From the Unité de Biochimie Structurale and
¶ Laboratoire de Chimie Structurale des Macromolécules,
URA 2185 du CNRS, Biologie Structurale et Agents Infectieux, Institut
Pasteur, 25 rue du Dr Roux, 75015 Paris, France, the
§ Laboratory of Medicinal Chemistry, Faculty of
Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium, and the
Laboratory of Medicinal
Chemistry, Rega Institute for Medical Research, Catholic University
of Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
Received for publication, September 19, 2002, and in revised form, November 25, 2002
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ABSTRACT |
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The chemical synthesis of new compounds designed
as inhibitors of Mycobacterium tuberculosis TMP kinase
(TMPK) is reported. The synthesis concerns TMP analogues
modified at the 5-position of the thymine ring as well as a novel
compound with a six-membered sugar ring. The binding properties of the
analogues are compared with the known inhibitor
azido-TMP, which is postulated here to work by excluding the
TMP-bound Mg2+ ion. The crystallographic structure of the
complex of one of the compounds, 5-CH2OH-dUMP, with TMPK
has been determined at 2.0 Å. It reveals a major conformation for the
hydroxyl group in contact with a water molecule and a minor
conformation pointing toward Ser99. Looking for a
role for Ser99, we have identified an unusual catalytic
triad, or a proton wire, made of strictly conserved residues (including
Glu6, Ser99, Arg95, and
Asp9) that probably serves to protonate the transferred
PO3 group. The crystallographic structure of the
commercially available bisubstrate analogue
P1-(adenosine-5')-P5-(thymidine-5')-pentaphosphate
bound to TMPK is also reported at 2.45 Å and reveals an alternative
binding pocket for the adenine moiety of the molecule compared with
what is observed either in the Escherichia coli or in the
yeast enzyme structures. This alternative binding pocket opens a
way for the design of a new family of specific inhibitors.
The incidence of tuberculosis has been increasing during the last
20 years; it is now the first cause of mortality among infectious diseases in the world (1). The combination of four active drugs (rifampicin, isoniazid, pyrazinamide, and ethambutol or streptomycin) is currently used in Mycobacterium tuberculosis treatment,
but this has led to the appearance of resistant bacterial strains (2).
These resistant strains are alarming for two reasons. First, as
there are only a few effective drugs available, infection with
drug-resistant strains could give rise to a potentially untreatable form of the disease. Second, although only 5% of immunocompetent people infected with M. tuberculosis succumb to the disease,
it is nevertheless highly contagious (3). Therefore, a large effort is
necessary to identify potential new targets and inhibitors.
An attractive potential target is thymidylate kinase (EC
2.7.4.9, ATP:TMP phosphotransferase,
TMPK),1 an essential enzyme
that catalyzes an obligatory step in the synthesis of TTP either
from thymidine via thymidine kinase (salvage pathway) or from dUMP via
thymidylate synthase in all living cells (4). This enzyme
phosphorylates TMP into TDP using ATP as the preferred phosphoryl donor.
In the case of the herpes simplex virus (HSV), the most successful
antiviral drug (acyclovir) available on the market is directed against
thymidine kinase. Acyclovir is phosphorylated by several viral or host
kinases into acyclovir triphosphate, which terminates DNA synthesis
when incorporated into the viral DNA (5, 6). The comparative x-ray
structures of different enzyme-ligand complexes of HSV
type 1 thymidine kinase (7-10) revealed a number of interesting structural features and paved the way for rational structure-based drug
design of antiviral compounds (11, 12). A similar approach might lead
to potent antituberculosis agents.
TMPK from M. tuberculosis is a homodimer with 214 amino
acids per monomer (13). The x-ray three-dimensional structure has been
recently solved at 1.95-Å resolution (14, 15) as a complex with
TMP, thereby making it possible to initiate structure-based drug design
studies. The global folding of the protein is similar to that of the
others TMPKs and NMP kinases despite the low similarity of their amino
acid sequences. The TMP kinase backbone is characterized by nine
solvent-exposed The active site of M. tuberculosis TMP kinase complexed with
TMP differs from the other known TMPKs in the following ways (15). It
is in a fully closed conformation with the ATP binding site being
already preformed and the LID region well ordered into a
In this study we have synthesized five TMP analogues (1-5,
Fig. 1), modified at the thymine moiety and focused on target 2. In
addition, one member of a new class of nucleotide analogues has been
tested (23, 24), namely a 1,5-anhydrohexitol analogue (6) of TMP, where the 3'-OH and/or the
5'-O-phosphate positions are expected to depend on the sugar
conformation. The inhibitory effect of these TMP analogues has been
measured in vitro by a novel direct specific enzymatic
activity test using HPLC and compared with the reference compound
AZTMP, which is a good inhibitor (13).
All compounds have been subjected to co-crystallization experiments as
well as soaking experiments for exchange with TMP in TMP-TMPK crystals
(M. tuberculosis TMPK does not crystallize in the absence of
TMP). We solved the structure of one promising enzyme-inhibitor complex
by x-ray diffraction and have determined the rearrangement of side
chains in the active site and the concomitant modification of the water
molecule network around the thymine moiety of the TMP substrate. In
addition, we have co-crystallized and solved the structure of the
complex between TMP kinase and the bisubstrate analogue
Ap5T. Altogether, considerable new insight into the
possible mechanism of phosphoryl transfer has been gained as described
at the end of the "Discussion."
