From the Scuola Internazionale Superiore di Studi
Aranzati, International School for Advanced Studies, via
Beirut 2-4, 34013 Trieste, Italy, the § Istituto Nazionale
di Fisica della Materia, 34014 Trieste Italy, the
¶ Department of Applied BioSciences,, Institute of Pharmaceutical
Sciences, Swiss Federal Institute of Technology, Winterthurerstrasse
190, CH-8057 Zürich, Switzerland, and the ** International Centre
for Genetic Engineering and Biotechnology, AREA Science Park,
Padriciano 99, 34012 Trieste, Italy
Received for publication, November 9, 2000, and in revised form, January 18, 2001
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ABSTRACT |
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Most antiherpes therapies exploit the large
substrate acceptance of herpes simplex virus type 1 thymidine kinase
(TKHSV1) relative to the human isoenzyme. The enzyme
selectively phosphorylates nucleoside analogs that can either inhibit
viral DNA polymerase or cause toxic effects when incorporated into
viral DNA. To relate structural properties of TKHSV1
ligands to their chemical reactivity we have carried out ab
initio quantum chemistry calculations within the density
functional theory framework in combination with biochemical studies. Calculations have focused on a set of ligands carrying a
representative set of the large spectrum of sugar-mimicking moieties
and for which structural information of the TKHSV1-ligand complex is available. The kcat values of these
ligands have been measured under the same experimental conditions using
an UV spectrophotometric assay. The calculations point to the crucial
role of electric dipole moment of ligands and its interaction with the
negatively charged residue Glu225. A striking
correlation is found between the energetics associated with this
interaction and the kcat values measured under
homogeneous conditions. This finding uncovers a fundamental aspect of
the mechanism governing substrate diversity and catalytic turnover and
thus represents a significant step toward the rational design of novel
and powerful prodrugs for antiviral and TKHSV1-linked suicide gene therapies.
The thymidine kinase from herpes simplex virus type 1 (TKHSV1)1 salvages thymine into the metabolism
of the virus by converting it to
thymidine monophosphate (1). In contrast to the human isoenzyme,
TKHSV1 acts as phosphorylating agent toward a large variety
of nucleoside analogs such as (North)-methanocarba-thymidine (n-MCT),
aciclovir (ACV), ganciclovir (GCV), and penciclovir (PCV). These
analogs exhibit chemical diversity for the nucleobase as well as for
the sugar-like chain moiety (2, 3) (Fig.
1). Substrate diversity of
TKHSV1 provides the molecular basis for effective and
selective treatment of virus infections. It is also exploited for gene
therapy approaches involving TKHSV1 as suicide gene by
anticancer intervention (4) or the control of graft versus
host disease by allogeneic bone marrow transplantation (5).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Chemical formulas of selected (fraudulent)
substrates and inhibitors of TKHSV1. dT is the natural
substrate; n-MCT, ACV, PCV, GCV, AHIU, AHTMU, and AZT are prodrugs; and
HBPG is an inhibitor. The 5'-OH and 3'-OH groups belonging to dT and
their mimics are labeled.
The recent determination of the x-ray structure of a large spectrum of
ligand-enzyme complexes (6-11) has opened a new avenue for the
understanding structure-function relationships as well as of the
functional role of amino acids involved in binding. It has emerged that
the enzyme accommodates the nucleobase and sugar-like chain moieties in
two different pockets (P1 and P2 in Fig.
2), interacting with the ligand by a
specific and extensive H-bond network.
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In the pocket P1, the nucleobase moiety (either a thymine or a guanine ring) is stabilized by direct H bonds with the highly conserved Gln125 and with Arg176 by means of two ordered water molecules. The pyrimidine ring is further fixed by a peculiar interaction with Tyr172 and Met128 (11) in a sandwich-like orientation (Refs. 12 and 13 and Fig. 2).
In P2, the sugar-like chains interact with the protein via its hydroxyl
groups. The 3'-OH (and its mimics) forms specific H bonds with
Tyr101 and/or Glu225 (Figs. 2 and
3), whereas 5'-OH forms a direct H bond
or water-mediated interactions with Arg163,
Glu83, and Arg222 (Figs. 2 and 3). In contrast,
the polar C-1'-O-4'-C-4' function belonging to dT and the
correspondent ether groups of ACV and GCV interact neither with polar
or charged groups of the protein nor with the solvent; instead, they
point toward a hydrophobic region made up of the Trp88,
Ile97, and Met128 side chains (Fig.
4).
