Potent inhibitors of a shikimate pathway enzyme from Mycobacterium tuberculosis: Combining mechanism- and modeling-based design

Article abstract of DOI:10.1074/jbc.M110.211649

Tuberculosis remains a serious global health threat, with the emergence of multidrug-resistant strains highlighting the urgent need for novel antituberculosis drugs. The enzyme 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyzes the first step of the shikimate pathway for the biosynthesis of aromatic compounds. This pathway has been shown to be essential in Mycobacterium tuberculosis, the pathogen responsible for tuberculosis. DAH7PS catalyzes a condensation reaction between P-enolpyruvate and erythrose 4-phosphate to give 3-deoxy-D-arabino-heptulosonate 7-phosphate. The enzyme reaction mechanism is proposed to include a tetrahedral intermediate, which is formed by attack of an active site water on the central carbon of P-enolpyruvate during the course of the reaction. Molecular modeling of this intermediate into the active site reported in this study shows a configurational preference consistent with water attack from the re face of P-enolpyruvate. Based on this model, we designed and synthesized an inhibitor of DAH7PS that mimics this reaction intermediate. Both enantiomers of this intermediate mimic were potent inhibitors of M. tuberculosis DAH7PS, with inhibitory constants in the nanomolar range. The crystal structure of the DAH7PS-inhibitor complex was solved to 2.35 A. Both the position of the inhibitor and the conformational changes of active site residues observed in this structure correspond closely to the predictions from the intermediate modeling. This structure also identifies a water molecule that is located in the appropriate position to attack the re face of P-enolpyruvate during the course of the reaction, allowing the catalytic mechanism for this enzyme to be clearly defined.

Full text of DOI:10.1074/jbc.M110.211649

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 18, pp. 16197–16207, May 6, 2011
© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Potent Inhibitors of a Shikimate Pathway Enzyme from
Mycobacterium tuberculosis
□
S
COMBINING MECHANISM- AND MODELING-BASED DESIGN*
Received for publication, December 13, 2010, and in revised form, February 7, 2011 Published, JBC Papers in Press, March 15, 2011, DOI 10.1074/jbc.M110.211649
Sebastian Reichau‡1, Wanting Jiao‡2, Scott R. Walker‡, Richard D. Hutton‡, Edward N. Baker§, and Emily J. Parker‡3
From the ‡Biomolecular Interaction Centre and Department of Chemistry, University of Canterbury, Christchurch 8140 and the
§Maurice Wilkins Centre for Molecular Biodiscovery and School of Biological Sciences, University of Auckland,
Auckland 1010, New Zealand
Tuberculosis remains a serious global health threat, with
the emergence of multidrug-resistant strains highlighting the
urgent need for novel antituberculosis drugs. The enzyme 3-de-
oxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS)
catalyzes the first step of the shikimate pathway for the biosyn-
thesis of aromatic compounds. This pathway has been shown to
be essential in Mycobacterium tuberculosis, the pathogen
responsible for tuberculosis. DAH7PS catalyzes a condensation
reaction between P-enolpyruvate and erythrose 4-phosphate to
give 3-deoxy-D-arabino-heptulosonate 7-phosphate. The
enzyme reaction mechanism is proposed to include a tetrahe-
dral intermediate, which is formed by attack of an active site
water on the central carbon of P-enolpyruvate during the course
of the reaction. Molecular modeling of this intermediate into
the active site reported in this study shows a configurational
preference consistent with water attack from the re face of
P-enolpyruvate. Based on this model, we designed and synthe-
sized an inhibitor of DAH7PS that mimics this reaction inter-
mediate. Both enantiomers of this intermediate mimic were
potent inhibitors of M. tuberculosis DAH7PS, with inhibitory
constants in the nanomolar range. The crystal structure of the
resistant strains of Mycobacterium tuberculosis, the pathogen
that causes the lung disease, highlights the need for rapid devel-
opment of new antibacterial drugs to combat tuberculosis
(1–4).
3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase
(DAH7PS)4 is an important enzyme in M. tuberculosis and
other pathogens. It catalyzes the first committed step in the
shikimate pathway, which is responsible for the biosynthesis of
aromatic amino acids and other essential aromatic metabolites
in microorganisms, plants, and apicomplexan parasites (5–7).
This pathway is absent in humans, and inhibitors of amino acid
biosynthesis have been shown to be effective antimicrobial and
herbicidal agents (8, 9). Gene disruption studies have demon-
strated that M. tuberculosis is not viable if the shikimate path-
way is not operational (10). These findings make DAH7PS an
attractive target for drug development.
DAH7PS catalyzes the aldol-like condensation of P-enolpy-
ruvate and D-erythrose 4-phosphate (E4P) to yield 3-deoxy-D-
arabino-heptulusonate 7-phosphate (Fig. 1a). The reaction
mechanism has been subject to extensive study, and many of
the key details of the mechanism have been elucidated (11–16).
The reaction occurs stereospecifically with respect to both sub-
strates, with the si face of P-enolpyruvate attacking the re face of
E4P. A divalent metal ion present in the active site is essential
for activity. The reaction takes place with cleavage of the C–O
bond of P-enolpyruvate rather than the O–P bond, requiring
water to attack C2 of P-enolpyruvate at some stage during the
reaction.
˚
DAH7PS-inhibitor complex was solved to 2.35 A. Both the posi-
tion of the inhibitor and the conformational changes of active
site residues observed in this structure correspond closely to the
predictions from the intermediate modeling. This structure also
identifies a water molecule that is located in the appropriate
position to attack the re face of P-enolpyruvate during the
course of the reaction, allowing the catalytic mechanism for this
enzyme to be clearly defined.
A mechanism consistent with the data published to date
starts with nucleophilic attack of P-enolpyruvate at the E4P
aldehyde moiety, resulting in the formation of oxocarbenium
species 1 (Fig. 1b). This oxocarbenium ion 1 can be attacked by
an active site water to form phosphohemiketal 2. It is notewor-
thy that water can potentially attack from either face of 1, giving
rise to two possible diastereoisomers of tetrahedral intermedi-
ate 2, differing in their absolute configuration at C2. Although
this stereogenic center is transient and the stereochemical
information is lost by elimination of phosphate in the final step
to generate the product DAH7P (3), the geometry of the
enzyme active site is likely to favor stereoselective attack of
Tuberculosis remains a serious global health threat with over
a million deaths per year. The recent emergence of multidrug-
* This research was funded in part by the Maurice Wilkins Centre for Molecu-
lar Biodiscovery.
