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* Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520-8066; USA; and
Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine/VAMC, Decatur, Georgia 30033, USA
1Correspondence: Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520-8066, USA. E-mail:karen.anderson{at}yale.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: transient kinetics • rapid chemical quench • pre-steady-state analysis • RNA-dependent DNA polymerization • incorporation efficiency
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The FDA-approved nucleoside analog, 3TC
[(-)-2',3'-deoxy-3'-thiacytidine, lamivudine], falls into this
category (2
3
4)
(Fig. 1
). Further structural modifications to 3TC have been made to optimize
the therapeutic index (5)
. Earlier studies examining
structure–activity relationships with pyrimidine nucleosides suggested
that toxicity may be substantially reduced by modifying the 5-position
of the cytosine ring with a small group, such as a halogen
(6)
. Based on this strategy, the 5-fluorinated derivative
of 3TC,
(-)-ß-L-2',3'-dideoxy-5-fluoro-3'-thiacytidine3
[(-)-FTC], and its (+)-ß-D-enantiomer (+)-FTC were synthesized and
examined for antiretroviral activity (Fig. 1)
. While cell culture
studies showed that both FTC isomers have antiretroviral activity, the
(-) isomer of FTC appeared to have higher potency against HIV-1 as well
as HBV, in accord with earlier studies of the isomers of 3TC (3
, 5)
. Thus, it is the unnatural ß-L-enantiomer (-)-FTC that has
been developed for clinical use. Like other nucleoside analogs, the
mechanism of inhibition of HIV-1 RT by the 3TC and FTC isomers involves
in vivo phosphorylation to the triphosphate form and
subsequent incorporation into the DNA primer strand, resulting in chain
termination.
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The mechanistic basis for the stereochemical selectivity and
differential toxicity of the isomeric 3TC and FTC compounds is not
completely understood, although a number of factors may clearly come
into play. The differences in potency and toxicity may be a combination
of factors including uptake (7
, 8)
, transport
(9)
, metabolic activation (7
, 8
, 10
, 11)
,
incorporation, and degradation (7
, 8
, 10
, 12)
. In
addition, toxicity to the host may also be related to inhibition of
human mitochondrial DNA polymerase 
(9
, 10
, 13)
.
To reverse transcribe the RNA genome into a double-stranded DNA copy that can be integrated into the host cell genome, HIV-1 RT possesses several unique catalytic activities. These enzymatic activities include DNA-dependent DNA polymerization, RNA-dependent DNA polymerization, RNase H cleavage, and the ability to initiate DNA synthesis using a primer-template substrate consisting of the human tRNALys annealed to the primer binding site of the viral RNA genome. Mechanistic studies of these catalytic processes can aid in a fundamental understanding of the molecular mechanism of catalysis for HIV-1 RT as well as mechanisms of inhibition and drug resistance.
In general, most studies have used a steady-state kinetic analysis to
examine catalysis by HIV-1 RT as well as to examine inhibition by
various nucleoside and non-nucleoside analogs and thereby extract
mechanistic information. These studies are limited to the measurement
of the steady-state kinetic parameters Km and
kcat. Although these parameters represent a
useful beginning, they are insufficient to establish detailed kinetic
and mechanistic information. On the other hand, a rapid transient
kinetic approach toward extracting mechanistic information offers the
important advantage of the ability to observe directly
events occurring at the enzyme active site. These include binding
events, protein conformational changes, and chemical catalysis. The
rate constants of individual steps can be measured and any enzyme
intermediate can be identified, including conformational species, which
may be important in governing catalysis (14
, 15)
.
The kinetic reaction pathway for HIV-1 RT has been established by using
a transient kinetic approach (16
17
18
19
20
21)
. This approach has
also been used to examine the molecular mechanism for AZT drug
resistance (22
23
24)
. As
illustrated in Scheme I ,
the HIV-1 RT kinetic reaction pathway for DNA polymerization involves an
ordered mechanism in which the DNA substrate
(DNAn) binds to the enzyme first to form a tight
E.DNAn complex with a
Kd value in the nanomolar range. This is followed by
binding of the dNTP to form an initial collision complex,
E.DNAn.dNTP,
with a Kd value in the micromolar range. At this
point, the selectivity involved in incorporating the correct nucleotide
is controlled by the base-pairing free energy upon recognition of the
correct dNTP over the incorrect dNTP. Binding of the correct dNTP
induces a rate-limiting change in protein conformation to form a very
tight ternary complex designated by
E*.DNAn.dNTP.
