Interaction of α-Thymidine Inhibitors with Thymidylate Kinase from Plasmodium falciparum

Article abstract of DOI:10.1021/acs.biochem.8b00162

Plasmodium falciparum thymidylate kinase (PfTMK) is a critical enzyme in the de novo biosynthesis pathway of pyrimidine nucleotides. N-(5′-Deoxy-α-thymidin-5′-yl)-N′-[4-(2-chlorobenzyloxy)phenyl]urea was developed as an inhibitor of PfTMK and has been reported as an effective inhibitor of P. falciparum growth with an EC₅₀ of 28 nM [Cui, H., et al. (2012) J. Med. Chem. 55, 10948-10957]. Using this compound as a scaffold, a number of derivatives were developed and, along with the original compound, were characterized in terms of their enzyme inhibition (K_i) and binding affinity (K_D). Furthermore, the binding site of the synthesized compounds was investigated by a combination of mutagenesis and docking simulations. Although the reported compound is indicated to be highly effective in its inhibition of parasite growth, we observed significantly lower binding affinity and weaker inhibition of PfTMK than expected from the reported EC₅₀. This suggests that significant structural optimization will be required for the use of this scaffold as an effective PfTMK inhibitor and that the inhibition of parasite growth is due to an off-target effect.

Full text of DOI:10.1021/acs.biochem.8b00162

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Interaction of #-Thymidine Inhibitors with
Thymidylate Kinase from Plasmodium falciparum
Mengshen Chen, Kaustubh Sinha, Gordon S. Rule, and Danith H. Ly
Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00162 • Publication Date (Web): 23 Apr 2018
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Interaction of αꢀThymidine Inhibitors with Thymidylate Kinase from Plasmodium falciparum.
Mengshen (David) Chena, Kaustubh Sinhaa, Gordon S. Rulea, Danith H. Lyb*
a Department of Biological Sciences, Carnegie Mellon University, 4400 5th Ave, Pittsburgh PA.
b Department of Chemistry, Carnegie Mellon University, 4400 5th Ave, Pittsburgh PA.
* Corresponding author, dly@andrew.cmu.edu. Tel: (412) 268ꢀ4010.
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Abstract
Plasmodium falciparum thymidylate kinase (PfTMK) is a critical enzyme in the de novo
biosynthesis pathway of pyrimidine nucleotides. Nꢀ(5'ꢀdeoxyꢀαꢀthymidinꢀ5'ꢀyl)ꢀN'ꢀ(4ꢀ(2ꢀ
chlorobenzyloxy)phenyl)urea was developed as an inhibitor of PfTMK and has been reported as
an effective inhibitor of P. falciparum growth with an EC50 of 28 nM (Cui et al, (2012) Journal of
Medicinal Chemistry, 55, 10948ꢀ10957). Using this compound as a scaffold, a number of
derivatives were developed, and, along with the original compound, were characterized in terms
of their enzyme inhibition (Ki) and binding affinity (KD). Furthermore, the binding site of the
synthesized compounds was investigated by a combination of mutagenesis and docking
simulations. Although the reported compound is indicated to be highly effective in its inhibition of
parasite growth, we observed significantly weaker binding affinity and inhibition of PfTMK than
expected from the reported EC50. This suggests that significant structural optimization will be
required for the use of this scaffold as an effective PfTMK inhibitor and that the inhibition of
parasite growth is due to an offꢀtarget effect.
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Introduction
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Malaria is one of the most devastating parasiteꢀborne infectious diseases with over 200 million
infections and over 500,000 deaths each year. Plasmodium falciparum, the deadliest form of
malaria parasite, is responsible for the mortality and morbidity of malaria infection. Current
treatments against P. falciparum malaria include Artemisinin Combination Therapies (ACTs).
Although ACTs work quite well against malaria, there is widespread resistance to many antiꢀ
malarial drugs. The resistance has arisen due to counterfeit or substandard treatments,
unregulated administration of drugs, and the lack of complementary combination treatment,
such as lumefantrine1. Due to increased drug resistance there is a continuing need to develop
additional antiꢀmalarial compounds.