Theatomiccoordinatesandstructurefactors(code3PFP)havebeendepositedin
the Protein Data Bank, Research Collaboratory for Structural Bioinformatics,
Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
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The on-line version of this article (available at http://www.jbc.org) contains
supplemental text and Figs. S1–S4.
1 Supported by a University of Canterbury Doctoral Scholarship and a New
Zealand International Doctoral Research Scholarship (NZIDRS).
2 Supported by a New Zealand Tertiary Education Commission Top Achiever
Doctoral Scholarship.
4 The abbreviations used are: DAH7PS, 3-deoxy-D-arabino-heptulosonate
7-phosphate synthase; MtuDAH7PS, M. tuberculosis DAH7PS; E4P, D-
erythrose 4-phosphate; G3P, glycerol-3-phosphate; KDO8PS, 3-deoxy-D-
manno-octulosonate-8-phosphate synthase; iPr, isopropyl.
3 To whom correspondence should be addressed: Dept. of Chemistry, Univer-
sity of Canterbury, Private Bag 4800, Christchurch. Tel.: 64-3-364-2871; Fax:
64-3-364-2110; E-mail: emily.parker@canterbury.ac.nz.
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Potent Active Site Inhibitors for M. tuberculosis DAH7PS
FIGURE 1. Catalysis by DAH7PS. a, overall reaction catalyzed by DAH7PS. b, proposed mechanism of condensation of P-enolpyruvate (PEP) with E4P. H-Enz,
H-enzyme.
water to form one diastereoisomer of 2 preferentially. In this ing one coordination site potentially free for the carbonyl moi-
way, DAH7P is formed in its acyclic form and cyclizes into its ety of the E4P aldehyde moiety, thereby activating this func-
cyclic pyranose form following release from the enzyme.
tionality to nucleophilic attack. The proposed E4P binding site
3-Deoxy-D-manno-octulosonate 8-phosphate synthase is constituted mostly by a 133KPRS136 motif that is highly con-
(KDO8PS), an enzyme involved in the synthesis of the cell-wall served in the type II subfamily, whereas members of the type I
lipopolysaccharide of Gram-negative bacteria, is structurally DAH7P synthase subfamily display a very similar, also highly
and evolutionary related to DAH7PS. KDO8PS catalyzes an conserved KPRT motif at the equivalent position. The best
analogous reaction between P-enolpyruvate and the five-car- indication of how E4P is bound to the enzyme can be found
bon sugar arabinose 5-phosphate (17). Studies of the reaction in the crystal structure of S. cerevisiae type I DAH7PS
mechanism of KDO8PS suggest that water is activated by the (SceDAH7PS) in complex with the E4P analog, glycerol 3-phos-
active site metal prior to attack at the reaction intermediate phate (G3P) (16). Arginine and threonine from the KPRT motif
(18). Computational and structural studies of KDO8PS indicate interact with the G3P phosphate moiety, whereas the primary
that after activation, water or hydroxide attacks from the si hydroxyl group of G3P forms a hydrogen bond to the backbone
face of P-enolpyruvate, resulting in the overall syn addition of carbonyl of the proline residue of the KPRT motif, and the
arabinose-5-phosphate and a hydroxyl group to the double secondary hydroxyl interacts with the metal-coordinating
bond of P-enolpyruvate (19). Due to lack of comparable data for aspartate.
the reaction catalyzed by DAH7PS, it is unclear whether these
Despite the many similarities in active site architecture and
findings also apply to the reaction catalyzed by DAH7PS. reaction chemistry between DAH7P synthases from different
Despite the similarities in their reaction chemistry, a number of organisms and families, there are some significant distinctions
key structural and mechanistic differences of DAH7PS and of type II DAH7P synthases. For example, Trp280 forms part of
KDO8PS such as divalent metal ion requirement and substrate the P-enolpyruvate binding site in MtuDAH7PS, whereas in the
specificity have also been identified (20).
E. coli and S. cerevisiae enzymes, this residue is replaced by Ala.
M. tuberculosis DAH7PS (MtuDAH7PS) is the only member However, in the type I enzymes, the space occupied by Trp280 is
of the DAH7PS type II family that has been structurally char- occupied by a Tyr residue from another part of the structure.
acterized (21–23). Type II DAH7PS enzymes show very little Another significant distinction between MtuDAH7PS and all
sequence similarity with their type I DAH7PS counterparts, other DAH7P synthases characterized to date is its complex
which are relatively well characterized and are found in organ- mechanism of allosteric regulation, which we have recently
isms such as Escherichia coli (14, 15) and Saccharomyces cerevi- investigated in detail (22). Furthermore, the recent discovery
siae (16, 24). Both type I and type II DAH7PS enzymes share the that M. tuberculosis chorismate mutase is activated in complex
common triosephosphate isomerase (TIM barrel) fold, and with DAH7PS suggests a key role of DAH7PS in the regulatory
mechanistic studies have suggested that the key details of the network of aromatic metabolism of M. tuberculosis (26).