Protein and Reagents
The M. tuberculosis (Mtub) TMP kinase was
overexpressed in E. coli and purified as described
previously (13). The protein was stored at Inhibitor Synthesis
Several methods have already been reported for the synthesis of
5-hydroxymethyl-2'-deoxyuridine. We found that
hydroxymethylation of 2'-deoxyuridine with formaldehyde under acidic
(25) or basic (26) catalysis gave only low yields. Therefore, we
decided to couple 5-benzyloxymethyluracil with an appropriate sugar. In
Ref. 27, the base is coupled with 1,2,3,5-tetracetylribose. Since this
method requires subsequent 2'-deoxygenation, we chose to glycosylate
the silylated base with
2-deoxy-3,5-O-di-(toluoyl)-
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helices surrounding a central
-sheet made of
five
-strands, typical of the so-called Rossmann-fold (16-20).
However, the dimerization mode of the M. tuberculosis enzyme
differs from that reported in the yeast, human, and Escherichia coli enzymes (15).
-helical conformation even though the second substrate ATP (or
non-hydrolyzable ATP) is absent from the structure. In the TMP binding
site, the protein-TMP interaction shows three specific features when
compared with yeast, human, or E. coli enzyme structures. The first feature involves both a magnesium ion and Tyr39;
they interact with two opposite non-bridging oxygens of the phosphate
moiety of TMP. The second feature involves Asn100 in
contact with atom N-3 of the base moiety (15). The third feature, perhaps more amenable to the design of new inhibitors, is the interaction of the 3'-OH atom of TMP with both the side chain of
Asp9 and a water molecule, W9, which is a ligand of the
Mg2+ ion. In addition, there is a high concentration of
positively charged side chains both from the LID region and from the
P-loop with arginine residues 14, 95, 149, and 160 as well as
Lys13 (15). Examination of the structure therefore
suggested the following targets for species-specific inhibitors.
In target 1, the 3'-OH and 2'-OH groups of the ribose ring could
be systematically replaced with different chemical groups; this has
been recently reported (21). In target 2, the 5-position of the thymine
ring is another possibility that has also been considered in the past for HSV thymidine kinase inhibitors (e.g. Refs. 11 and 12). Preliminary results of compounds modified at this position have already
been reported (13). Here we report results on other compounds with the
aim of adding an extra hydrogen bond with water molecule W12 detected
in the three-dimensional structure. W12 is located close to
Pro37 (which is in a cis conformation) and forms hydrogen
bonds with residues building up the thymidine binding cavity, such as
Phe70, Asp73, and Arg74. In target
3, the 2-position of the thymine ring could also be explored, and
preliminary but encouraging results have been reported for one compound
modified at this position (13). The aim here is to replace the inserted
(space-filling) water molecules W2 and W3 that, if removed, give rise
to a well defined cavity that can be readily materialized with computer
programs such as VOIDOO (22).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C in aliquots of
100 µl at 4 mg/ml in a buffer containing 20 mM Tris-HCl,
pH 7.5, 0.5 mM dithiothreitol, and 1 mM EDTA,
conditions at which it is stable over several months. Ap5T
was purchased from Jena Bioscience (Germany). All other reagents used
were purchased from Sigma, including the reference inhibitor AZTMP. All
solutions were made with pyrolyzed water.
-D-erythro-pentafuranosylchloride (28). Unfortunately racemization of the sugar prior to coupling led to
a hardly separable mixture of the
- and
-anomers of the protected
nucleoside. After alkaline removal of the acyl groups, however, the
anomeric mixture could be separated via column chromatography to give
the
- and
-anomers 10 and 11 as white foams. Both isomers were then phosphorylated. We noticed that the
5-O-benzyl group got partly removed during this
step. Thus phosphorylation of the
-nucleoside gave three compounds:
2'-deoxy-5-hydroxymethyl-5'-O-phosphoryluridine (1), its benzyl-protected analogue (2), and
2'-deoxy-5-hydroxymethyluridine (12). Phosphorylation of the
-nucleoside gave the corresponding benzylated and non-benzylated
nucleotides 13 and 3 (Scheme
1).
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Scheme 1.
Reagents and conditions. a,
5-bensyloxymethyl-2,4-bis[(trimethylsilyl)oxy]pyrimidine,
CH3CN; b, NH3, MeOH; c,
POCl3, (MeO)3PO.
2'-Deoxy-5-(2-furyl)uridine (15) and
2'-deoxy-5-(thien-2-yl)uridine (16) were synthesized
according to a published procedure from unprotected
2'-deoxy-5-iodouridine (14) (29). Phosphorylation of these
two nucleosides yielded the two desired 5-heteroaryl-substituted
nucleotides 4 and 5 (Scheme
2).