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Key aspects of nucleoside binding have been recently elucidated by
theoretical (12) and experimental (6-11) approaches. In contrast,
fundamental questions regarding the nature of the interactions between
the ribose-like moiety and the enzyme are still open. First, it
is rather intriguing that the accessibility of the 5'-OH mimic
to the ATP cofactor is roughly the same (Fig. 5), yet the HBPG molecule, which shares
the same binding mode as the structurally related prodrug aciclovir
(6), inhibits the enzyme. Furthermore, it is not known why
the kcat values of prodrugs are much smaller
than that of the natural substrate (see Table
I), although the protein-sugar mimicking
H bonding interactions is very similar (Fig. 5). Finally, as
discussed above, it is rather surprising that the environment
accommodating the inner sugar ring C-1'-O-4'-C-4' group of the
natural substrate is totally hydrophobic (Fig. 4).
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In this paper we address these fundamental issues by performing a
combined quantum chemical and biochemical investigation of sugar and
sugar-like moieties of substrates and inhibitors of TKHSV1.
The theoretical methodology is based on gradient-corrected density
functional theory. This approach allows an accurate description of
electrostatic and hydrogen-bonded complexes (12, 14-17), such as those
investigated here. The biochemical study involves the measurement of
the catalytic constant (kcat) of dT and prodrugs using a modified UV spectrophotometric assay based on pyruvate kinase
and lactate dehydrogenase (3). This assay does not require the use of
radioactively labeled compounds. This provided a set of homogenous
data, which is necessary for a proper comparison with the quantum
chemical calculation. As we will show, the interaction between the
electric dipole of the sugar mimicking moiety and the
negative charge of the conserved residue Glu225
correlates with the kcat measurements and thus
is a critical factor for the phosphorylation reaction.
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EXPERIMENTAL PROCEDURES |
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Biochemical Essays
Materials-- PreScission protease was purchased from Amersham Pharmacia Biotech. AZT and reagents for enzyme assays were obtained from Sigma. Strain BL21 (Amersham Pharmacia Biotech) served as the expression host. n-MCT and PCV were kindly provided by Dr. Marquez (National Institutes of Health, Bethesda, MD) and Dr. Johannsen (Forschungszentrum Rossendorf Institute of Bioinorganic and Radiopharmaceutical Chemistry Research Center, Rossendorf, Germany), respectively. 5-Trifluoromethyl anhydrohexitoluridine (AHTMU), AHIU analogs, and HBPG were gifts of Dr. De Clercq (Rega Institute Katholike Universiteit Leuven, Belgium) and Dr. Wright (University of Massachussets, Worcester, MA), respectively. ACV and GCV were purchased by Glaxo-Wellcome and Roche, respectively. dT was obtained from Fluka.
Expression and Purification of TKHSV1-- TKHSV1 was expressed as gluthathione S-transferase fusion protein in competent Escherichia coli BL21 using the vector pGEX-6P-2-TK (9). The protein was purified by gluthathione-affinity chromatography followed by on-column PreScission protease cleavage using a previously described protocol (9). Purification was monitored by SDS-polyacrylamide gel electrophoresis and led to a >90% pure TKHSV1, which was directly used for kcat determination. Total protein concentration was measured using the Bio-Rad protein assay.
Spectrophotometric Assay for kcat Determination-- An UV spectrophotometric test based on a lactate dehydrogenase-pyruvate kinase coupled assay (3) was employed to monitor ADP formation during substrate phosphorylation. The concentrations of ATP and the ligands were 5 and 1 mM, respectively. These concentrations are at least five time higher than the binding affinities of the studied compounds allowing the measurement of Vmax and thus of kcat (kcat = Vmax/[E]). The change in absorbance at 340 nm was recorded over time and correlates with the kcat of the analyzed substrates for which values were known from the literature and allows the determination of kcat for compounds that are not available in radiolabeled form (see Table I).
Quantum Chemical Calculations
Our structural models for quantum chemical calculation on the sugar moiety of dT-, ACV-, GCV-, PCV-, n-MCT-, and HBPG-TKHSV1 were constructed from the correspondent crystal structures (Protein Data Bank codes 1kim, 2ki5, 1ki2, 1ki3, 1e2k, and 1qhi) (6, 8, 9). The resolution of the structures ranges from 1.7 to 2.4 Å.