reaction chemistry are similar for enzymes of both DAH7PS
In vitro studies with mechanism-based inhibitors have been
types (25). Despite the low sequence similarity, the active site previously reported for E. coli DAH7PS (27, 28). Recent results
architecture of MtuDAH7PS shows remarkable correspond- in our laboratory have shown that extension of inhibitors tar-
ence to that of type I enzymes (Fig. 2). P-enolpyruvate is held in geting the P-enolpyruvate binding site to pick up interactions in
place by a tightly knit network of interactions. The P-enolpyru- the E4P binding site leads to increased inhibitor potency (49). In
vate phosphate forms salt bridges to Lys306 (MtuDAH7PS num- addition, carbohydrate-derived compounds, designed as
bering) and Arg337 and forms a hydrogen bond to the backbone mechanism-based inhibitors of both KDO8PS and DAH7PS,
N–H of Glu283, whereas the P-enolpyruvate carboxylate forms were shown to have mild antibacterial properties when tested
a salt bridge to Arg126. The metal ion is coordinated by His369
,
against E. coli, Yersinia enterocolitica, Pseudomonas aerugi-
Glu411, Cys87, and Asp441 in a trigonal pyramidal fashion, leav- nosa, Staphylococcus aureus, and Bacillus subtilis, although
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Potent Active Site Inhibitors for M. tuberculosis DAH7PS
FIGURE 2. Active site architecture of DAH7PS. a, stereo view of the active site of M. tuberculosis DAH7PS (PDB code 3NV8) (22) showing P-enolpyruvate (PEP,
yellow carbons) in its binding site. The manganese iron is represented as a purple sphere. Major interactions between P-enolpyruvate and the enzyme and the
metal coordination sphere are shown as dashed lines. b, stereo view of the active site of S. cerevisiae DAH7PS in complex with P-enolpyruvate, G3P (yellow
carbons), and a Co2ϩ ion (magenta sphere)(PDB code 1OF8) (16). A water molecule (WAT) on the re side of P-enolpyruvate (left in this orientation), which is
proposed to play the role of the substrate water, is shown as a red sphere.
further in vitro studies were suggested to confirm that these cells. The temperature was continuously controlled at 303 K
compounds indeed target KDO8PS and/or DAH7PS (29).
However, to date, no inhibitors for the DAH7PS from the
pathogen M. tuberculosis have been reported. Herein we report
the design, synthesis, and testing of an intermediate mimic as
the first potent inhibitor of MtuDAH7PS in both its racemic
and its enantiomerically pure forms. The crystal structure of
the target enzyme in complex with the inhibitor shows that the
simplified intermediate analog binds in a fashion in accordance
with its design and thus provides valuable insight into both the
DAH7PS reaction mechanism and a lead structure for novel
potent antimycobacterial drugs targeting MtuDAH7PS.
(30 °C) by the use of a jacketed multicell holder, connected to an
external Varian Peltier temperature controller filled with either
water or ethylene glycol.
Expression, Purification, and Crystallization of MtuDAH7PS—
M. tuberculosis DAH7PS was overexpressed and purified as
described previously (21–23). A reservoir solution containing
400 ␮M inhibitor was prepared by dissolving an appropriate
amount of solid rac-4 in crystallization buffer (0.1 M Tris-HCl
buffer at pH 8, 1.5 M ammonium sulfate, and 12% v/v glycerol).
500 ␮l of enzyme stock solution was concentrated to ϳ50 ␮l
using centrifugation with a 10-kDa cutoff membrane, and the
resulting solution was diluted to ϳ500 ␮l using the inhibitor
solution. This process was repeated twice to buffer-exchange
the solution as completely as possible. 1 ␮l of the buffer-ex-
changed enzyme solution (concentration 3–5 mg/ml) was
mixed with 1 ␮l of crystallization buffer. Crystals were grown by
sitting drop vapor diffusion over 300 ␮l of reservoir solution
and appeared within 24 h at 25 °C. Crystals were flash-frozen in
liquid nitrogen in a cryoprotectant solution consisting of crys-
tallization buffer and 20% v/v glycerol.
EXPERIMENTAL PROCEDURES
Analytical Methods—Optical rotations [␣]2D0 were measured
at room temperature on a PerkinElmer Life Sciences Model 341
polarimeter; analyte concentrations are given in g/100 ml. Spe-
cific rotations are reported. NMR spectroscopy was carried out
on a Varian UNITY 300 MHz or Varian INOVA 500 MHz NMR
spectrometer. 1H and 13C NMR spectra are referenced to exter-
nal tetramethylsilane; 31P NMR spectra are referenced to exter-
nal 85% phosphoric acid. Mass spectrometry was performed on
Bruker maXis 3G or Micromass LCT by electrospray ionization
in positive or negative ionization modes.
Structure Determination—X-ray diffraction data were col-
lected at the MX2 beamline at the Australian Synchrotron (see
Table 1). The dataset was integrated using iMosflm (30). The
UV-visible spectrophotometry was carried out on a Varian
Caryꢀ One UV-visible spectrophotometer, in stoppered quartz space group and cell parameters were identical to those of pre-
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Potent Active Site Inhibitors for M. tuberculosis DAH7PS
viously reported structures of MtuDAH7PS allowing phases nium molybdate in 4 M HCl, 0.1 parts 1.5% v/v Triton X-100 in
calculated from the original structure (Protein Data Bank water. The components were mixed in the dark and stirred for
(PDB) code 2B7O) (21) to be used to solve the structure. The 1 h before the solution was filtered through a 0.45-␮m syringe
structure was refined using the CCP4 software package (30) by filter. For the qualitative detection of phosphate-containing
methods analogous to the ones described for the native struc- fractions after anion exchange chromatography, a 20-␮l sample
ture (21). Both enantiomers of inhibitor 4 were built using the of each fraction was mixed with 250 ␮l of Lanzetta reagent, and
Dundee PRODRG2 server (31). The two enantiomers of 4 were the color change was judged by optical inspection. For the
initially positioned manually into the electron density observed quantitative determination of inhibitor concentration, 300 ␮l
in the active site using COOT and then refined at half-occu- of the inhibitor solutions was incubated with 10 ␮l of calf alka-
pancy. The model was optimized using repetitive cycles of line phosphatase solution (5 units/ml in 4 mM MgCl2) for at
model building with COOT and refinement with REFMAC5. least 2 h. To 100 ␮l of the digested sample was added 700 ␮l of
Water molecules were added automatically in COOT and ver- Lanzetta reagent, and the absorbance at 630 nm was deter-
ified using ͉2Fo Ϫ Fc͉ and ͉Fo Ϫ Fc͉ maps and potential to hydro- mined after 20 min. A calibration curve for the determination of
gen-bond to at least one protein atom or other water molecule. phosphate concentration was obtained from analogous analysis
A bias-removed ͉Fo Ϫ Fc͉ omit map (see Fig. 5a) was generated of solutions of appropriate concentrations (6–150 ␮M) of
by deleting the ligand and active site waters from the model and KH2PO4, which had been dried in high vacuum for at least 3 h
subjecting the model to simulated annealing and refinement in before use. As a control, a glucose-6-phosphate solution of
PHENIX. The ͉2Fo Ϫ Fc͉ map obtained by this procedure (see known concentration was also digested with calf alkaline phos-
Fig. 5a) shows clear continuous electron density for ligand 4. phatase and analyzed.