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Synthesis: General-- NMR spectra were obtained with a Varian Mercury 300 or 500 spectrometer using the solvent signal of Me2SO-d6 as a secondary reference. All signals assigned to amino and hydroxyl groups were exchangeable with D2O. Mass spectra and exact mass measurements were performed on a quadrupole/orthogonal-acceleration time-of-flight tandem mass spectrometer (qTof 2, Micromass, Manchester, UK) equipped with a standard electrospray ionization interface. Samples were infused in a 2-propanol:water (1:1) mixture at 3 µl/min. If necessary, nucleoside 5-O-monophosphates were ultimately purified using a Gilson HPLC system with a Gilson 322 pump, a UV/VIS-156 detector on a C18 column (10 µM, Altech, Altima, 250 × 22 mm). Precoated Merck silica gel F254 plates were used for TLC, and spots were examined with UV light at 254 nm and sulfuric acid-anisaldehyde spray or phosphomolybdic acid (0.5% in EtOH) solution. Column chromatography was performed on Uetikon 560 silica (0.2-0.06 mm) and Amersham Biosciences DEAE-SephadexTM A-25.
The 1H (and 31P, if appropriate) NMR spectra allowed the characterization of all purified intermediates in the synthesis and final products and are available from the authors upon request. In all instances, mass spectra were found to give the calculated mass within experimental error.
5-Benzyloxymethyl-1-[2-deoxy-3,5-O-di-(toluoyl)--D-erythro-pentafuranosyl]thymine
(8) and
5-Benzyloxymethyl-1-[2-deoxy-3-5-O-di-(toluoyl)-
-D-erythro-pentafuranosyl]thymine
(9)--
5-Benzyloxymethyluracil (680 mg, 2.93 mmol)
was suspended in a mixture of hexamethyldisilazane (62 ml),
trimethylsilyl chloride (0.5 ml, 3.94 mmol), and pyridine (5 ml). The
mixture was refluxed overnight. The resulting solution was evaporated
and co-evaporated with toluene. The obtained residue was suspended in
anhydrous CH3CN (3.5 ml), and
2-deoxy-3,5-O-di-(toluoyl)-
-D-erythro-pentofuranosyl chloride (1 g, 2.58 mmol) was added. The reaction mixture was stirred
for 3 h at room temperature. CH2Cl2 (25 ml) was added, and the organic layer was washed with a 7% solution of
NaHCO3 (25 ml). The water layer was washed twice with
CH2Cl2 (25 ml). The combined organic layers
were dried over MgSO4 and evaporated. The residue was
purified by column chromatography (silica,
CH2Cl2:MeOH 98:2) to give a mixture of
- and
-anomers (0.932 g, 62%). The anomers were partly separated
by a combination of precipitation (ether:MeOH 13:8) and column
chromatography (silica, CH2Cl2:MeOH 100:0
99:1
98:2). The two mixtures, enriched in either anomer, were used
without further purification in the next step.
1-(2-Deoxy--D-erythro-pentofuranosyl)-5-(benzyloxymethyl)thymine
(10) and
1-(2-Deoxy-
-D-erythro-pentofuranosyl)-5-(benzyloxymethyl)thymine
(11)--
A mixture of 8 and 9 (932 mg, 1.70 mmol) was dissolved in EtOH (60 ml), and 2 N NaOH
(37 ml) was added. The mixture was stirred at room temperature for 15 min and evaporated. The obtained residue was dissolved in 5 ml of
H2O and neutralized with HCl. The precipitate was filtered,
and the filtrate was evaporated and co-evaporated with EtOH. The
obtained residue was purified by column chromatography (silica,
CH2Cl2:MeOH 93:7) to give 10 (261 mg, 44%) and 11 (284 mg, 48%) as white foams.
1-(2-Deoxy-5-O-phosphoryl--D-erythro-pentofuranosyl)-5-(hydroxymethyl)thymine
(1),
1-(2-Deoxy-5-O-phosphoryl-
-D-erythro-pentofuranosyl)-5-(benzyloxymethyl)thymine
(2), and
1-(2-Deoxy-
-D-erythro-pentofuranosyl)-5-(hydroxymethyl)thymine
(12)--
A solution of 10 (261 mg, 0.75 mmol)
in trimethyl phosphate (3.5 ml) was cooled to 0 °C,
POCl3 (0.22 ml, 2.4 mmol) was added dropwise, and the
mixture was stirred for 4 h at 0 °C. The mixture was poured
into crushed ice-water (20 ml), neutralized with concentrated
NH4OH, and evaporated to dryness. The residue was subjected
to column chromatography (silica,
iPrOH:NH4OH:H2O 77.5:15:2.5
60:30:5)
yielding 12 (38.7 mg, 20%) as a white foam as well as an
oily mixture of 1 and 2. This mixture was further
purified by HPLC (C18, CH3CN:MeOH:0.05% HCOOH in
H2O 45:45:10, 3 ml/min), and the fractions containing the
nucleotides were lyophilized yielding 2 (47 mg, 14%) and
1 (109 mg, 41%) as white powders.