The structure of AHTMU-TKHSV1 has not been solved yet, whereas the structure of the complex AHIU-TKHSV1 is known (Protein Data Bank code 1ki6) (8). AHIU is chemically and structurally extremely similar to AHTMU (Fig. 1); the two analogs differ only for the group at position 5 of the nucleobase ring (a iodine in AHIU and a CF3 in AHTMU). All available structural data show that this substitution does not affect the binding orientation of the sugar mimicking moiety (8). The initial configuration of AHTMU is therefore built from this x-ray structure.
The x-ray structure of the AZT-TKHSV1 complex is not available, and therefore one has to resort to theoretical structural models. Here we used molecular dynamics-based models reported earlier (18).
The complexes included the sugar-like moieties of the ligands and part of side chains of all of the groups directly interacting with it or forming water-mediated hydrogen bonds (Tyr101, Arg163, Glu83, Glu225, and Arg222). Trp88, Ile97, and Met128 side chains were not included because (i) they would have dramatically increased the size of our model complexes, (ii) they do not contribute significantly to the electrostatic interactions (the focus of the present work) because they form only weak hydrophobic contacts with the sugar, and (iii) the position of these groups is essentially the same in all of the complexes investigated here (6, 8, 9) and therefore their contribution is expected to be rather constant. Hydrogen atoms were added assuming standard bond lengths and bond angles. In Fig. 3 the quantum mechanical model for dT, the natural substrate, is shown in detail. The overall charge of our complexes is 0.
The calculations were performed within the framework of density
functional theory (19) in its Kohn and Sham (20) formulation. In this
approach the use of gradient corrections is crucial for correctly
describe hydrogen bond interactions. Here we used the prescription of
Becke and Lee (41, 43) because it has been shown to provide an
accurate description of water and hydrogen bonding in biological
systems (14, 15, 21, 22). The basis set consisted of a plane wave basis
set up to a cut-off of 70 Rydberg, the interactions between valence
electrons and ionic cores being described by pseudopotentials of the
Martins-Troullier type (23). Only the point was used. The
dT-TK complex was inserted in orthorombic box with edges 14 × 15 × 16 Å3. Similar box sizes were used for all of the
other complexes. In all circumstances the separation between periodic
images was at least 6 Å. However, the Coulombic interaction between
images was screened using the approach of Barnett and Landman (24). Geometry optimization was performed using the direct inversion iterative subspace (25). C
atoms were constrained to
crystallographic positions to take into account the reduced mobility of
residues caused by protein environment. A similar procedure has been
reported elsewhere (14).
The electronic structure was described with the geometrical
analysis of centers of maximally localized Wannier functions (WFC) (26-28). This analysis is useful for investigating the polarity of
chemical bonds, because WFC shifts are related quantitatively to the
differences of Pauling electronegativities (29) with respect to
the noninteracting compounds (22); an increase of
corresponds to
an increase of polarization of the electronic density in the X-Y bond
toward the most electronegative atom X. The effects of the protein
electric field were estimated through a comparison of Wannier center
shifts for the system in vacuum or in the presence of a protein
electric field. The procedure was identical to that of Piana and
Carloni (14). Interaction energies between the substrate and the active
site are calculated within the central dipole approximation (30) where
the dipole is the result of the quantum calculation. Interaction energy
was calculated as Echarge-dipole= (
µ·R)/(4
0R3), where
R is the distance vector between the centers of charges of
the sugar moiety and the residue of charge
and µ is the dipole moment for the sugar moiety.
Even if the expansion is limited to large distances between two
interacting groups, this model can provide quantitative results for a
comparison between systems that have similar geometries. We have
reported the energetics in arbitrary units.
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RESULTS |
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Catalytic Activity of TKHSV1
The catalytic activity of TKHSV1 with the compounds
listed in Fig. 1 was measured under the same conditions. Table I shows that the measured kcat of dT agrees with those
reported in literature (31, 32). Also the kcat
of ACV corresponds to that measured in two independent laboratories
(33, 34). In the literature a value of 0.015 s1 is also
reported for ACV (31), but, considering the newly reported values, this
seems to be an outliner. The kcat values of GCV
and PCV are also congruent with the literature values (31, 34). The
same agreement has been found for AZT whose kcat
value is 0.044 s
1. The kcat values
of the new compounds AHTMU and n-MCT have been reported here for the
first time.
Quantum Chemistry Calculations
Overall, the geometry of the complexes are in very good agreement with the experimental structural data as shown by the low root mean squared deviation values between experimental and optimized structures (Table II). In particular Table III shows that the structural parameters of the geometry optimized systems are in good agreement with experimental data. Thus our first principle approach appears to reproduce very reliably the structural properties of the adducts. Geometry optimization permits also us to establish the H bond network between the substrate and the enzyme that is crucial in determining the electrostatic properties of the adducts and that is only indirectly deducible from the crystal structure where hydrogen atoms are missing.