Further evidence for the tight ternary complex observed by kinetic
studies is provided by the recently solved 3-dimensional structure of
HIV-1 RT ternary complex poised for catalysis (25)
. The
formation of this ternary complex allows the critical transition state
to be reached, leading to very rapid chemistry to produce the elongated
DNAn+1. After nucleotide incorporation, the
enzyme releases the pyrophosphate (PPi) and the
elongated DNA (DNAn+1) sequentially. This final
product release of the elongated DNA is the slowest step in the overall
reaction pathway and the step that is examined by using steady-state
analysis (14
, 15)
. The crucial information regarding
events occurring at the enzyme active site, including chemical
catalysis and conformational changes, lies in the boxed area of
Scheme I
, and is that which is discerned using a transient kinetic approach.
Thus, key kinetic parameters required to define the dNTP interaction
with HIV-1 RT are the binding affinity, Kd; the
maximum rate of polymerization, kpol; and the
efficiency of incorporation,
kpol/Kd.
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Very recently, preliminary results of clinical trials suggest that
(-)-FTC is 6- to 10-fold more potent than
3TC,4however, the molecular mechanism for the enhanced potency of (-)-FTC to
inhibit viral replication is not understood (26)
. A
detailed side-by-side comparison of pharmacokinetic parameters for 3TC
and (-)-FTC has not been conducted. Nevertheless, initial
pharmacokinetic studies indicate that (-)-FTC is phosphorylated to its
active form, (-)-FTC-TP, at a rate similar to that for 3TC-TP, and both
serve as very poor substrates for degrading enzymes such as cytidine
deaminase, which use the corresponding (+) isomers more efficiently
(11
, 27
, 28)
. These results suggest that the (-) isomers
of 3TC and FTC may be similar with respect to their uptake, activation,
and metabolism. Therefore, the differences in potency may lie in part
in the efficiency with which the triphosphate form of the analogs is
incorporated into DNA by HIV-1 RT. The steady-state kinetic analysis of
incorporation of 3TC-TP, (-)-FTC-TP, and their (+) isomers by HIV-1 RT
has been reported (8
, 10
, 29)
, and a pre-steady-state
kinetic analysis has examined the incorporation of the (-) isomer
3TC-TP into DNA by wild-type and mutant HIV-1 RT (23)
.
We recently used a transient kinetic analysis to examine the
incorporation of both the (-) and (+) isomers of 3TC-TP into DNA by
HIV-1 RT (30)
. Our studies have shown that HIV-1 RT
incorporates both isomers with a much higher efficiency during
RNA-dependent DNA synthesis, and therefore this may be the most
biologically relevant step in terms of inhibition. In view of these
findings, the present studies were designed to extend the transient
kinetic analysis to examine the efficiency of incorporation of the (-)
and (+) isomers of FTC-TP by HIV-1 RT during RNA-dependent DNA
synthesis. In addition, we sought to understand the effect of
structural and stereochemical modifications on the kinetic and
thermodynamic properties for these analogs at the RT enzyme active
site.
The mechanistic studies described in this report provide a direct comparison of the incorporation of 3TC-TP and (-)-FTC-TP and the corresponding (+) isomers into DNA during RNA-dependent DNA synthesis by HIV-1 RT. More important, our results illuminate key mechanistic features that may play a role in the differential potency observed in the clinical trials comparing 3TC and (-)-FTC.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 18)
. The
protein concentration of purified RT was measured
spectrophotometrically at 280 nm, using an extinction coefficient
280 = 260,450 M-1
cm-1. The concentration of active RT was
determined as described previously with pre-steady-state burst
experiments (16)
, which gave burst amplitudes of 40%, and
the experiments described here were performed using the corrected
active site concentration. dCTP was purchased from Pharmacia LKB
Biotechnology Inc. (Piscataway, N.J.). The (+) and (-)-FTC-TP isomers
were synthesized in Dr. R. F. Schinazi's laboratory. These
isomers were found to be greater than 98% pure as determined by
high-performance liquid chromatography as well as LC/ESI mass
spectrometry. [-32P] ATP was purchased from
Amersham Pharmacia Biotech (Piscataway, N.J.). The DNA oligonucleotide
(23-mer, Table 1
) was synthesized on an Applied Biosystems 380A DNA synthesizer (DNA
synthesis facility, Yale University) and purified using denaturing
polyacrylamide gel electrophoresis (16% acrylamide, 8 M urea). The RNA
oligonucleotide (45-mer, Table 1
was synthesized and gel purified by
New England Biolabs (Beverly, Mass.). Concentrations of the
oligonucleotides were determined spectrophotometrically at 260 nm using
the following calculated extinction coefficients: DNA 23-mer, =
226,750 M-1 cm-1; RNA
45-mer, = 507,960 M-1
cm-1. All experiments using HIV-1 RT were
carried out in 50 mM Tris-Cl, 50 mM NaCl buffer at pH 7.8, and a
temperature of 37°C.