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The development of malaria infection is divided into two phases: exoerythrocytic phase and
erythrocytic phase2, both of which require large amounts of deoxythymidineꢀ5’ꢀtriphosphate
(dTTP) for DNA replication. P. falciparum lacks the ability to salvage pyrimidines3, consequently
it relies solely on de novo pyrimidine nucleoside synthesis to support the high dTTP demand
during rapid DNA replication in the erythrocytic phase. One of the enzymes in the biosynthetic
pathway to dTTP is thymidylate kinase (TMK). Thymidylate kinases are unique in that they are
the only nucleotide kinase that facilitates the phosphorylation of deoxythymidineꢀ5’ꢀ
monophosphate (dTMP) to deoxythymidineꢀ5’ꢀdiphosphate (dTDP).
Xꢀray crystal structures of ligandꢀbound TMKs have been solved in various organisms, such as
human4,Saccharomyces cerevisiae5, Mycobacterium tuberculosis6, Vaccinia virus7, P.
falciparum8, and Candida albicans9. All TMKs are homodimers, each subunit consists of five
stranded parallel β–sheets and 7 to 11 α–helices. There are two conserved motifs in the
nucleotide binding domain: a Pꢀloop, which is involved with substrate recognition and enzyme
catalysis, and a LID domain, which plays a part in catalysis10.
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The low sequence similarity between huTMK and TMKs from bacteria and fungi suggests that
inhibitors of nonꢀhuman TMKs are potential antibiotics because they would selectively interfere
with DNA replication in the pathogen without inhibition of huTMK. Correspondingly, a number of
groups have developed inhibitors of TMKs from a variety of pathogens, including gram positive
bacteria11, 12, Mycobacterium tuberculosis13, 14, and Pseudomonas aeruginosa15.
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The superposition of the enzymeꢀsubstrate complex for human TMK (huTMK) and PfTMK
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Figure 1. Xꢀray crystal structure of PfTMK. Left Panel: PfTMK dimer complexed with
ADP, dTMP, and Na+ (pdb 2WWF). Right Panel: Overlay of huTMK (green) and PfTMK
(cyan). Xꢀray crystal structure of huTMK (pdb 1NN0) is superimposable on that of PfTMK
(pdb 2WWF), except for the LID domain. Purple spheres represent bound Mg+2 ions.
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suggests that the human and Plasmodium enzymes are highly similar in their topology (see
Figure 1). However, the low sequence similarity (40%) between huTMK and PfTMK (see Figure
2), and the fact that PfTMK is one of the few TMKs that can also phosphorylate dGMP, indicates
that it should be feasible to develop effective antiꢀmalarial drugs that are specific inhibitors of
PfTMK3. Kato et al16 explored racemic carbocyclic derivatives of thymidine and obtained KI
values for PfTMK which were as low as 20 ꢀM for compound A (Chart 1). Noguchi et al17
subsequently prepared pure enantiomers of derivative of compound A, producing compounds
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Figure 2. Sequence alignment of PfTMK and HuTMK.. The Pꢀloop motif is well conserved between PfTMK
(top, Pf) and HuTMK (lower, Hu), but not the LID domain, which explains the superimposable Pꢀloop and
nonꢀsuperimposable LID domain in the overlay structure. The conserved Mg+2 binding site is labeled and
highlighted.
with slightly higher affinity. Cui et al reported the development of a large number of inhibitors of
PfTMK8. These compounds were based on an αꢀthymidine thiourea inhibitor of TMK from
Mycobacterium tuberculosis. They established that αꢀthymine inhibitors are more potent than βꢀ
thymidine, and that replacement of the thiourea with urea improved inhibition. Cui reported that
Nꢀ(5'ꢀdeoxyꢀαꢀthymidinꢀ5'ꢀyl)ꢀN'ꢀ(4ꢀ(2ꢀchlorobenzyloxy)phenyl)urea (compound B, chart 1) was
the most effective inhibitor of P. falciparum growth, with an EC50 value of 28 nM and a CC50 of
29 ꢀM. However, the ability of compound B to inhibit PfTMK was not reported, consequently it is
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not possible to determine whether its effectiveness is due to inhibition of PfTMK, or some other
target in the organism.
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Although an effective inhibitor of growth, compound B is metabolically unstable, likely due to the
presence of the chlorobenzene substituent. In the liver, chlorobenzene quickly goes through
oxidation to form an epoxide, and the epoxide is either hydrolyzed or conjugated to glutathione
to form preꢀmercapturic acid18.