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Enzyme-Sugar Interactions--
One of the most important physical
properties of polar molecules such as ribose and its analogs is given
by their electric dipole moment. Fig. 6
offers a visual representation of the calculated dipoles projected to
the geometry-optimized structure. The dipole of the sugar moiety of the
natural substrate aligns strikingly to the negatively charged
Glu225 group. Interestingly, the dipoles of most of the
prodrugs investigated here also point to these residues. The calculated
stabilization energy resulting from the electrostatic interaction is
the maximum for the natural substrate (Table III). The dipoles of AZT
and the inhibitor HBPG do not point toward the negative residue and as a consequence the interaction energy is repulsive.
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In an attempt at correlating electrostatic interactions with kinetics data, we first subdivide the compounds into two major classes. The first class includes the compounds bearing a thymine or a thymine-substituted base (namely dT, AHTMU, n-MCT, and AZT); the second class includes the compounds bearing guanine (ACV, HBPG, GCV, and PCV).
The subdivision is necessary as the computational model did not
include the base and consequently the electrostatic calculations do not
consider the contributions of the nucleobases (12), whose dipole
moments are different but
additive.2 In Fig.
7, the correlation between the
interaction energy dipole-Glu225 and the catalytic constant
of the studied ligands appears clearly. The slope of the linear fit
depends on the type of sugar mimicking moiety of the ligand. The
additivity of the sugar-like chains and the nucleobase dipoles is
confirmed by a rigid shift between the linear fit for the guanine and
thymine derivatives (Fig. 7).
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The Roles of Arg222, Arg163, and Glu83 Interacting Groups-- Although there is no direct correlation between the kcat and the Arg163-dipole interaction, the calculated interaction between Arg163 and the sugar is important, consistent with previously published mutagenesis experiments (35). From the crystallographic and mutagenesis studies, Arg222 and Glu83 are essential elements of the kinase machinery; Arg222 forms the anion hole with Arg220 to make the phosphate atom more electrophilic, and Glu83 behaves as base on the O-5' atom, causing an increase in nucleophily. Thus it is not surprising that the Arg222- and Glu83-dipole interactions appear not to have any direct correlation with kcat (data not shown), because their influence is based on a totally different mechanisms.
Sugar-TKHSV1 Hydrophobic Interactions-- Our calculations have pointed to the crucial role of the electric dipole moment of the sugar moieties. We now address the following question. In the natural substrate, is the polar C-1'-O-4'-C-4' (which faces a hydrophobic pocket; Fig. 4a) important for the correct orientation of the dipole? To answer this question, we calculate the change of electric dipole associated with replacement of O-4' with the CH2 apolar group (Fig. 4b). Our calculations show that the resulting dipole is both smaller and different in orientation relative to the sugar moiety (Fig. 4, b and c). We conclude that the polar function is essential for a correct alignment of the dipole to the Glu225 charge.
Protein Environment Effects--
The field of the protein may be
very important for the chemistry of the active site of this and other
enzymes (36). Here we estimate the effect of the protein frame by
comparing the electronic structure of the complexes in a vacuum with
those in the presence of the protein. A convenient representation of
the electronic structure is given by the Wannier functions, whose
centers (WFC) represent chemical concepts such as lone pairs and
chemical bonds. Fig. 8 shows that there
is no appreciable displacement of the WFC, so that it is possible to
conclude that the main contribution to the interaction is included in
the model we have chosen for quantum mechanics calculations.
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DISCUSSION |
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Deciphering the binding mode of prodrugs to the enzyme and their catalytic turnover may help to rationally design more and potent prodrugs for antiviral therapy to overcome the problems of resistance or to be used in enzyme-prodrug gene therapy as well as improve variants of TKHSV1 for gene therapeutic approaches (37).
The mechanisms ruling binding affinities toward TKHSV1 have been previously presented (8, 9, 12, 38). However, the intriguing question posed by the recently published crystal structure of the inhibitor HBPG (6, 9) as to what is the structural basis for the different properties between inhibitor and substrate sharing the same binding mode remained open. This issue prompted us to perform a combined biochemical and quantum chemical study. Two key parameters have been considered: on the one hand binding affinity and on the other hand catalytic turnover.
Because not all kcat values were available and some conflicting kinetics results had been reported in the literature (Table I), the catalytic activity of TKHSV1 toward all ligands has been measured under the same experimental conditions. In this way, a homogeneous set of measured kcat values have been obtained and compared with the calculations.