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5'-32P-labeling and annealing of the DNA/RNA
23/45-mers
Before annealing both primer and template strands of the DNA/RNA
23/45-mer, each strand was 5'-radiolabeled with T4 polynucleotide
kinase (New England Biolabs) according to previously described
procedures (16)
. The heteroduplex 23/45-mer
primer-template of DNA/RNA were formed by annealing ~1: 1.3 molar
ratio of pure 23- and 45-mer at 80°C for 4 min and 50°C for 30 min.
The heteroduplex mixtures were analyzed by nondenaturing polyacrylamide
gel electrophoresis (15%) to ensure that proper annealing had taken
place.
Rapid chemical quench experiments
Transient kinetic experiments using rapid chemical quench
methodology were performed as described previously using a KinTek
Instruments Model RQF-3 rapid-quench-flow apparatus (16
, 18)
. Unless noted otherwise, all concentrations refer to the
final concentrations after mixing.
Pre-steady-state kinetic analysis for incorporation of next
correct nucleotide to DNA/RNA heteroduplex at 37°C
The pre-steady-state analysis was conducted under conditions in
which the duplex concentration was in threefold excess relative to the
enzyme concentration. The reaction was carried out by mixing a solution
containing the preincubated complex of 100 nM HIV-1 RT and 5'-labeled
300 nM DNA/RNA heteroduplex with a solution of 10 mM
Mg2+ and varying concentrations of dNTP (in the
range of 0.5 to 500 µM). Polymerization reactions were quenched with
0.3 M EDTA at time intervals ranging from 3 ms to 3 min. DNA
polymerization products and RNA cleavage products were quantified by
sequencing gel analysis. The product formation occurred in a fast
exponential phase, followed by a slower linear phase (Fig. 2
). The data were fitted to a burst equation (see Data analysis below).
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Product analysis
The products were analyzed by sequencing gel electrophoresis
(20% acrylamide, 8 M urea, 1 x TBE running buffer) and the
products were quantified using a Bio-Rad GS525 Molecular Imager
(Bio-Rad Laboratories, Inc., Hercules, Calif.).
Data analysis
Data were fitted by nonlinear regression using the
program KaleidaGraph version 3.09 (Synergy Software, Reading, Pa.).
Data from burst experiments were fitted to a burst equation:
[product] = A[1-exp(-kobsdt) +
ksst], where A represents the
amplitude of the burst that correlates with the concentration of enzyme
in active form, kobsd is the observed first-order
rate constant for dNTP incorporation, and kss the
observed steady-state rate constant. The dissociation constant,
Kd, for dNTP binding to the complex of RT and 23/45
heteroduplex is calculated by fitting the data into the following
hyperbolic equation: kobsd =
(kpol x [dNTP])/(Kd +
[dNTP]), where kpol is the maximum rate of dNTP
incorporation, [dNTP] is the corresponding concentration of dNTP, and
Kd is the equilibrium dissociation constant for the
interaction of dNTP with the E.DNA complex.
Standard errors are reported in Table 2
and figure legends (32)
.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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.
Kinetics of incorporation into 23/45-mer DNA/RNA
primer-template by HIV-RT
Pre-steady-state burst experiments were performed by mixing a
preincubated solution of RT (100 nM) and
5'-[32P]-labeled DNA/RNA 23/45-mer (300 nM)
with Mg2+ (10 mM) and various concentrations of
dNTP (dCTP, 3TC-TP, and FTC-TP) under rapid quench conditions. Table 1
shows the template (RNA 45-mer) and primer (DNA 23-mer) used in these
experiments. Representative pre-steady-state burst experiments using a
20 µM solution of (+) and (-)-FTC-TP are shown in Fig. 2A
and B, respectively. The time courses illustrated in Fig. 2A
, 2B
show a faster exponential phase
corresponding to an initial burst of elongated DNA primer (D 24-mer)
product (kburst), followed by a slower linear
phase of product formation (kss).