1-(2-Deoxy-5-O-phosphoryl--D-erythro-pentofuranosyl)-5-(hydroxymethyl)thymine
(3) and
1-(2-Deoxy-5-O-phosphoryl-
-D-erythro-pentofuranosyl)-5-(benzyloxymethyl)thymine
(13)--
A solution of 11 (248 mg, 0.71 mmol)
in trimethyl phosphate (3.5 ml) was cooled to 0 °C,
POCl3 (0.21 ml, 2.3 mmol) was added dropwise, and the
mixture was stirred for 4 h at 0 °C. The mixture was poured
into crushed ice-water (20 ml), neutralized with NH4OH, and
evaporated to dryness. 3 and 13 were separated by
column chromatography (silica,
iPrOH:NH4OH:H2O 77.5:15:2.5
60:30:5). Further purification was accomplished by HPLC (C18,
CH3CN:MeOH:0.05% HCOOH in H2O 45:45:10, 3 ml/min). The fractions containing the principal nucleotides
were lyophilized yielding 3 (106 mg, 42%) and 13 (47 mg, 15%) as white powders.
2'-Deoxy-5-(2-furyl)-5'-O-phosphoryluridine
(4)--
A solution of 15 (382 mg, 1.22 mmol)
in trimethyl phosphate (5.8 ml) was cooled to 0 °C.
POCl3 (0.4 ml, 4.29 mmol) was added dropwise, and the
mixture was stirred for 4 h at 0 °C. It was poured into crushed
ice-water (40 ml), neutralized with NH4OH, and evaporated
to dryness. The resulting residue was purified by column chromatography
(silica, iPrOH:NH4OH:H2O 77.5:15:2.5 60:30:5). The obtained white powder was further purified on DEAE-Sephadex A-25 (triethylammoniumbicarbonate 0
0.5 M) yielding the triethylammonium salt of 4. This
was converted to its corresponding sodium salt (NaI, acetone) (248 mg,
51%) as a white powder.
2'-Deoxy-5-(thien-2-yl)-5'-O-phosphoryluridine
(5)--
A solution of 16 (320 mg, 1.03 mmol)
in trimethyl phosphate (4.6 ml) was cooled to 0 °C,
POCl3 (0.31 ml, 3.3 mmol) was added dropwise, and the
mixture was stirred for 4 h at 0 °C. The mixture was poured
into crushed ice-water (20 ml), neutralized with 28% ammonia, and
evaporated to dryness. The resulting residue was purified by column
chromatography (silica, iPrOH:NH4OH:H2O 77.5:15:2.5 60:30:5). The obtained white powder was further purified on DEAE-Sephadex A-25 (triethylammonium bicarbonate 0
0.5 M) yielding the triethylammonium salt of 5. This was converted to its corresponding sodium salt (NaI, acetone) (240 mg,
57%) as a white powder.
Enzymatic Assay
The TMP kinase activity was measured by HPLC separation of nucleotide substrates and products as described below. The major reason for using this test instead of the more rapid coupled spectrophotometric assay (30) is that some inhibitors absorb light at 340 nm (4 and 5), thereby rendering difficult the evaluation of NADH concentration in the coupled reaction. Also, coupled enzymatic tests might be error-prone in the determination of the true value of Ki and Km of inhibitors and substrate, respectively, through the recycling of some substrate or product during the reaction coupling (31). The reaction is carried out in a 1-ml final volume of a solution of 50 mM Tris-HCl, pH 7.5, 20 mM magnesium acetate, 100 mM KCl, 1 mM EDTA, 0.5 mM dithiothreitol using different initial concentrations of ATP and TMP in the presence or absence of various inhibitors. To follow the enzymatic kinetics an aliquot of 100 µl is taken at different times after enzyme addition, and the reaction is quenched by adding 900 µl of 100 mM sodium phosphate, pH 7. The concentration of each nucleotide at different times during the reaction is measured at 260 nm by HPLC (isocratic mode) using a SephasilTM C18, 5-µm SC 2.1/10 (Amersham Biosciences) column. The buffer used for elution is 50 mM sodium phosphate, pH 6.5, 2.5% (v/v) ethanol, 40 mM tetrabutylammonium bromide with a flow rate of 250 µl/min. All the reaction velocities are calculated by monitoring the production of TDP expressed in terms of optical absorbance per minute at 260 nm. It was checked that all four sources of information (appearance of TDP or ADP, disappearance of ATP or TMP) can be used and lead to the same results (see also Ref. 11).
Crystallization and Diffraction Data Collection
Co-crystals of M. tuberculosis TMPK in complex with
compound 1 (see Fig. 1) were obtained as described for
crystals with the TMP substrate (14). Briefly a 6-µl drop of a 1:1
mixture of the protein solution (3.5 mg/ml) incubated overnight with 5 mM analogue 1 and the reservoir solution was
equilibrated with 34% (w/v) ammonium sulfate solution, 100 mM HEPES, pH 6.0, containing 2% (w/v) polyethylene glycol
2000, 20 mM magnesium acetate, and 0.5 mM
-mercaptoethanol. Crystals grew in 1-3 weeks to bipyramids of
400 × 200 × 200 µm3. X-ray data were
collected from cryo-cooled crystals using 25% (w/v) glycerol as cryoprotectant.
Soaking experiments were performed by first transferring TMP-TMPK co-crystals to a stabilizing solution containing 70% ammonium sulfate and 1 mM TMP and then to a fresh solution containing 70% ammonium sulfate and 10 mM inhibitor (but no TMP). This last solution was replaced three times, each soaking time lasting 24 h.