The calculated sugar dipole points to Glu225, and the resulting stabilization energy correlates strikingly with the catalytic activity (Fig. 7). These results are consistent with a previously published study on E225L TKHSV1. Indeed this mutant exhibits a loss in binding affinity proportional to the loss of one hydrogen bond and, more interestingly, a dramatic drop in catalytic activity (32).
Glu225 contributes to the binding affinity of the prodrug by H bond interactions with the 3'-OH group (7-11). Our calculations point to its important role in catalysis derived from its interaction with the dipole. In contrast no correlation have been found between the calculated terms and binding affinity expressed as Ki or Km. This type of calculations are very straightforward and can be extended in the future to new compounds.
Because the dominating component of the sugar-like chain-protein interactions and correlations is electrostatic, one might think about using molecular mechanics calculation for performing similar studies (42). We performed such a calculation on some of the complexes, and indeed the results are very promising (data not shown). These calculations, however, required the development of the force field parameters of the prodrugs, which in turn required quantum chemical calculation. Therefore, the calculation effort using force field methodologies is not reduced compared with the quantum chemical calculation. On the other hand, the latter, being a parameter-free approach, allows an automatic procedure studying prodrug-enzyme interactions.
Our study also provides a rationale for the presence of the polar group in the hydrophobic pocket of the enzyme (Fig. 4). Indeed, replacement of the O-4' with a CH2 group ether provides a dramatic reduction and a change of orientation of the dipole (Fig. 4, b and c), which in turn could decrease the stabilizing dipole-Glu225 interactions.
In conclusion we indicated a substrate-enzyme molecular interaction
that has been shown to be relevant to catalysis. We have shown a nice
correlation between the dipole-charge (substrate-Glu225)
interaction and the catalytic activity, which suggests this electrostatic contribution as a stabilizing mechanism for catalysis. Up
to now all prodrugs have been the results of nucleobase or sugar moiety
substitution with the only rational guide of mimicking the sugar moiety
assuming the importance of elements such as the O-4' but not knowing
the exact role. With our work we suggest a guide element in the
rational design of novel nucleosides analogs having an increased
phosphorylating activity.
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ACKNOWLEDGEMENTS |
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We are thankful to Drs. E. De Clercq, B. Johannsen, G. E. Wright, and V. Marquez for providing several of the studied compounds. Calculations were carried out with CPMD version 3.0h (39) on cray t3e in Bologna. The C4 Project is also acknowledged.
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FOOTNOTES |
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* This work was supported in part by an Instituto Nazionale di Fisica della Materia-Consorzio Interuniversitario Nord Est Calcolo Avanzato grant ("Substrate Diversity in Herpes Simplex Type 1 Thymidine Kinase: an Ab-initio Approach") and by progetto "Giovani Ricercatori" by Repione Friuli Venezia Giulia.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.
Supported by the Stipendienfonds der Basler Chemischen Industrie.
To whom correspondence should be addressed: SISSA,
International School for Advanced Studies, via Beirut 2-4, 34013 Trieste, Italy. Tel.: 39-040-3787-407; Fax: 39-040-3787-528;
E-mail: carloni@sissa.it.
§§ To whom correspondence should be addressed: Inst. of Pharmaceutical Sciences, Swiss Federal Institute of Technology (ETH), Winterthurerstr. 190, CH-8057 Zurich, Switzerland. Tel.: 41-1-635-6036; Fax: 41-1-635-6884; E-mail: scapozza@pharma.anbi.ethz.ch.
Published, JBC Papers in Press, March 21, 2001, DOI 10.1074/jbc.M010223200
2 The contribution of the nucleobase of the nucleotides to the dipole interaction was additive, even if different for thymine and guanine. Because the chemical bond between the nucleobase and the sugar moiety is nonpolar, we have verified the additivity of the nucleobase and sugar moiety dipoles.
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ABBREVIATIONS |
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The abbreviations used are: TKHSV1, thymidine kinase from herpes simplex virus type 1; n-MCT, 2'-exo-methanocarba-thymidine, (North)-methanocarba-thymidine; dT, (2'-deoxy)thymidine; ACV, aciclovir; PCV, penciclovir; GCV, ganciclovir; AHIU, 5-iodouracil anhydrohexitoluridine; AHTMU, 5-Trifluoromethyl anhydrohexitoluridine; AZT, 3'Azido-thymidine; HBPG, 9-(4-hydroxybutyl)-N2-phenylguanine; WFC, Wannier functions centers.
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