = 340 and 180 nM, respectively) against the 3D7 strain.
Moreover, regardless of the drug resistance of the Dd2 strain,
these two compounds possess even more pronounced activity
against this strain of the parasite with EC50 values of 180 and
170 nM. The acetyl-containing compounds 5m and 5p have,
however, relatively lowered antimalarial activity, as compared to
4m and 4p with a formyl group. The EC50 values of 5m are 440
and 630 nM against 3D7 and Dd2, respectively, and those of 5p
are 310 and 460 nM. Compound 7 with a phenyl group was
found have a relatively weak antimalarial activity against P.
falciparum strains 3D7 and Dd2 (EC50 = 3.5 and 1.7 μM,
respectively).
It is noteworthy that all of these DXR inhibitors including 1
exhibit more activity against chloroquine-resistant P. falciparum
Dd2 strain, demonstrating that these compounds working in a
different mechanism have the potential to become a new class
of antimalarials without cross-drug resistance. It is also
interesting that compounds 4m and 4p possess enhanced
activities against the proliferation of P. falciparum, although
they are relatively less active against the recombinant enzyme
than the acetyl-containing inhibitor 5m. This could be
attributed to lowered cell membrane permeability of 5m,
since these inhibitors need to pass both membranes of
erythrocyte and the parasite to reach their cellular target
PfDXR.
Potential toxicities of the newly synthesized compounds were
assessed using human noncancerous fibroblast WI-38 cells. All
of the pyridine-containing compounds 4 and 5 have essentially
no cytotoxicity with EC50 values of >300 μM against the growth
of WI-38 cells, showing a very high selectivity index of >1700-
fold. These compounds therefore represent promising leads for
further antimalarial drug development.
The next question of interest is how these pyridine-
containing inhibitors bind to P. falciparum DXR. Prior
structural studies were mostly focused on EcDXR.17,19,22−25
Currently, only two crystal structures of PfDXR in complex
with 1 and 2 are available.26 However, it is known that the
conformation of DXR, especially for the flexible loop at the
active site, changes considerably when a ligand having a
different property (e.g., hydrophobicity) from 1 and 2 binds.19
Therefore, crystallographic studies for the binding of these
pyridine-containing inhibitors to PfDXR are needed.
We determined the crystal structure of a quaternary complex
of Pf DXR with compound 5p, NADPH, and a Mn2+, at 2.3 Å.
Parameters for data processing and refinement are listed in
Table S1 in the Supporting Information. As shown in Figure
2A, the protein crystallizes as a homodimer, with each
monomer being almost identical to the other. The binding
conformation of NADPH is very similar to that in previous
reported structures. Each of the two Mn2+ ions is anchored to
Pf DXR via coordination to three carboxylate groups of the
residues Asp231, Glu233, and Glu315 (Figure 2B). The
electron density and omit maps of inhibitor 5p are shown in
Figure S3 in the Supporting Information. In addition, the
racemic form of 5p was used in the study, and at the 2.3 Å
resolution, it is not unambiguous to assign the absolute
configuration of the α-carbon atom of compound 5p (Figure S3
in the Supporting Information). Fifty percent occupancy for
each enantiomer was therefore assigned in the final structure.
Figures 2B and S4 in the Supporting Information show the
close-up view of the PfDXR active site and detailed protein−
ligand interactions. As always can be seen in other DXR−
hydroxamate inhibitor complexes,22−25 the two O atoms of the
hydroxamate group of 5p chelate the central Mn2+, with their
distances being 2.1 and 2.4 Å. The acetyl O atom also forms a
hydrogen bond with the side chain −OH of Ser232. The
negatively charged phosphonate group of compound 5p is
recognized by a polar environment, forming five hydrogen
bonds as well as electrostatic interactions with Lys312, Asn311,
Ser306, Ser270, and Ser269. The pyridine group of 5p was
found to be favorably located in a mainly hydrophobic pocket
defined by residues His293, Trp296, Met298, Cys338, and
Pro358, which is not present in the reported crystal structure of
the Pf DXR:1 complex.26 In addition, a hydrogen bond between
the N of the pyridine ring of 5p and the −SH of Cys338 was
observed, with the distance of N···H−S being 3.4 Å. Because
the structurally similar compound 7 cannot form a hydrogen
bond with Cys338, the interaction could therefore contribute to
the observed ∼6× more inhibitory potency of 5p. Moreover,
this hydrogen bond, together with the favorable hydrophobic
interactions described above, could be responsible for the
enhanced activity of 5p against Pf DXR as compared to 1. It is
also remarkable that the methyl group of 5p has favorable
hydrophobic interactions with Met298 and Trp296. This could
explain that the acetyl-containing DXR inhibitors (e.g., 5m and
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ACS Medicinal Chemistry Letters
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7) in general possess increased enzyme activity than the
corresponding compounds having a formyl group.