Diffraction data were processed using the DENZO/SCALEPACK (32) package. The CCP4 package (33) was used to calculate structure factors from the observed intensities (TRUNCATE). Reflections in the resolution ranges 2.28-2.22 and 2.70-2.64 Å had to be suppressed due to the presence of ice rings during data collection for the 1-TMPK complex.
Model Building and Refinement
Refinement was performed up to 2.0-Å resolution
with CNS (34). Standards protocols, including maximum likelihood
target, bulk solvent correction, and isotropic B-factors, were used
(34, 35). The model was inspected manually with SIGMAA-weighted
2Fo Fc and
Fo
Fc maps (36), and
progress in the model refinement was evaluated by the decrease in the
free R-factor. Manual rebuilding in the electron density
maps was done with O (37). Stereochemistry of the final model was
assessed using PROCHECK (38). Coordinates of the
1-TMPKMtub binary complex have been deposited in the Research Collaboratory for Structural Bioinformatics (RSCB) Protein
Data Bank (accession code 1MRS). Similar structural details
apply to the Ap5T-TMPK complex where all the dictionaries necessary for O and CNS come from the data base of G. Kleywegt.2 The coordinates
have been deposited in the RSCB Protein Data Bank (accession code
1MRN).
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Enzymatic Test: Calibration with the Natural Substrates--
In
all experiments, the enzyme concentration was set to a value at least
200 times lower than the substrates concentrations, i.e.
typically 5 × 108 M. The
reaction catalyzed by TMP kinase was found to follow the Michaelis
model (39). The initial velocity of the reaction at different fixed
(saturating) concentrations of ATP and different concentrations of TMP
allows for the determination of the apparent Km for
TMP and for ATP. With this protocol we find an apparent
Km = 40 µM TMP. For ATP, we measured
Km = 100 µM. Both values compare well
with the values obtained with the coupled assay (13). The
Km of 40 µM for TMP has been measured
at the fixed concentration of ATP of 0.5 mM and shows some
dependence upon ATP concentration. The Km value of
TMP extrapolated at zero ATP concentration is 4-5 µM in
agreement with results reported earlier (13).
The role of ATP as the phosphoryl donor has been explored in Ref. 13 where other NTPs were tried and found to be less efficient than ATP. Similar results were obtained using the direct assay method described here (data not shown).
Inhibition of TMP Kinase Activity with Synthetic Compounds-- Measuring the initial velocity of the reaction at a fixed saturating concentration of ATP and different concentrations of TMP, both in the absence and presence of inhibitors, allows the testing of the nature of the inhibition (39, 40). In our case, the reciprocal plot confirms that all inhibitors are competitive with TMP because the least-squares-fitted best lines in the absence and presence of the inhibitor cross exactly on the y axis. In the classical competitive inhibition model (with the Lineweaver-Burk representation), we can use Equation 1 (39).
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(Eq. 1) |
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For AZTMP, the dependence of the Ki value upon ATP concentration was found to be significant in accordance with Ref. 13. However, for all the inhibitors reported here, we report only the apparent Ki value at the fixed concentration of ATP of 1.0 mM. AZTMP will serve as a reference compound here since our aim is to do better than this compound.
Exploring the 5-Position of the Thymine Ring-- Systematic substitution of the methyl group in the 5-position of the base into halogen atoms was explored in Ref. 8. Bromine, which has the same size as CH3, was found previously to be very similar to TMP in terms of both Ki and Vmax (13). The difference in the kinetic parameters of these compounds reflects essentially a size effect with the halogen atoms serving as cavity-filling atoms. In this work other compounds substituted at the 5-position of the base moiety of TMP have been synthesized and will be described here.
Nucleotide analogue 1 was designed in an effort to induce an additional contact between the 5-CH2OH group and the side chain of Arg74. The value of the Ki constant indicates a moderately active inhibitor as compared with AZTMP (Table I).
Other TMP analogues were synthesized in which the volume of the
substituent at this position was further increased while maintaining one polar atom, namely a thiophene and a furane derivative. The resulting compounds (4 and 5) were less active
than the 5-CH2OH-dUMP, indicating that the volume of this
cavity cannot be stretched too much. Another compound (2)
aiming toward a stacking interaction with the Phe36 residue
resulted in a less active compound (Table I). The unnatural -isomer
of 1 was also synthesized (3, see Fig. 1) and assayed but proved less active
than 1 (see Table I).
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Another possibility would be to increase the length of the linker from the C-5 position up to a polar atom to replace directly the W12 water molecule, which is located at 3.95 Å from the C-5 atom of TMP behind the pyrrolidine ring of Pro37. Pro37 has a cis-peptide bond conformation that is maintained through a hydrogen bond between its carbonyl atom and a water molecule, W26, which is also in contact with one of the oxygens of the phosphate group of TMP. Therefore, the exact position of Pro37 is expected to be important for catalysis. This cis-peptide bond is also present in the three other known structures of TMP kinases (16-20). The deoxyribose analogue of dU with a substituent of exactly the size to reach out for the water molecule W12 (-CH=CH-Br) has been studied and compared with dT. This compound, (E)-5-(2-bromovinyl)-2'-deoxyuridine, has already proved efficient for inhibition of HSV type 1 thymidine kinase (12). It was predicted that the electronegative bromine atom would play the role of W12. However, this compound proved relatively inefficient for M. tuberculosis TMPK (Table I).