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Here, we report the synthesis of a number of derivatives of compound B (see scheme 1). We
have fully characterized the original compound and two of these derivatives, where the chlorine
was replaced by with fluorine or a methoxy group. These compounds were characterized to test
whether the chlorobenzene group is unique in conferring compound B the ability to potentially
inhibit PfTMK. We also wanted to explore the effect of replacing the chlorine atom with
metabolically more stable fluorine19 on the interaction with PfTMK. We have quantified the
binding of these inhibitors to PfTMK (KD) and measured their ability to inhibit PfTMK and report
Ki values. Although the Clꢀderivative has a low EC50 value (28 nM), the experimentally
measured Ki and KD values reported here are significantly higher (200 ꢀM), and unaffected by
the substitution of chlorine with fluorine or methoxy. Docking simulations show that these
compounds bind to the same region of the active site as dTMP and the benzyloxyꢀphenyl group
Chart 1: Inhibitors of PfTMK. The chlorobenzene
group in B was replaced by H, F, CH3 and -O-CH3 in
this work.
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approaches the binding site for ATP. We verified the binding site of these compounds to PfTMK
by generating a mutant PfTMK and show that this mutation greatly reduces the binding affinity.
Our data suggest that compound B will require significant modification to increase its
effectiveness as a PfTMK inhibitor and that the intracellular target of this compound is unlikely to
be PfTMK and the inhibition of parasite growth is likely due to an offꢀtarget effect.
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Materials and Methods
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Synthesis of prospective inhibitors:
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The general synthetic approach is outlined in Scheme 1 and additional details are provided in
the supporting information. The 5’ hydroxyl group of βꢀthymidine is protected with diphenylacetyl
group (compound 10), this was followed by protection of the 3’ hydroxyl group with a pꢀtoluoyl
group (compound 11). These two specific protecting groups were chosen because they are
beneficial for the crystallization of C1’ꢀepimerization product20. Racemization was then carried
out to convert βꢀanomer to αꢀanomer (compound 12)21, 22. After the racemization, the protecting
groups were removed (compound 13), this was followed by a 3ꢀstep amination of 5’ hydroxyl
(selective sulfonylation of 5’ hydroxyl, azidation of sulfonyl group, and hydrogenation) to yield
the amine compound 16. Meanwhile, various benzyl bromide derivatives (5aꢀd) were coupled to
Bocꢀprotected 4ꢀaminophenol (compound 6aꢀ6d), this was followed by the removal of the Boc
protecting group (compound 7aꢀ7d), and synthesis of isocyanate derivatives using triphosgene
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a. R = H
b. R = Cl
c. R= F
d. R = CH3
e. R = OMe
Scheme 1. Library synthesis. Reagents and conditions: (a) NaH, NꢀBoc protected 4ꢀaminophenol, DMF,
0°C, 1.5 hrs; (b) TMSꢀCl, MeOH, RT, 3 hrs; (c) Et3N, triphosgene, EtOAc, 77°C, 3 hrs; (d) Diphenylacetyl
chloride, pyridine, DCM, 0°C, overnight; (e) pꢀtoluoyl chloride, pyridine, DCM, RT, overnight; (f) Acetic
anhydride, H2SO4, ACN, RT, 3 hrs; (g) NaOMe, MeOH, RT, 3 hrs; (h) MsCl, pyridine, ꢀ38°C, 1 hr, 0°C, 3hrs;
(i) NaN3, DMF, RT, 3 hrs; (j) 10% Pd/C, MeOH, RT, 3 hrs; (k) DMF, RT, 3 hrs.
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(compound 8aꢀ8d). Lastly, the two halves were coupled together to yield final compounds (17aꢀ
17d).
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Synthesis of the methoxy derivative (17e) did not fully follow this general synthetic scheme as
the starting materials for this derivative was not commercially available. Instead, 3ꢀ
methoxybenzyl alcohol was reacted with phosphotribromide in dichloromethane (DCM) for 3
hours at 0 ºC to generate compound 5e, and the rest of the synthesis procedure to the final
compound 17e followed the general synthetic scheme.
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Our synthesis procedures generally followed that of Cui et al8. However, some of the published
synthetic strategies had unacceptably low yields. Consequently, conditions and reagent
optimizations were made for the synthesis of the prospective inhibitors: the removal of Bocꢀ
protecting group from compounds 6a – e were carried out by dissolving the starting materials in
methanol, then slowly titrating in one equivalence of trimethylsilane chloride, the reaction was
carried out at room temperature for two hours, giving the following yields: 6a (96%), 6b (92%),
6c (92%), 6d (86%), and 6e (96%).