promise to overcome the widespread drug resistance of the
malaria parasites. The crystal structure of the PfDXR:5p
complex reveals the exact binding pose of the highly potent
inhibitor. The backbone of 5p binds to Pf DXR in a similar
fashion as 1. Conformational changes were observed for the
flexible loop of PfDXR to form a mainly hydrophobic pocket
that holds and has favorable interactions with the pyridine
group of the inhibitor. In addition, the hydrogen bond between
the pyridine N atom and the Cys338 should also contribute
considerably to the increased potency.
Alignment of the structures of PfDXR:5p and PfDXR:1 was
performed to find the conformational differences of the protein.
As shown in Figures S5a in the Supporting Information, these
two structures can be aligned very well with a root-mean-square
(rms) deviation for the protein backbone being 0.5 Å. The only
notable difference is the conformation of the flexible loop
consisting of residues 290−299, which is part of the active site
of PfDXR. As shown in Figure 2C, the loop is located much
closer to the ligand-binding site in the PfDXR:1 structure, with
the big, hydrophobic indole ring of Trp296 being right beneath
the carbon skeleton of 1. This conformation allows the active
site to be largely separated from the solvent, which is
considered to be important for the enzyme activity or for the
tight binding of polar inhibitor 1. For example, a previous
mutagenesis study showed that replacing the corresponding
Trp204 in Synechocystis DXR with an Ala, Val, or Leu residue
renders the enzyme completely inactive.27 The only weakly
active mutation is Trp204Phe having a smaller, aromatic phenyl
side chain. In the Pf DXR:5p structure, the flexible loop was
observed to move up to 4.5 Å away from the center of the
active site to hold the pyridine of 5p and allow the Trp296
indole and several other residues to have favorable hydrophobic
interactions with the inhibitor.
We next compared the binding of inhibitor 5p to Pf DXR
with that of another pyridine-containing inhibitor 3 to
EcDXR.19 Protein alignment indicated that these two structures
have a rms deviation for the protein backbone of 1.9 Å (Figure
S5b in the Supporting Information). The close-up view of the
active site is shown in Figure 2D, in which the two
phosphonate groups of 5p and 3 are very close to each
other, with the distance between the two P atoms being 0.5 Å.
The two pyridine rings were found also to be located in the
same hydrophobic pocket. Relatively large differences can be
observed with respect to the conformation of the flexible loops
of these two enzymes. Not only the backbones of the loops
exhibit an average distance for the Cα atoms of ∼4 Å between
the two structures, but the orientations of the side chains of
several residues are different as well. These conformational
alterations could be driven mainly by two factors. First,
compound 3 does not have a hydroxamate moiety. Second, the
indole ring of Trp211 of EcDXR has a π−π interaction with the
pyridine group of 3. The steric as well as π−π stacking
interactions allow the EcDXR flexible loop to shift ∼4 Å on
average toward the pyridine binding site. For the Pf DXR:5p
complex, the presence of the hydroxamate moiety seems not to
favor the formation of a similar π−π interaction between
Trp296 and the pyridine, due likely to the steric congestion
between the hydroxamate moiety and the Trp296 indole group.
Rather, the hydrophobic side chains of the flexible loop, such as
Met298, Trp296, and His293, together with those of residues
Pro358 and Cys338 from other chains, are orientated to create
a generally hydrophobic environment to have favorable
interactions with the pyridine ring as well as the methyl
group of compound 5p.
ASSOCIATED CONTENT
* Supporting Information
■
S
Supplementary Figures S1−S5, Supplementary Table, and
Experimental Section. This material is available free of charge
via the Internet at http://pubs.acs.org.
Accession Codes
The coordinates for the PfDXR:5p complex have been
deposited in Protein Data Bank as entry 4GAE.
AUTHOR INFORMATION
Corresponding Author
*Tel: 713-798-7415. E-mail: ysong@bcm.edu.
■
Present Address
‡Department of Parasitology, Wuhan University School of
Basic Medical Science, Wuhan 430071, China.
Author Contributions
†These authors contributed equally.
Funding
This work was supported by a grant (R21AI088123) from the
National Institute of Health (NIH) to Y.S. We thank the staff of
the X-ray Crystallography Facility at Baylor College of
Medicine for assistance in data collection.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
DXR, 1-deoxy-D-xylulose-5-phosphate reductoisomerase;
EcDXR, Escherichia coli DXR; Pf DXR, Plasmodium falciparum
DXR
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