A new family of inhibitors of Mycobacterium tuberculosis thymidine monophosphate kinase

Full text of DOI:10.1080/15257770701513349

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A New Family of Inhibitors of
Mycobacterium Tuberculosis Thymidine
Monophosphate Kinase
C. Gasse a , V. Huteau a , D. Douguet b , H. Munier-Lehmann a & S.
Pochet a
a Unité de Chimie Organique, URA CNRS 2128, Institut Pasteur, Paris
c, France
b CBS, CNRS, U5048, INSERM, U554, Université Montpellier1,
Montpellier, France
Published online: 10 Dec 2007.
To cite this article: C. Gasse , V. Huteau , D. Douguet , H. Munier-Lehmann & S. Pochet (2007) A New
Family of Inhibitors of Mycobacterium Tuberculosis Thymidine Monophosphate Kinase, Nucleosides,
Nucleotides and Nucleic Acids, 26:8-9, 1057-1061, DOI: 10.1080/15257770701513349
To link to this article: http://dx.doi.org/10.1080/15257770701513349
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Nucleosides, Nucleotides, and Nucleic Acids, 26:1057–1061, 2007
Copyright ꢀ Taylor & Francis Group, LLC
C
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770701513349
A NEW FAMILY OF INHIBITORS OF MYCOBACTERIUM
TUBERCULOSIS THYMIDINE MONOPHOSPHATE KINASE
C. Gasse and V. Huteau
Unite´ de Chimie Organique, URA CNRS 2128, Institut
2
Pasteur, Paris c, France
D. Douguet
Montpellier, France
CBS, CNRS, U5048, INSERM, U554, Universite´ Montpellier1,
2
H. Munier-Lehmann and S. Pochet
Unite´ de Chimie Organique, URA CNRS
2
2128, Institut Pasteur, Paris c, France
Tuberculosis (TB) ranks among the leading causes of death worldwide
from a single infectious agent, particularly in developing countries. The
emergence of multidrug resistant strains of Mycobacterium tuberculosis and
the unfortunate revival of TB due to HIV infection have rendered the quest
for new antimycobacterial drugs against TB a priority.
We currently are studying thymidine monophosphate kinase of M. tuber-
culosis (TMPKmt) as a potential target for developing new antituberculosis
agents.[1] TMPK catalyzes the transfer of γ -phosphate from ATP to dTMP.
TMPK is the last specific enzyme for the synthesis of dTTP and it repre-
sents a key enzyme in its metabolism. Biochemical and structural character-
ization of M. tuberculosis TMPK (TMPKmt) revealed distinct features when
compared to its counterpart from yeast, E. coli and human (TMPKh).[1−3]
As a part of our ongoing program to design inhibitors of TMPKmt.[4−7]
we applied a new fragment-based algorithm called LEA3D to the dTMP
binding site of TMPKmt with the aim to generate new ligand families.[8]
Molecule (1), thus, was identified as a potential inhibitor of TMPKmt, the
benzyl ring acting as a substitute for the sugar part of dTMP (Figure 1). The
target amide 1, as well as the carboxylic acid derivative 2, were synthesized.
The evaluation of their inhibitory potency (Ki) on recombinant TMPKmt
This work was supported by the Institut Pasteur, CNRS, INSERM. C.G. thanks the Ministe`re Franc¸ais
de la Recherche and Pasteur-Weizmann for a doctoral fellowship.
Address correspondence to S. Pochet, Unite´ de Chimie Organique, URA CNRS2128, De´partement
Biologie Structurale et Chimie, Institut Pasteur, 28, rue du Dr Roux, 75724 Paris cedex 15, France. E-mail:
spochet@pasteur.fr
1057

1058
C. Gasse et al.
FIGURE 1 Structures of dTMP and 3-(4-thymidylmethyl-phenyl)-propionamide (1).
concluded to the possible replacement of the sugar part by a benzyl group
(Table 1).
We decided to further explore this family of benzyl-thymine derivatives
by varying the arm length in para position of the benzyl ring, the nature
of the terminal function, as well as the substituent at C-5 position on the
pyrimidine ring (CH3, Br, or Cl). We report here the results obtained in the
thymine series.
CHEMISTRY
The benzyl-thymine derivatives were synthesized according to
palladium-catalyzed coupling reactions between an aryl halide and a
suitable alkene or alkyne. Typically, the first step is the N 1-arylation of
TABLE 1 Evaluation of benzyl thymine derivatives on M. tuberculosis TMPK and on
M. bovis (BCG) bacteria
Cpd
R
TMPKmt Ki (µM) FlexX score
MIC50 (µg/mL)
1
2
3
4
5
6
7
8
6
7
8
9
10
(CH2)2CONH2
(CH2)2COOH
CH=CHCOOH
CH=CHCONH2
(CH2)3COOH
CH=CHCH2COOH
C=CCH2CH2OH
(CH2)3CH2OH
(CH2)3CONH2
(CH2)4COOH
(CH2)4CONH2
(CH2)5COOH
(CH2)5CONH2
89
55
−32
−30
N.I.b
N.I.b
N.D.
N.D.
N.I.b
N.I.b
N.I.b
N.D.
N.I.b
120
N.I.a
195
13
−36
−28
−29
−33
−32
−41
39
70
51
112
58
55
47
27
N.D.
195
230
−48
−36
N.D.: not determined.
aN.I.: no inhibition until 2 mM.
b N.I.: no inhibition until 400 µg/mL.
The MIC50 values are the means of two independent assays performed in duplicate.