Additional changes in the synthesis including increasing the reaction time for the 3’ꢀ and 5’ꢀ
protection of βꢀthymidine to overnight for optimal yield; and the azidation reaction of compound
14 was performed at 100°C overnight, the reaction mixture was then subjected to highꢀvacuum
for solvent removal, and was reꢀdissolved in ethanol, the precipitate was then removed, and the
ethanol filtrate was concentrated and subjected to column chromatography to purify compound
15.
Protein expression and purification:
A DNA sequence for PfTMK that was optimized for expression in Escherichia coli was obtained
from DNA 2.0, with a (His)6 tag at the amino terminus for the purification purpose. This DNA was
cloned into the T7 expression plasmid pETꢀ22b23 and used to transform C3013 cells (New
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England Biolabs). Clones that expressed PfTMK were maintained with 50 mg/ml of ampicillin.
For the production of nonꢀisotopically labelled protein, a single colony was inoculated into 5ml of
Luria Broth (LB), grown overnight, transferred to larger volume of LB media, and induced for 3
hours with 1mM isopropylthiogalactoside (IPTG) after the optical density (OD600) at 600 nm
reached 0.7.
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Isotopically labeled PfTMK was produced by inoculating a colony into 1 ml of Studier’s PG
media24 with 50% H2O and 50% D2O for overnight growth. The D2O level was increased by
adding additional 4ml of the D2O media the next day and incubated for 5 hours. The cells were
then harvested and transferred to a larger volume of PG media containing 100% D2O. Once the
OD600 reached 1.4, precursors were added to the culture (ketobutyric acid – methyl 13C (50
mg/L) and alpha – ketoisovaleric acid (100 mg/L)25). The culture was then incubated at 30°C for
1 hour, and then induced with 1mM IPTG for 18 hours.
The induced cells were harvested and resuspended in 20 ml of lysis buffer (50 mM potassium
phosphate, 250 mM NaCl, 5 mM imidazole, 0.02% azide, 10% glycerol, 0.1 mM ADP, 0.1 mM
TMP, 10 mM MgSO4, pH 7.4) followed by the addition of lysozyme (1mg/ml) followed by
incubation for 30 min at room temperature. This was followed by the addition of 1% TritonꢀX and
sonication with Fisher Scientific 550 Sonicator. The lysate was subjected to centrifugation
(19,000 g for 30 minutes) and the supernatant was collected and incubated with 5ml Hispur
cobalt resin (Thermal Scientific) for 1 hour at 4 ºC. The supernatantꢀresin mixture was loaded
onto a gravity column and was washed with 10ꢀcolumn volumes of wash buffer (50 mM
potassium phosphate, 250 mM NaCl, 5 mM imidazole, 0.02% azide, pH 7.4). Lastly, the protein
was eluted with 6ꢀcolumn volumes of elution buffer (50 mM sodium phosphate, 300 mM NaCl,
450 mM imidazole, pH 7.4). Buffer exchange was performed with Amicon Ultra concentrator to
remove the high concentration of imidazole in the protein sample. The protein sample was then
loaded on to a QꢀSepharose column that was preꢀequilibrated with column buffer (20 mM
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potassium phosphate, 0.92% sodium azide, 1 mM EDTA, pH 8.0). The protein was then eluted
with a linear salt gradient from 0M to 2 M NaCl in column buffer, and the eluate was collected on
fraction collector. The purified protein was then concentrated and stored at 4 ºC, typical yields
are 50 mg of PfTMK from 1 L of culture.
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NADH Coupled Assay
The NADH coupled assay26 was performed to measure the KM of PfTMK for TMP, and to
estimate the Ki of the Clꢀ, Fꢀ, and OMeꢀderivatives (17b, 17c, 17e) against PfTMK.
In the measurement of KM of PfTMK for TMP, 1 ml kinase reactions contained 50 mM TrisꢀHCl,
50 mM KCl, 0.5 mM phosphoenolpyruvate, 1 mM ATP, 20 mM Mg2+, 5 U/ml lactate
dehydrogenase, 5 U/ml pyruvate kinase, 0.15 mM NADH, and a range of concentrations of TMP
from 2 to 150 ꢁM. The reaction was initiated by the addition of 200 nM of PfTMK and the
absorbance at 340 nm was measured as a function of time.