The structure was validated using SFCHECK, with 97.5% of
Modeling Procedure for Linear Intermediate and the
residues in the most favored region of the Ramachandran plot Designed Inhibitors—Modeling studies were carried out using
(Table 1). The final model included two protein chains each software packages from Schro¨dinger Suite 2006 software pack-
containing all 462 natural residues and two residues from His age (34–37); more detailed computational methods can be
tag cleavage, 554 water molecules, two manganese ions, one obtained from the supplemental material. The previously
sulfate ion, one chloride ion, and (R)- and (S)-4.
reported crystal structure of MtuDAH7PS (PDB code 2B7O)
Inhibition Assays—Enzyme activity was monitored by fol- (21) was used as the receptor. As a starting point for docking
Ϫ1
lowing the loss of absorbance at 232 nm (⑀ ϭ 2800 liters mol
studies, an ensemble of low-energy conformations of the
cmϪ1) due to the consumption of P-enolpyruvate (32). Assays ligands (R)- and (S)-4 and both epimers of the tetrahedral inter-
were carried out in 50 mM 1,3-bis(tris(hydroxymethyl)methyl- mediate 2 were identified by conformational searches. The
amino)propane buffer at pH 7.5 containing 1 mM tris(2-car- modeling of the tetrahedral intermediate epimers into
boxyethyl)phosphine at 30 °C. The buffer solution was pre- the active site of MtuDAH7PS was carried out with the
pared using ultrapure water, which was stirred over Chelex Schro¨dinger Suite 2006 Induced Fit Docking protocol (36). The
resin (Bio-Rad) and filtered before use. Manganese(II) sulfate induced fit docking procedure lead to an intermediate adapted
stock solution (10 mM) was made up in ultrapure water pre- protein receptor in which several residues in the active site
treated with Chelex resin. P-enolpyruvate and E4P substrate adopted conformations different from the ones observed in the
solutions were made up to ϳ10 mM by dissolving the P-enolpy- native crystal structure (PDB code 2B7O). This intermediate
ruvate monopotassium salt and E4P monosodium salt adapted structure of MtuDAH7PS was then used as a rigid
(Aldrich) in buffer solution. Accurate substrate concentrations receptor for docking studies of compounds (R)- and (S)-4,
were determined by enzymatic reaction; the absorbance which were conducted using Glide (37).
changes of solutions containing one substrate in excess were
Synthesis—All reactions were carried out under an inert
determined before and after conversion of the limiting sub- atmosphere of nitrogen in flame-dried glassware unless other-
strate by added DAH7PS had occurred, and the extinction coef- wise stated. Organic solvents were dried before use by standard
ficient of P-enolpyruvate was used to calculate the concentra- methods (38). Dess-Martin periodinane (39, 40) was prepared
tion of the limiting substrate. The absorbance change was according to literature procedures. m-Chloroperbenzoic acid
corrected by the absorbance change caused when DAH7PS was was recrystallized from dichloromethane before use, and all
added to a control solution that did not contain E4P. For inhi- other reagents were purchased from Sigma-Aldrich and used
bition studies, assay solutions containing manganese (II), vary- without further purification.
ing concentrations of P-enolpyruvate and inhibitors, and buffer
Preparative procedures and characterization of previously
were equilibrated at 30 °C. 2 ␮l (1.3 mg/ml) of M. tuberculosis unreported compounds can be found in the supplemental
DAH7PS was added, and the mixture was equilibrated for 2 min material. A representative procedure for the final step of the
before the reaction was initiated by the addition of E4P (11 ␮l). synthesis and characterization of inhibitor 4 is outlined in detail
Final assay conditions were 10 ␮M Mn2ϩ, 5.2 nM DAH7PS, 100 below.
␮M E4P, 57.6–115.2 ␮M P-enolpyruvate, 0–940 nM inhibitor
Heptanoic Acid 2,7-Bisphosphate 4—Protected bisphosphate
with an appropriate amount of buffer to make up a final volume 11 (177 mg, 0.24 mmol) was dissolved in ethyl acetate (15 ml),
of 1 ml.
and palladium on charcoal (10% palladium/carbon, 57 mg,
Lanzetta Phosphate Assay—Lanzetta reagent (33) was pre- 0.053 mmol of palladium) was added. The reaction flask was
pared fresh as required from the following components: 3 parts purged with hydrogen gas with three freeze-pump-thaw cycles.
0.045% w/v malachite green in water, 1 part 4.2% w/v ammo- The reaction mixture was stirred for 16 h, after which TLC
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Potent Active Site Inhibitors for M. tuberculosis DAH7PS
FIGURE 3. Molecular modeling studies and inhibitor design. a, the modeled best pose of the tetrahedral open chain intermediate 2 (S-isomer) from induced
fit docking to the active site of MtuDAH7PS superimposed on the crystal structure of SceDAH7PS (PDB code 1OF8). The active site carbons from the modeled
structure are in green, and the active site residues of SceDAH7PS are shown with yellow carbons. The modeled linear intermediate molecule is shown with
magenta carbons, and the C2-hydroxyl group is pointing behind the plane. The residues are labeled according to the numbering in MtuDAH7PS. The metal ion
is displayed as a purple sphere. Hydrogen bonds are represented by dashed lines. b, left, linear intermediate 2. Center, molecular model of linear intermediate 2
showing the preferred 2S-configuration. Right, simplified intermediate analog 4, synthetic target for this study.
indicated complete consumption of starting material 11. The side the water attacks the P-enolpyruvate substrate during the
suspension was filtered through a Celite pad, the Celite was
washed with ethyl acetate and methanol, and the solvent was
evaporated under reduced pressure. The resulting residue
was dissolved in 1 M aqueous potassium hydroxide solution (7
ml) and stirred vigorously for 2.5 h. The reaction mixture was
passed down a column consisting of freshly regenerated
DOWEX 50WX4-200 (Hϩ) resin (ϳ5 ϫ 1 cm), and the column
was eluted with 50 ml of water. The eluate was neutralized with
1 M potassium hydroxide solution and lyophilized. The crude
product was purified by anion exchange chromatography on
SOURCE-Q resin eluting with a gradient of water/aqueous
ammonium bicarbonate solution. Fractions containing the
desired product were identified using the qualitative Lanzetta
assay (see below), pooled, and lyophilized to give 42.5 mg of a
white powder consisting of the product and residual ammo-
nium bicarbonate. Lanzetta phosphate assay (see above) estab-
lished that the purity of the white powder yielded was 80% by
weight; the corrected yield for this step is thus 34%.
reaction, both possible epimers of the linear intermediate were
modeled.