Inhibition by Ap5T-- The bisubstrate analogue Ap5T has been found to be more efficient than AZTMP with an inhibition constant Ki = 30 µM, making it one of the most powerful inhibitors of M. tuberculosis TMP kinase known to date. This compares well with similar inhibitors designed against other NMP kinases (18, 41). The inhibition mechanism inferred from the reciprocal plot analysis is compatible with the one described for adenylate kinase where it is a competitive inhibitor for the forward reaction and a mixed noncompetitive inhibitor for the backward reaction (42). To understand the chemical nature of the interaction of this inhibitor with its target and to further increase the efficiency of this compound, the structure determination of its complex with its TMPK was undertaken (see below).
Inhibition by the 1,5-Anhydrohexitol Analogue of TMP-- Modification of the sugar moiety of TMP into a 1,5-anhydrohexitol was performed (6). This analogue is actually a member of a novel series of inhibitors already tested against HSV type 1 thymidine kinase (24). The 5'-O-monophosphate was tested against M. tuberculosis TMPK but proved to be a moderately potent competitive inhibitor, again using AZTMP as a reference (see Table I).
Structure Determination of Bound Drugs in the TMP Binding Site of TMPK-- To better understand the molecular origin of the differences in the Ki inhibition constants of all these compounds, we tried either to co-crystallize them with TMPK or to exchange them with TMP by soaking TMPK-TMP crystals with an excess of inhibitor.
No exchange of TMP by any of the analogues studied here was observed even after extensive soaking experiments on TMPK-TMP crystals in contrast with the control experiment with I-dUMP, which worked well as already reported in Ref. 15. However, one should bear in mind that I-dUMP is a substrate, whereas none of the compounds tried here are substrates.
No co-crystals were obtained except with compound 1,
whose structure was determined at 2.0-Å resolution (see Tables II and III
for data collection and refinement statistics, respectively). The
Fourier difference map clearly shows density for the OH group of
CH2OH (Fig. 2). The hydroxyl
group makes an extra hydrogen bond with W12, which is also held in
place by the side chain of Asp73 and the essential
Arg74. After inspection of the residual SIGMAA-weighted
Fo Fc map, we found
density at the 2.5
level for a possible alternative conformation of
the hydroxyl group that reaches out for the side chain of the strictly
conserved Ser99 (for which no definite role has yet been
proposed). No attempt has been made to refine this alternative
conformation since the limited resolution of our data makes it
difficult to estimate their relative occupancies. Ser99 is
part of a hydrogen bond network involving both Glu6
(strictly conserved) and Tyr179 and helps to position
correctly Arg95 (Fig. 3),
which is strictly conserved among all known TMPKs except for the one
from African swine fever virus where it is a histidine (15).
Arg95 is the only residue of the whole protein
whose main chain dihedral angles lie outside the "allowed"
regions in a Ramachandran plot (Table III), an observation that
holds true in the other structures of TMPKs known, so it must have an
important functional role. The guanidinium group of Arg95
is located 3.2 Å away from the carboxylate group of Asp9.
Although the conformation of the side chain Arg95 is well
conserved in the different TMPK structures, it has been shown that it
can exist in another conformation in the complex with the products TDP
and ADP, pointing toward the carbonyl of residue 36 just before the
cis-peptide bond of Pro37 (17).
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Structure Determination of Bound Ap5T--
The crystal
structure of the complex of M. tuberculosis TMP kinase with
Ap5T has been determined at 2.45-Å resolution (see Tables
II and III for data collection and refinement statistics, respectively). The structure reveals an unexpected binding mode for the adenine moiety of this compound. While the thymidine moiety of
the bisubstrate analogue indeed occupies the binding pocket of the TMP
substrate, the rest of the molecule departs from its expected position
after the 5'-O-phosphate of the TMP moiety (Fig. 4). This is inferred from the
superimposed crystal structures of both the E. coli and
Homo sapiens enzymes complexed with the same bisubstrate
analogue (18, 19). The phosphate backbone of the bound Ap5T
is making an angle of almost 90° with the superposed same molecule
from E. coli at the -phosphate. The surprise comes from
the fact that the ADP moiety of the Ap5T molecule snugly fits into a cavity on the surface of TMP kinase that appears to be specific to the M. tuberculosis enzyme (Fig.
5). This cavity involves packing
interactions against helix
2 (residues 42-53) on one side and the
region 152-162 on the other side. His53 is nicely stacked
on the adenine ring.
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DISCUSSION |
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One of the most striking findings of this study is that none of
the 5-substituted dUMP analogues investigated here behave as a
substrate for M. tuberculosis TMPK, contrary to what had been observed for halogen-substituted compounds at the same position (13, 15). Our current interpretation is that any modification that
significantly perturbs the volume of the substituent at this position
will change the orientation of the sugar moiety of the TMP molecule as
well as that of the -phosphate. Indeed, recent results obtained on a
series of compounds modified at the 3'- and 2'-positions of the sugar
(21) indicated an extreme sensitivity of the reaction to the exact
positioning of at least the 3'-OH group of the sugar moiety, which is
in a C2'-endo conformation in the active site. This is also
illustrated in the present study with compound 6, which
behaves as an inhibitor probably because of its inability to place
correctly both the 5'-O-phosphate and the 3'-OH groups in a
favorable arrangement for catalysis.