The Ki values for Clꢀ, Fꢀ, and OMeꢀderivatives were obtained by adding the compound to the
reaction mixture prior to the addition of enzyme. We determined the initial velocities at various
TMP concentrations (2 to 150 µM) in the presence of saturated ATP concentrations, and in the
presence of various concentrations (100, 200, and 300 µM) of each compound. The Ki for each
compound were obtained using a Dixon plot.
Tryptophan fluorescence and titration assay
Tryptophan fluorescence titration experiments were conducted to determine the binding affinity
of the Clꢀ, Fꢀ, and OMeꢀderivatives to PfTMK. The general experimental conditions were 300 nM
PfTMK and 20 mM Mg2+ in Tris buffer pH 7.4. 50 ꢁl of the reaction mixture was pipetted into the
wells on a 384ꢀwell plate. The Tryptophan fluorescence was measured with Tecan Infinite
M1000 plate reader with excitation at 280nm and recording the fluorescence intensity as a
function of wavelength (288 to 500 nm) for 80, 120, 160, 200, 240, 280, 320, 360, and 400 ꢁM
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of each compound. The KD for each of the synthesized compound was estimated by fitting the
experimental values of ∆F to the following equation: ∆F = ∆Fmax[C]/(KD+[C]) using the Solver
routine in Excel.
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Generation of F74A mutant
Siteꢀdirected mutagenesis was performed to generate F74A mutant PfTMK using standard PCR
methods27. The presence of the mutation was verified by Sanger sequencing of the entire
coding region and the protein preparation and purification followed the same procedure as
described above. The structural integrity of the F74A mutant was assessed by comparison of a
twoꢀdimensional HMQC methyl carbon spectra for the mutant and wildꢀtype enzymes. The
kinetic activity of the mutant protein was examined using the NADHꢀcoupled assay. The activity
of the F74A mutant was evaluated through NADHꢀcoupled assay and no enzymatic activity was
detected, consistent with the previous findings by Kandeel et al28. The binding properties to the
F74A protein were examined by tryptophan fluorescence titration assay.
Docking simulation
The docking simulation experiments were carried out using Autodock Vina29. In our docking
simulations, we used the Xꢀray crystal structure of PfTMK in complex with TMP (pdbid 2WWF)10.
The 3D structure of the ligands were prepared with Chemsketch version 14.0130 and the
PDBQT format of both the receptor and ligands were prepared with Autodock Tools31. All water
and solvent atoms were removed, and the polar hydrogens were added prior to docking
simulation. The grid box was generated centering the TMP binding site of PfTMK and the
coordinates of the grid box were then fed to Autodock Vina for the docking simulation
calculations. Molecular structure images were prepared using Pymol, version 1.832.
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Our synthetic procedure generally followed previous literature methods8, 21, 33 with modifications
as described above. One of the most crucial steps in this procedure is the racemization of 3’
and 5’ protected βꢀthymidine to its αꢀthymidine anomer. The reaction was closely followed by
thinꢀlayer chromatography (TLC), the reactant and the product exhibit distinguishable Rf values,
and the reaction was terminated after the reactant was fully consumed. The identities of
compounds 11 and 12 were determined by proton NMR. The end products: compounds 17a to
17e were confirmed by both proton NMR and massꢀspec (Supporting information Figure S1 to
S5). The Clꢀderivative (17b) was further evaluated through a series of 2D NMR experiments,
including HMBC, HSQC, and COSY (not shown).
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Compounds 17b, 17c, and 17e were utilized in subsequent kinetics and binding assay. Although
compound 17b was previously characterized in vivo8, its in vitro KD and Ki values were not
reported. Compounds 17c and 17e were characterized because 17b contains a
pharmacologically unstable chloroꢀbenzene substituent while 17c and 17e are substituted with
electronꢀwithdrawing and electronꢀdonating groups, i.e. fluoroꢀbenzene, and methoxyꢀbenzene.
PfTMK Inhibition:
NADHꢀcoupled assay was performed to experimentally measure the KM and kcat of PfTMK for
TMP, and to examine the inhibition effect of compounds 17b, c, and e. The Kcat of WT PfTMK
was measured to be 3.14±0.10 sꢀ1, and the KM for TMP for PfTMK was 25.5±0.49 µM (triplicate
measurements); our experimental values for both the kcat and KM are consistent with previously
published data28, 34
.