The modeling study produced a total of 30 possible poses for
the two possible C2 diastereoisomers of the tetrahedral inter-
mediate in the active site, out of which 17 retained the expected
P-enolpyruvate binding interactions. All 17 poses with correct
P-enolpyruvate orientation are from the S-isomer of the inter-
mediate, consistent with water attacking the C3 carbon of
P-enolpyruvate from its re face, which is the side opposite the
active site Mn2ϩ ion.
The best pose from the modeling study (Fig. 3a) was selected
based on two criteria. The first is that the P-enolpyruvate part of
the tetrahedral intermediate retains the same interactions as
observed in the original crystal structure (PDB code 2B7O). The
second is that the positioning of the E4P phosphate group
should be as close as possible to the phosphate group of G3P in
the crystal structure of SceDAH7PS (PDB code 1OF8), which is
considered to provide the best prediction for the E4P phosphate
position. In the best pose (Fig. 3a), the P-enolpyruvate part of
the molecule interacts with Arg126, Lys306, Arg337, Arg284, and
Glu283. Arg284 bridges the phosphate groups of P-enolpyruvate
and E4P through salt bridges and hydrogen bonds. The E4P
1H NMR (500 MHz, D2O) ␦ 4.34 (td, J ϭ 5.6, 9.0 Hz, 1H), 3.73
(q, J ϭ 6.4 Hz, 2H), 1.63–1.76 (m, 2H), 1.49–1.59 (m, 2H), 1.22–
1.42 (m, 4H). 13C NMR (126 MHz, D2O) ␦ 24.0 (br), 25.2 (br),
30.1 (br), 33.7 (br), 65.4 (br), 76.1 (br), 180.5 (br). 31P NMR (121
MHz, D2O) ␦ ppm 3.31 (t, J ϭ 6.0 Hz, 1P), 1.85 (d, J ϭ 8.8 Hz,
1P). HRMS[ESI,neg] calculated for C7H14O10P2 321.0140,
phosphate group interacts with Arg284, Arg135, and Ser136
,
)
which suggests that part of the KPRS motif (Arg135 and Ser136
found 321.0131 [M-H] . [␣]2D0 (R)-4, -0.9 (c 1.8, H2O), (S)-4,
Ϫ
plays a key role in positioning of the E4P phosphate, as expected
from the G3P-bound SceDAH7PS structure. The E4P aldehyde
group, now converted to a hydroxyl group in the linear inter-
mediate, is at a distance of 2.2 Å from the metal ion. This indi-
cates that the aldehyde of E4P may be held in place by coordi-
nation to the metal ion before reaction occurs. The hydroxyl
ϩ0.8 (c 1.9, H2O).
RESULTS
Modeling of the Reaction Intermediate into MtuDAH7PS
Active Site—To inform inhibitor design, the predicted tetrahe-
dral reaction intermediate (2, Fig. 3) was modeled into the
active site of MtuDAH7PS. Because it is unknown from which group that corresponds to the incoming water molecule forms
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Potent Active Site Inhibitors for M. tuberculosis DAH7PS
FIGURE 4. Synthesis of racemic inhibitor 4. (i) TBDMSCl, imidazole, dichloromethane, 61%. (ii) Dess-Martin periodinane, dichloromethane, 70% (crude). (iii)
i
i
isopropyl dichloroacetate, KOiPr, PrOH, Et2O. iv, NaCNBH3, PrOH, 60% (two steps). (v) tetrabutylammonium fluoride, tetrahydrofuran, 56%. (vi) 1)
(BnO)2PN(iPr)2, 1H-tetrazole, dichloromethane; 2) meta-chloroperbenzoic acid, 50%. (vii) 1) H2, palladium/carbon; 2) KOH, H2O; 3) DOWEX-Hϩ; 4) SOURCE-Q
anion exchange chromatography, 42%.
hydrogen bonds with Trp280, Glu248, and Lys133. Two of these TABLE 1
residues, Glu248 and Lys133, are conserved across the entire
DatacollectionandrefinementstatisticsforM. tuberculosisDAH7PSin
complex with 4
DAH7PS family, whereas Trp280 is only conserved in the type II
Data collection
enzymes.
Crystal system
Space group
Unit cell parameters
a (Å)
Trigonal
P3221
Inhibitor Design—Combining results from the intermediate
modeling and previous work on mechanism-based inhibitors
(27, 28), we designed bisphosphate 4 (Fig. 3b) as a simplified
analog of the tetrahedral intermediate. The 2-phosphorylcar-
boxylic acid moiety targets the P-enolpyruvate binding site and
resembles phospholactate, which has been reported as an effi-
cient inhibitor of E. coli DAH7PS (27). Bisphosphate 4 repre-
sents a simplified analog of tetrahedral intermediate 2; the
phosphoryl and carboxyl moieties of compound 4 are pro-
posed to act as charged anchors arranged in a similar geom-
etry to those of intermediate 2 and held together by a non-
functionalized hydrocarbon linker. Given its low degree of
functionalization, bisphosphate 4 may be used as a lead
structure for specific introduction of functionality to
enhance enzyme-ligand affinity. In light of the results for
molecular modeling of the tetrahedral intermediate 2, which
suggest a preference for the S-configuration at C2, we
devised two syntheses leading to target compound 4 to assess
possible enantio-discrimination: one racemic synthesis,
which had already been proven useful in our laboratory, as
well as a chiral pool synthesis, which allowed control of the
absolute configuration at C2.