Similarly, AZTMP is an inhibitor of the M. tuberculosis enzyme contrary to what is observed in TMPKs of other species (13) where it is either a weak substrate (in H. sapiens) or a good substrate (E. coli). This may be related to a unique feature of this enzyme revealed by its three-dimensional structure, namely the presence of a Mg2+ ion in the vicinity of Asp9 and the 3'-OH position. Inhibition by AZTMP in the M. tuberculosis TMPK is probably due to a steric and/or electrostatic effect of the azido group, which would prevent the binding of the Mg2+ ion.
No Mg2+ ion has been observed in this position in any of
the three other structures of TMPK reported so far (E. coli,
yeast, and human enzymes) in various complexes (16-20). This is
especially intriguing since most of the side chain ligands for the
divalent cation observed in the M. tuberculosis are
conserved in the three other known TMPK structures. A possible
explanation for this phenomenon may be the peculiar coordination of the
5'-O-phosphate of TMP, which involves not only the
Mg2+ ion but also Tyr39, a specific feature of
the M. tuberculosis sequence. Normally, at this
Tyr39 position, an arginine or a lysine residue, as seen in
the human, yeast, and E. coli enzyme structures and in the
multialignment, is expected to neutralize the TMP phosphate negative
charge. Since Tyr39 cannot play this role, it is
tempting to postulate that this role is fulfilled by the
Mg2+ cation in both M. tuberculosis and
Mycobacterium leprae enzymes, but then the positive charge
is located on the opposite side of the phosphate compared with the
other known TMPKs, i.e. close to the essential
Asp9. Mg2+ may also contribute to deprotonate
both the Asp9 residue and the phosphate of TMP. We note
that one of the water molecule ligands of the octahedral environment of
the Mg2+ ion is likely to be replaced by one of the
non-bridging oxygens of the -phosphate of ATP as seen in the
structure of the complex with Ap5T, thereby contributing to
the exact positioning of all the reactants and stabilizing the
transition state. This is inferred from the superimposed structures of
the complex of Ap5T with the enzymes from E. coli (19) and/or H. sapiens (17) with the M. tuberculosis TMPK-TMP structure (15).
In the human enzyme, a magnesium ion is found on the ATP molecule.
Since M. tuberculosis TMP kinase has been crystallized only
in the presence of TMP, one can only speculate about the presence of a
second magnesium ion in the active site when ATP is bound. This
(ATP-bound) second magnesium ion, weakening the bond between the -
and
-phosphates and therefore assisting in the departure of the
leaving group, is a general feature of various kinases and is therefore
expected also in the M. tuberculosis enzyme (43). Two
magnesium ions in the active site of kinases would not be surprising as
this has been observed in several instances already. However, looking
again at the multialignment of all known TMP kinases sequences (15),
one notices that Thr14 of the Walker A motif, which is
essential in the coordination of this second magnesium ion, is not
conserved in both M. leprae and M. tuberculosis
sequences. Instead it is replaced by an arginine residue whose
guanidinium group can easily be brought, through a simple
-1
rotation, to the expected binding site of the ATP-bound Mg2+ ion.
The next surprising point is that most crystal soaking experiments did not work, indicating that the TMP molecule could not be exchanged at least in the conformation of the enzyme that has been captured in the crystal. In very much the same way, most co-crystallizations did not work (except for 1), indicating the inability of the analogues tried here to induce the conformational change leading to the form of the enzyme that crystallizes, namely the closed form. Our current interpretation is that the crystal structure has trapped an intermediate already well engaged along the reaction coordinate pathway: most of the inhibitors described here exert their binding abilities on a more open form of the enzyme but are unable to induce the transition from this open form to the closed form. In that sense, the transition to the closed form deserves the name of an induced fit mechanism (44, 45). M. tuberculosis TMP kinase is unique among the other known crystal structures of TMP kinases in that the closed form is already formed upon TMP binding with the ATP binding site clearly preformed, whereas virtually all other NMP kinases need the binding of the two substrates to be in the fully closed form (16-20). Two extrinsic factors contribute to this phenomenon: one is the Mg2+ ion, and the other is the sulfate ion, which drives the closing and the disorder-order transition of the LID region and which comes from the precipitating agent used to obtain crystals. The Mg2+ role has been mentioned above while discussing the inhibition by AZTMP. The role of the sulfate is worth discussing in light of the Ap5T complex structure.