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Compound
KD (ꢁM)
Ki (ꢁM)
225
Clꢀderivative (17b)
Fꢀderivative (17c)
OMeꢀderivative (17e)
175 ± 2
177 ± 15
181 ± 1
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Table 1. KD and KI values for compounds 17b, 17c, 17e.
The Clꢀ, the Fꢀ, and the OMeꢀderivatives (17b, 17c, 17e) all behaved as competitive inhibitors;
showing no effect on apparent kcat, while significantly increased the apparent KM value. The
measured Ki values for these compounds are shown in Table 1. The Dixon plot for 17c is shown
in Figure 3B and in the supporting information for compounds 17c (Figure S6) and 17e (Figure
S7).
Tryptophan fluorescence titration:
To validate the KI values from the NADH coupled assay, KD values were obtained from
tryptophan fluorescence as a function of inhibitor concentration for each of the synthesized
compounds (17b, 17c, 17e). The fluorescence changes due to the binding of 17b are shown in
Figure 3b. The KD values for each of the compounds were measured in duplicate and are given
in Table 1. The measured KD values are similar to the measured Ki values.
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Figure 3. Characterization of Clꢀderivative (17b). Panel A: KD estimation: ∆F is plotted
against the concentration of the synthesized compound and fitted to a hyperbolic curve for
the estimation of the KD value. The KD of compound 17b is estimated to be 175.08±1.56 ꢁM.
Panel B: Dixon plot for the estimation of Ki: 1/v is plotted against the concentration of the
synthesized compound; the xꢀcoordinate of the intercept of the trend lines = ꢀKi. The Ki for
compound 17b is estimated to be ~225 ꢁM. Panel C: Left: Tryptophan fluorescence titration
curve for F74A mutant. Right: Tryptophan fluorescence titration curve for Wildꢀtype PfTMK.
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Docking Simulation:
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On the basis of docking simulations, the synthesized compounds share the same binding
pocket with TMP and exhibit similar binding mode (Figure 4, S8, S9). The Clꢀ, Fꢀ, and OMeꢀ
derivatives exhibited favorable and similar binding energies (Clꢀderivative (17b): ꢀ9.3 Kcal/mol;
Fꢀderivative (17c): ꢀ9.3 Kcal/mol; OMeꢀderivative (17e): ꢀ9.1 Kcal/mol), this suggests that
substituting the functional groups on the terminal benzyl group has relatively small effect on the
binding, as indicated by our KD measurements presented in table 1. The three compounds fit
into the TMP binding pocket and retain the base stacking between the pyrimidine base and that
of the aromatic ring of Phe74 residue, which is consistent with their activity as competitive
inhibitors. The docking simulations also show that the alpha configuration facilitates interactions
with His71 and Arg78 residues, which improves the interaction of the 3’OH with Tyr107 and
Arg99. In addition to these residues, the docking simulation revealed that Asp17, Arg18, Lys21,
Arg47, and Glu151 potentially interact with the synthesized compounds (see Figure 3 and Table
2).
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The thymidine ring of the inhibitors stacks on the phenyl group of Phe74, in a similar manner to
the substrate (TMP). The critical role of Phe74 in binding thymidine is shown by the fact that the
F74A mutant has no enzymatic activity28. Consequently, the replacement of Phe74 with Ala
(F74A) should significantly reduce the binding of the inhibitors to PfTMK.
The structural integrity of the F74A mutant was previously investigated using circular
dichroism28. Those experiments indicated that there were no significant changes in the overall
secondary structure of the F74A protein. To characterize the effect of this mutation on the
tertiary structure of PfTMK, we compared 2D protonꢀcarbon HSQC spectra of ILV labeled F74A
protein to the ILV spectrum of wildꢀtype protein (Figure. 4). A small number of methyl peak shifts
were observed in the isoleucine region (presumably I55, and I70, as they are 4.7 Å and 5.9 Å
away from Phe74), however the majority of the peaks were in similar positions.