New Family of Inhibitors of Mycobacterium tuberculosis
1059
SCHEME 1 General synthetic scheme.
thymine (Scheme 1). For selectivity purpose, the pyrimidine moiety was
suitably N 3 protected by a benzoyl group. Starting from this key halide
intermediate (I), various commercially available carboxylic acids, esters or
alcohols (according to the selected chain length) were introduced by Heck
or Sonogashira coupling reaction. The resulting coupling products (II)
were further hydrogenated into saturated derivatives (III).
INHIBITORY ACTIVITY ON TMPKmt
The ability of synthesized compounds to inhibit in vitro TMPKmt in the
presence of ATP and dTMP was examined on recombinant enzyme using
an enzymatic assay as previously described.[1] Selectivity of the most active
compounds was measured against TMPKh. All molecules tested exhibited
inhibitory potency with Ki values ranged between 13 and 195 µM (except
for compound 3 that is devoid of activity) (Table 1). The highest inhibitor
corresponds to molecule 5 having a chain length of 4 carbons with a termi-
nal carboxylic acid function.
STRUCTURAL ANALYSIS
In an attempt to rationalize these results, the inhibitors were docked
into the active site of TMPKmt. The crystal structure of TMPKmt in com-
plex with dTMP (PDB structure 1G3U) was selected for the docking using
the program FlexX as in our previous studies (Table 1).[8] Molecular mod-
elling suggested possible orientations of the ligands: due to the arm flex-
ibility, two binding modes are predicted, the alkyl chain adopting either
an elbowed or extended conformation (Figure 2). An interaction between
Arg95 and the terminal COOH of molecule 5 can explain that the opti-
mal length within the carboxylic acid series corresponds to an alkyl chain

1060
C. Gasse et al.
FIGURE 2 The predicted binding mode of molecules 5 and 10. The hydrogen bond network is depicted
in yellow. The thymine base is able to form a significant π-stack with Phe70 and three hydrogen bonds
with Arg74, Asn100 and a water molecule (W in magenta). The carboxylate group of 5 is able to interact
with Arg 95, the nitrogen of the amide function of 10 is able to interact with Glu 166 and the backbone
carboxyl of Ala 161.
of 4 carbons. Within the carboxamide series, the strongest inhibitors have
a 6 carbons chain, that corresponds to an optimal interaction between the
nitrogen of the amide function with Glu166 and the backbone carboxyl of
Ala161 (Figure 2, molecule 10).
ANTIMYCOBACTERIAL ACTIVITY AND CYTOTOXICITY
These thymine derivatives were evaluated for inhibitory activity against
whole-cell M. bovis (BCG strain) (Table 1). Compounds 7, 9, and 10 having
a chain length of 5 or 6 carbons exhibited moderate activity (MIC50 up to
120 µg/ml) without any cytotoxicity at concentrations up to 400 µg/ml
(Vero cells).
In conclusion, among the molecules investigated we identified a new
family of benzyl-thymines as potent and selective inhibitors of recombinant
TMPKmt. Some members of these series showed moderate inhibitory po-
tency on the growth of M. bovis (BCG) bacteria while no cytotoxiciy was de-
tected. We are currently working on further modification and optimization
of this new class of molecules.
REFERENCES
1. Munier-Lehmann, H.; Chaffotte, A.; Pochet, S.; Labesse, G. Thymidylate kinase of Mycobacterium
tuberculosis: A chimera sharing properties common to eukaryotic and bacterial enzymes. Protein Sci.
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New Family of Inhibitors of Mycobacterium tuberculosis
1061
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TMP at 1.95 A resolution. J. Mol. Biol. 2001, 311, 87–100.
3. Fioravanti, E.; et al. Mycobacterium tuberculosis thymidylate kinase: structural studies of intermediates
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