203.58
b (Å)
203.58
c (Å)
66.49
Resolution range (Å)
Measurements
Unique reflections
Redundancy
Completeness (%)
I/␴(I)
55.641–2.35 Å (2.48–2.35)
726,170
65,888
11.0 (11.2)
100 (100)
5.0 (1.9)
R
merge (%)
13.0 (39.2)
26.5
Wilson B-value (Å2)
Refinement
Resolution (Å)
51.16–2.35 (2.41–2.35)
R
R
cryst (%)
16.4
free (%)
19.6
Amino acids (chain length
464 residues)
Water molecules
Other
464 ϩ 464 res.; 7136 atom sites
554
2 Mn2ϩa, 1 SO24Ϫ, 1 Cl , (R)-4a, (S)-4a
Ϫ
Mean B (Å2)
Protein
25.13
32.06
12.12
Water
Other
r.m.s.d.b from target values
Bond lengths (Å)
Bond angles (°)
Dihedral angles (°)
Ramachandran
Most favored (%)
Allowed (%)
0.010
1.214
5.634
98.3
1.2
Synthesis of Racemic 4—The synthetic strategy toward rac-4
relies on the establishment of the key ␣-hydroxyester moiety by
a Darzens-type chain elongation of an aldehyde, a methodology
that has been used for the synthesis of related compounds (Fig.
4) (29, 41). The final deprotection was achieved by hydrog-
Disallowed (%)
PDB code
0.5
3PFP
a Refined at reduced occupancy.
b r.m.s.d., root mean square deviation.
enolysis of the benzyl groups followed by hydrolysis of the iso- itor 4 (Table 1). The crystal structure was solved by molecular
propyl ester. The final product, rac-4, was purified by anion replacement using the previously determined MtuDAH7PS
exchange chromatography eluting with an ammonium bicar- structure (PDB code 2B7O) (21) as search model and was
bonate gradient.
refined using x-ray diffraction to 2.35 Å (Table 1) The asym-
Crystal Structure—The racemic inhibitor 4 was co-crystal- metric unit contains two enzyme molecules as in other
lized with DAH7PS from M. tuberculosis. Although generally MtuDAH7PS crystal structures; tertiary and quaternary struc-
both purification of the enzyme and crystal growth are difficult tures are essentially unaffected by binding of inhibitor 4. Super-
in the absence of P-enolpyruvate in the buffer solution, x-ray imposition on the wild-type protein (PDB code 3NV8) (22)
diffraction quality crystals were obtained by buffer exchange of showed that 5029 atoms could be matched with a root mean
the purified enzyme solution with a solution containing inhib- square positional difference of 0.22 Å.
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Potent Active Site Inhibitors for M. tuberculosis DAH7PS
Clear continuous electron density for inhibitor 4 was found
Synthesis of (R)-4 and (S)-4—Because the previously de-
in both active sites. The ͉Fo Ϫ Fc͉ omit map generated by remov- scribed synthesis of rac-4 offers little potential with respect to
ing the inhibitor and active site waters from the model and stereoselective introduction of the ␣-hydroxyester moiety,
subjecting the resulting model to simulated annealing and another synthetic route was developed in which the chirality is
refinement shows clear continuous density for inhibitor 4 derived from a cheap and readily available chiral pool starting
and for a water molecule in the active site (Fig. 5a). Based on the material (Fig. 6). The chiral epoxyester 14 of appropriate con-
electron density maps obtained, it could not be concluded figuration was prepared from D- or L-serine, respectively, fol-
whether there is preferential binding of one of the enantiomers lowing a previously reported method (42). Ethyl glycidate 14
from the racemic mixture. To represent this ambiguity, our was ring-opened regioselectively to yield ␣-hydroxyester 15.
final model includes both (R)-4 and (S)-4 at half-occupancy.
Hydrogenolysis of ester 15 liberated the 7-hydroxyl group to
Inhibitor 4 binds as predicted with its 2-phosphoryl and car- generate dihydroxyester 16, which could then be converted to
boxylate moieties in the P-enolpyruvate binding site and in an (R)-4 or (S)-4 using the same procedures as described above for
extended conformation so that the phosphate moiety on C7 is the racemic synthesis.
located near the proposed E4P phosphate binding site (Fig. 5b).
Inhibition Studies—(R)-4 and (S)-4 were tested for inhibition
Active site residues interacting with the P-enolpyruvate-mim- of MtuDAH7PS. Although inhibitor 4 was designed as a bisub-
icking portion of ligand 4 show little movement, consistent strate inhibitor, it is known that DAH7PS operates by a sequen-
with this portion of the inhibitor being a good mimic of P-enol- tial ordered reaction mechanism in which P-enolpyruvate has
pyruvate. As expected, the conformations of the metal binding to bind to the enzyme first (25). This means that although com-
residues were unaffected by binding of compound 4. The petition with P-enolpyruvate is most important to assess the
2-phosphate moiety of inhibitor 4 forms salt bridges with inhibitory potency, the fixed concentration of the second sub-
Arg284, Arg337, and Lys306 and a hydrogen bond to the back- strate can influence the inhibition. The kinetic assays were car-
bone N–H of Glu283. The carboxyl group forms salt bridges ried out with P-enolpyruvate concentrations ranging from 57 to
with Lys133 and Arg126 and coordinates to the manganese ion 115 ␮M at a fixed E4P concentration of 100 ␮M, which corre-
present in the active site. The 7-phosphate moiety extends into sponds to four times the value determined for the Michaelis
the tentative E4P phosphate binding site, forming salt bridges constant for E4P (Km(E4P)). The concentrations of (R)-4 and
with Lys380 and Arg135 and hydrogen bonds with the Ser136 (S)-4 were varied from 0 to 940 nM. Even at these high levels of
hydroxyl group and backbone N–H. Conformational changes E4P, (R)-4 and (S)-4 gave inhibitory constants of 360 Ϯ 50 and
relative to the P-enolpyruvate-liganded structure were 620 Ϯ 110 nM respectively (see supplemental Figs. S1 and S2).
observed for the KPRS motif associated with inhibitor binding (R)-4 and (S)-4 represent the first potent inhibitors reported for
(Fig. 5c). Arg135 and Ser136 interact with the 7-phosphate moi- MtuDAH7PS and the first inhibitors with substantially greater
ety of inhibitor 4, moving closer into the active site pocket. The affinity than the substrate P-enolpyruvate (Km(PEP) ϭ 29 ␮M
side chain of Lys133 shows the biggest conformational change of for MtuDAH7PS) for any DAH7P synthase.