Although the ATP binding site is preformed in our crystal form, the
"ADP" part of Ap5T was found to bind to an unexpected site. This probably results from the inability of Ap5T to
displace the strongly bound sulfate ion that is located at the ATP
-phosphate binding site. Since this sulfate ion comes
from the mother liquor of crystallization, the alternative
binding mode of the ADP moiety of Ap5T might be seen as an
artifact of crystallization. However, since it already binds well into
a cavity on the surface of the molecule for which it has not been
especially designed, it is easy to imagine how one might improve its
binding energy. For instance, it might be possible to take advantage of
the close proximity of Glu55 to C-2 of the adenine
ring and of Glu50 and Asp46 to two different
water molecules bound to the 2'- and 3'-position of ADP. It should then
be possible to design new drugs against TMP kinase by synthesizing
branched molecules at the
-phosphate of Ap5T with an
extra chemical group reaching out to this cavity that is present only
in the M. tuberculosis enzyme and designed specifically to
bind there.
An important finding of the present study is the structural characterization of the binding of TMPK to compound 1. The structure partly verifies our expectation, by showing an anticipated hydrogen bond between 1 and a known water molecule (W12 in Protein Data Bank structure 1G3U), and partly brings a surprise, by displaying an alternative binding mode with the hydroxyl group pointing toward the network of interactions between Glu6, Arg95, Tyr179, and Ser99. Both binding modes contribute to the rigidification of the TMP binding pocket and explain why this compound is an inhibitor rather than a substrate, compared with 5-I-dUMP (13, 15), of comparable volume.
We now discuss in more detail the role of Ser99, a strictly conserved residue whose function has remained unclear up to now. First, we observe that the network of interactions in which Ser99 is involved contains Glu6, the only other non-glycine strictly conserved residue among all known TMPK sequences (15), as well as Arg95, which is almost universally conserved (except for one exception of 32 sequences where it is an histidine). It is tempting therefore to assume a central role in catalysis for this cluster of interacting residues.
In fact, we can describe this structural catalytic core as a "proton
wire" in the active site of TMP kinases, i.e. a
continuous chain of several side chains in line and sharing a proton
including W13, Glu6, Ser99, Arg95,
and Asp9 (Fig. 6). W13 is in
contact with the solvent, so this proton wire couples Asp9
to a reservoir of water molecules (and protons). Asp9,
which is always either an aspartate or a glutamate in all known TMPK
sequences, is within hydrogen bond distance of two non-bridging oxygens
of phosphates, one from the -phosphate of TMP (acceptor) and the
other from the
-phosphate of ATP (donor). The distances between the
residues in this proton wire are within 2.55-2.85 Å with the
exception of one definitely weak link between Arg95 and
Asp9 (3.19 Å). We suggest that this distance becomes
shorter upon ATP binding, making it possible to consider the
Ser99-Arg95-Asp9 set as yet another
variation of the well known catalytic triad of serine proteases
(Ser-His-Asp/Glu). Asp9 could get a proton from
Arg95 (Fig. 6a), which immediately gets
reprotonated from Ser99 and Glu6 (Fig. 6,
b and c). The role of Asp9 would then
be to provide the proton for the transferred phosphoryl group, playing
the role of a general acid (Fig. 6c). We believe that the
proton from the hydroxyl group of the 5'-phosphate of TMP has already
been removed by the Mg2+ ion. In that sense, we are one
step further along the reaction pathway compared with all other
transition-state analogue complex structures of other TMPKs. To sustain
this hypothesis, we note that catalytic triads show considerable
variation of sequence in the three different positions of the classical
catalytic triad (46). There is even one case where the histidine
residue is replaced by a lysine, so arginine is not inconceivable
between a serine and an aspartate (46). Second, it has recently been observed that a previously hitherto unidentified catalytic triad is
present in different families of cellulases where the Asp/Glu residue
plays the role of a proton donor (47). Third, the recent work of Hutter
and Helms (48) urges one to find a proton donor since their quantum
molecular mechanics calculations point to a concerted
phosphoryl transfer mechanism involving the synchronous shift of a
proton from the nucleotide monophosphate to the transferred PO3 in the related enzyme UMP/CMP kinase. According to
these authors (48), most of the activation energy of the
phosphoryl transfer is spent on moving the proton toward its
destination on the
-phosphate group, so it is perhaps not surprising
to find that TMP kinases have evolved such a sophisticated network of
four interacting highly conserved residues to accomplish this
process.
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ACKNOWLEDGEMENTS |
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We thank D. Bourgeois for useful discussions concerning the catalytic mechanism. We thank O. Bârzu for carefully reading the manuscript. V. Vanheusden is indebted to the Fonds voor Wetenschappelijk Onderzoek-Flaanderen for a position of Aspirant.
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FOOTNOTES |
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* This work was supported by Grant BIO-CT98-0354 from the European Economic Community (to both M. D. and P. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1MRS and 1MRN) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
** To whom correspondence should be addressed. Tel.: 33-1-45-68-86-01; Fax: 33-1-45-68-86-04; E-mail: marc.delarue@pasteur.fr.
Published, JBC Papers in Press, November 25, 2002, DOI 10.1074/jbc.M209630200
2 HIC-Up, available at alpha2.bmc.uu.se/hicup/t5a.
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ABBREVIATIONS |
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The abbreviations used are: TMPK, TMP kinase; Ap5T, P1-(adenosine-5')-P5-(thymidine-5')-pentaphosphate; HSV, herpes simplex virus; W, water; HPLC, high pressure liquid chromatography; AZTMP, azido-TMP; Mtub, M. tuberculosis; iPrOH, isopropanol.
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