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Tryptophan fluorescence was used to determine the binding constant of the inhibitors to the
F74A protein for the chlorine derivative (Figure 3C), as well as the fluoro (Figure S6C) and the
methoxy derivative (Figure S7C). These data indicate that the affinity of the F74A protein for the
inhibitors is considerably reduced, as would be expected due to loss of stacking between the
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B
A
R99
Y107
D17
H71
F74
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K21
K21
K21
Figure 4. Simulated Docking. Panel A: Chloroꢀbenzene substituent (17b) fits tightly into the
TMP binding pocket with a binding energy of ꢀ9.3 Kcal/mol. Panel B: Atomic level detail of
the αꢀthymidine moiety interacting with PfTMK. Panel C: Chloroꢀbenzene substituent
(compound 17b) interacting with Lys21. Panel D: Fluoroꢀbenzene substituent (compound
17c) interacting with Lys21. Panel C: Methoxyꢀbenzene substituent (compound 17e)
interacting with Lys21.
thymidine base and F74.
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Figure 5. Overlay of PfTMK HMQC spectra of ILV methyls. Wildꢀtype PfTMK spectrum
(black) is overlaid with the F74A mutant spectrum (red).
Table 2. Compound interaction with PfTMK
Amino acid residue
Tyr107
Arg99
Ligand
3’OH of TMP
3’OH of TMP
Asp17
3’OH, urea linkage
Pyrimidine base
Pyrimidine base
Pyrimidine base
Urea linkage
Phe74
His71
Arg78
Glu151
Arg47
Urea linkage
Arg18
Phenol group
Lys21
Terminal benzyl functional group
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Conclusions
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Here, we present strong evidence from enzyme inhibition, binding studies, docking simulation,
and mutational studies that show that the Clꢀ, Fꢀ, and OMeꢀderivatives (17b, 17c, 17e) are all
competitive inhibitors against PfTMK with comparable Ki and KD values. Consequently, the
chloro group can be substituted with electron withdrawing or donating groups (i.e. fluorine and
methoxy) without significant changes in binding affinity.
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Although the Clꢀderivative (17b) is an efficient inhibitor of parasite growth, with an impressive
EC50 value of 28 nM8, our experimental data show that the binding to PfTMK is relative weak,
with Ki values around 200 ꢁM. This suggests that the physiological target of the Clꢀderivative is
unlikely to be PfTMK, but some other enzyme in the cell. Alternative explanations are that the
Clꢀderivative may accumulate to high concentrations intraꢀcellularly. Conversely, the effective
growth inhibition could also be explained by the fact that the metabolite of the Clꢀderivative
consists of an epoxide, which could lethal to the organism.
Although the results from the docking simulation suggest that modifications could be made to
the terminal benzyl group or the urea linkage that would improve binding of these inhibitors, the
relatively high KI values for these compounds suggest that significant structural modifications
will be required to increase their binding affinity. It is unclear if these changes would provide
sufficient improvement to the binding energy such that they would become effective at inhibiting
PfTMK and consequently growth of the parasite. Moreover, considering that the inhibition of
growth by the Clꢀderivative is likely due to offꢀtarget effects, the Clꢀderivative may not serve as
an optimal scaffold to investigate structure activity relationships (SAR) for this group of
compounds with respect to PfTMK. Rather, it will be necessary to identify the true intracellular
target of these compounds before attempting more detailed SAR studies.
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Lastly, the high quality of the methyl NMR spectrum of the PfTMK enzyme indicates that it will
be possible to utilize chemical shift perturbation of methyl groups to screen fragment libraries to
identify new synthetic routes to more effective PfTMK inhibitors.
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Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This research was supported by funding from the Mellon College of Science to GSR. We thank
Roberto Gil and Virgil Simplacenu for support of the NMR instrumentation.
Supporting Information
Details on chemical synthesis schemes to generate compounds. Oneꢀdimensional proton NMR
spectra of compounds 17aꢀ17e, with assignments. Interaction of Fꢀderivative (17c) and OꢀMet
derivative (17e) with wildꢀtype and mutant (F74A) PfTMK, characterized by tryptophan
fluorescence and enzyme inhibition. Images of docked compounds for Fꢀ and OꢀMe derivatives.
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For Table of Contents Use Only
Interaction of ꢀThymidine Inhibitors with Thymidylate Kinase from Plasmodium falciparum
Mengshen (David) Chena, Kaustubh Sinhaa, Gordon S. Rulea, Danith H. Lyb*
α
.
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a Department of Biological Sciences, Carnegie Mellon University, 4400 5th Ave, Pittsburgh PA.
b Department of Chemistry, Carnegie Mellon University, 4400 5th Ave, Pittsburgh PA.
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References:
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