all active site residues, establishing a salt bridge to the carbox-
Modeling of (R)-4 and (S)-4 into MtuDAH7PS Active Site—
ylate group of ligand 4. This big movement may also be partly Both (R)-4 and (S)-4 were modeled into the MtuDAH7PS active
due to increased electrostatic repulsion with the guanidinium site, and the results showed that there is little preference for
group of Arg135, which has moved closer into the active site. binding between the two isomers of inhibitor 4. Both isomers
Interestingly, the side chain movement of Lys133 correlates well adopt similar binding modes, which are also very similar to that
with the movement that the induced fit molecular modeling of the linear intermediate in the active site, with the R-isomer
results predict for this residue to accommodate the proposed having a slightly better docking score than the S-isomer. The
reaction intermediate (Fig. 5d).
interactions found in the best poses from modeling of the two
The superposition of structures of the DAH7PS-4-complex isomers are the same as those observed in the crystal structure
and the induced fit-modeling results (Fig. 5d) show clearly that of the MtuDAH7PS-4-complex (supplemental Fig. S3). These
the experimental binding mode of bisphosphate 4 closely modeling results in conjunction with the inhibition data
matches the in silico modeling of the proposed reaction inter- obtained for (R)- and (S)-4 and the tetrahedral intermediate
mediate into the active site. The carboxylate and both phos- modeling results suggest that a fully functionalized C2-carbon
phate groups of inhibitor 4 occupy positions that were pre- of the tetrahedral intermediate bearing carboxylate, phosphate,
dicted for the corresponding moieties of the intermediate and hydroxyl substituents is necessary to observe stereoselec-
model, with minor deviations found in the carbon chain. A tive recognition in the enzyme active site.
crystallographically located water molecule occupies a position
DISCUSSION
very close to the predicted position of the intermediate 2-
hydroxyl group. This water is held in place by hydrogen bonds
The study presented here demonstrates the successful com-
to Trp280 and Glu248. The distance of this water to C2 of the bination of synthetic, structural, and computational methods to
ligand is 3.4 Å when (R)-4 is modeled into the active site and 3.2 inform the design of novel lead structures of inhibitors for
Å for the model containing (S)-4. Preliminary testing of rac-4 MtuDAH7PS. Although both the substrate and the product of
indicated that its Ki against MtuDAH7PS was in the submicro- the enzymatic transformation, P-enolpyruvate and DAH7P,
molar range. Encouraged by these results, and to assess whether possess planar geometry at C2, mechanistic considerations sug-
one enantiomer of 4 is preferentially bound by the enzyme, we gest that the enzyme active site is also set up to accommodate
synthesized compounds (R)-4 and (S)-4 for inhibition assays.
preferentially a tetrahedral reaction intermediate. The high
MAY 6, 2011•VOLUME 286•NUMBER 18
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Potent Active Site Inhibitors for M. tuberculosis DAH7PS
FIGURE 5. Stereo view of the active site of the enzyme in complex with 4 (only the (R)-enantiomer is shown for clarity). a, ͉Fo Ϫ Fc͉ simulated
annealing omit map contoured at 3␴ showing the electron density for inhibitor 4 (blue carbons) in the active site. WAT, water. b, interactions of 4 with
the active site residues (green carbons) and metal ion (purple sphere). c, overlay of P-enolpyruvate (yellow carbons)-DAH7PS structure 3NV8 (cyan carbons)
with DAH7PS-4-complex (green carbons). d, overlay of the preferred pose of the linear intermediate (magenta carbons) and 4 (blue carbons; the oxygens
on the C2-phosphate were omitted for clarity). The enzyme structure experimentally obtained from the DAH7PS-4-complex (green carbons) superim-
poses well onto the enzyme structure obtained by induced fit modeling of the linear intermediate (yellow carbons, peptide; magenta carbons,
intermediate).
16204 JOURNAL OF BIOLOGICAL CHEMISTRY
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Potent Active Site Inhibitors for M. tuberculosis DAH7PS
FIGURE 6. Synthesis of enantiopure inhibitor 4. Yields are given for the synthesis of (R)-4; analogous procedures using L-serine as the starting material were
used for the synthesis of (S)-4 in comparable yields. Please refer to the supplemental material for more detail. (i) HNO2, KBr, H2O then KOH/EtOH, 37% (two
steps). (ii) EtBr, Bn(Et)3NCl, CH2Cl2, 66%. (iii) BnO(CH2)4Br, magnesium, tetrahydrofuran, room temperature, then CuBrꢁSMe2, Ϫ78 °C, then 14, 32%. (iv) H2,
palladium/carbon, EtOAc, 66%. (v) (BnO)2P-N(iPr)2, 1H-tetrazole, dichloromethane, then meta-chloroperbenzoic acid 64%. (vi) H2, palladium/carbon, EtOAc,
then KOH/H2O, then DOWEX-Hϩ, anion exchange chromatography, 27%.
inhibitory potencies of (R)- and (S)-4 confirm that intermediate proposition that the substrate water attacks P-enolpyruvate
analogues with tetrahedral C2 are a promising scaffold for high- from the si side during the course of the reaction (18, 19, 46). It,
affinity inhibitors of DAH7PS.
however, so far remains unclear whether this mechanistic pro-
The fact that the experimentally determined structure and posal can be transferred to MtuDAH7PS or even DAH7P syn-
the induced-fit model are in good agreement firstly demon- thases in general. In the crystal structure reported in this study,
strates that inhibitor 4 is a good intermediate mimic, and sec- a water molecule was located in close proximity to C2 of the
ondly, verifies that our modeling approach is effective for pre- inhibitor on what would be the re side of P-enolpyruvate. The re-
dictions of enzyme-ligand interactions for MtuDAH7PS. The semblance of this arrangement of inhibitor and water to the
structural information from the enzyme-inhibitor complex can location of the 2-hydroxyl group of the modeled tetrahedral
be used to inform future inhibitor design; based on the binding reaction intermediate, combined with the configurational pref-
mode of inhibitor 4 in the active site, modifications to the lead erence observed for the docking of the intermediate, suggest
structure allow the introduction of specific new interactions that in MtuDAH7PS, water attack proceeds from the re side of
with the enzyme. For example, additional hydroxyl groups at P-enolpyruvate. This is further supported by studies of type I
C4 and C6 of inhibitor 4 with the appropriate absolute con- DAH7P synthases from S. cerevisiae (16), E. coli (14), and Ther-
figuration are expected to form hydrogen bonds to Asp441
,
motoga maritima (47), which located a water molecule that was
interactions that were observed in the modeling of the inter- suggested to attack P-enolpyruvate during the course of the
mediate (Fig. 3). Introduction of a C4-hydroxyl or other met- enzymatic reaction in a very similar environment. The glutamic
al-coordinating group such as an amine or thiol group could acid residue has been proposed as a general base catalyst to
also lead to additional enzyme-inhibitor interactions facilitate nucleophilic attack of water. The highly conserved
through metal coordination.
nature of the arrangement of water in the P-enolpyruvate bind-
Inhibitor 4 is a highly charged molecule, featuring two phos- ing site suggests a common catalytic mechanism for all DAH7P
phate groups and a carboxylate moiety. This characteristic of synthases that involves water attacking the re face of P-enolpy-
this molecule is expected to significantly limit membrane per- ruvate during the course of the reaction.
meability. Phosphates are abundant in naturally occurring mol-
The combined results from intermediate modeling and the
ecules, making them highly interesting moieties in the develop- structural and kinetic study of intermediate analog 4 allow sig-
ment of drugs. Strategies to generate phosphate prodrugs nificant refinements of the proposed catalytic mechanism of
containing suitable masking groups that facilitate membrane MtuDAH7PS (Fig. 7). P-enolpyruvate and E4P binding has
permeability and allow the release of the active drug inside the already been discussed in detail. We propose that the substrate
cell have been developed, and examples of these prodrugs are in water is bound on the re face of P-enolpyruvate through hydro-
clinical trials (43–45).
gen-bonding interactions with Trp280 and Glu248. Nucleophilic
Although both enantiomers of inhibitor 4 displayed a high attack of P-enolpyruvate at the E4P aldehyde moiety is facili-
affinity to the enzyme active site, the small difference in affinity tated by metal coordination, and the resulting oxyanion is pro-
of the two enantiomers provides valuable insight into the tonated by Lys133. Both the intermediate modeling results and
enzyme mechanism and its inhibition. The observation that the crystal structure of the intermediate analog show significant
(S)-4 proved to be a slightly less potent inhibitor may be due to movement of the Lys133 side chain upon ligand binding, posi-
the fact that the C2-hydrogen of (S)-4 points in the direction of tioning it in a suitable position to fulfill this role as a general
a water molecule also present in the active site, with interatom acid. The substrate water is activated for attack at C2 of P-enol-
distances that suggest a steric clash between these moieties pyruvate by deprotonation with Glu248
.
(supplemental Fig. S4). For the related enzyme KDO8PS, data
A similar mechanism has been previously suggested in stud-
from computational and crystallographic studies have led to the ies of S. cerevisiae and T. maritima type I DAH7PS (16, 47). The
MAY 6, 2011•VOLUME 286•NUMBER 18
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Potent Active Site Inhibitors for M. tuberculosis DAH7PS
FIGURE 7. Refined catalytic mechanism for MtuDAH7PS in accordance with the results obtained from this study.
CONCLUSION
fact that a glutamate residue is found at the equivalent position
for M. tuberculosis type II DAH7PS and the crystallographic
localization of a suitable substrate water molecule in the
MtuDAH7PS-4-complex in this study as well as our modeling
results further support this mechanistic proposition. In the
next step, the now neutral Lys133 can abstract the proton from
Glu248, either by direct deprotonation or relayed via an active
site water molecule. This step regenerates both the Lys133
ammonium and the Glu248 carboxylate group necessary for the
By combining molecular modeling-guided and mechanism-
based design, we have synthesized the first potent inhibitor of
MtuDAH7PS. The crystal structure of the enzyme-inhibitor
complex shows that the structural features of the inhibitor
designed to mimic features of the natural substrates and pro-
posed reaction intermediate indeed lead to the predicted spe-
cific enzyme inhibitor interactions. Inhibitor 4 represents a
promising lead structure for antimycobacterial drugs and offers
next catalytic cycle. Lys306, which is positioned within hydro- ample opportunity for further modification to establish struc-
ture-activity relationships. The crystallographic location of an
active site water molecule has clarified the enzyme catalytic
mechanism.
gen-bonding distance of the P-enolpyruvate phosphate, could
aid the elimination of phosphate from tetrahedral intermediate
2 by protonating the phosphate, thus making it a better leaving
group. Elimination of hydrogen phosphate yields DAH7PS pro-
tonated on the 2-keto moiety; this proton can then be removed
by Lys306. After dissociation of the product DAH7P, all active
site residues have returned to the protonation state that is
required for the next catalytic turnover. This mechanism is the
simplest that is in accordance with the data published to date,
and it accounts for all protonation and deprotonation steps
required on active site residues and intermediates. The only
major side chain movement of an active site residue is required
from the proton-shuttling Lys133, a residue that is not well
defined or possesses high B-factor values in some crystal struc-
tures of DAH7P synthases, indicating mobility of its side chain
(11, 21). Further support for the important role of both Lys133
and Lys306 comes from results in our laboratory that show that
both residues are essential for catalytic activity.5
Acknowledgments—This research was undertaken on the MX2 beam-
line at the Australian Synchrotron, Victoria, Australia (48). We
acknowledge travel funding provided by the International Synchro-
tron Access Program (ISAP) managed by the Australian Synchrotron
and the New Zealand Synchrotron Group.
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MAY 6, 2011•VOLUME 286•NUMBER 18
JOURNAL OF BIOLOGICAL CHEMISTRY 16207

Enzymology:
Potent Inhibitors of a Shikimate Pathway
Enzyme from Mycobacterium tuberculosis:
COMBINING MECHANISM- AND
MODELING-BASED DESIGN
Sebastian Reichau, Wanting Jiao, Scott R.
Walker, Richard D. Hutton, Edward N. Baker
and Emily J. Parker
J. Biol. Chem. 2011, 286:16197-16207.
doi: 10.1074/jbc.M110.211649 originally published online March 15, 2011
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