Determination of kinetic parameters, kpol,
Kd, and
kpol/Kd
The equilibrium dissociation constant, Kd, for
the interaction of the natural substrate dCTP, the (-) and (+) isomers
of the analogs 3TC-TP and FTC-TP with the E.DNA
complex, and the maximum rate of incorporation
(kpol) were determined by examining a series of
reaction time courses at varying concentrations of dNTP.
Kd and kpol were obtained by
examining the dNTP concentration dependence on the observed burst rate
of polymerization for incorporation into a DNA/RNA 23/45-mer
primer-template using HIV-1 RT. Representative Kd
curves for the (-) and (+)-FTC-TP isomers are shown in Fig. 3
A and B, respectively. A complete summary of the
kinetic data comparing the natural nucleotide substrate dCTP and the
(+) and (-) isomers of FTC-TP and 3TC-TP is provided in Table 2
. Shown
are the kinetic parameters Kd,
kpol, and the efficiency of incorporation
expressed as kpol/Kd. The
steady-state rate constant (kss) for each dNTP
was obtained from the rate constant of the linear phase of the burst
equation at saturating dNTP and is included in Table 2
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. This catalytic activity of HIV-1 RT may be
the most biologically relevant one to examine for understanding
inhibition by thiacytidine analogs at a basic and clinical level. This
study focused on examining the kinetics of incorporation of the (+) and
(-) isomers of 3TC-MP and FTC-MP into a DNA/RNA primer-template by
HIV-1 RT. As shown by a comparison of the kpol
values in Table 2
, all of the synthetic nucleotides are incorporated
much slower than that of the natural substrate dCTP. For both the
fluorinated and nonfluorinated thiacytidine analogs, the (+)-isomer is
consistently incorporated faster than the (-)-isomer. Although the
(+)-isomers of FTC-TP and 3TC-TP share similar
kpol values, the effect of a fluorine at the
5-position of cytosine ring is shown primarily through the (-)-isomers,
in which (-)-FTC-TP is incorporated twice as fast as that of
(-)-3TC-TP. The dissociation constants of dNTP with the preformed RT-DNA/RNA primer–template complex (Kd) reveal that the 3TC-TP and FTC-TP analogs all bind very tightly to the enzyme. The binding affinity for these analogs is ~6 to 30-fold higher than that of the natural substrate dCTP. Of all of the analogs, (-)-FTC-TP has the highest affinity, which is three- to fivefold higher than the others. The two isomers of 3TC-TP and the (+)-FTC-TP have a similar affinity for HIV-1 RT.
We found modest differences between the (+)- and (-)-isomers of FTC-TP
in single nucleotide incorporation by HIV-1 RT. These results are
similar to those previously observed for the 3TC-TP enantiomers in both
pre-steady-state studies (30)
and steady-state studies
(8)
. Pharmacokinetic studies indicate that the
differential potency between the two isomers observed in
vivo may be due to a combination of factors including uptake,
biological activation, and degradation (11
, 27
, 31)
.
Compared to the (+)-isomer, (-)-FTC is more efficiently taken up into
the cell and phosphorylated to its active triphosphate form. Also, by
serving as a much poorer substrate of cytidine deaminase than the
(+)-isomer, (-)-FTC is not degraded to an inactive species.
The incorporation efficiency of (+)-3TC-TP is comparable to that of the
corresponding fluorinated analog (+)-FTC-TP (Table 2)
. The important
question, from a clinical perspective, in understanding the enhanced
potency of (-)-FTC over 3TC is whether there are differences in the
incorporation efficiency for the 3TC-TP as compared with the
(-)-FTC-TP. Here we found that the differences between the two
(-)-isomers are quite striking (Table 2
, shown in boldface). The
(-)-FTC-TP is incorporated almost an order of magnitude more
efficiently than 3TC-TP during RNA-dependent DNA synthesis. This
higher incorporation efficiency results from a combination of a higher
rate of incorporation, kpol, and a tighter
dissociation constant, Kd. The effect of the
fluorine atom at the 5-position of the cytosine ring would be expected
to increase the lipophilicity and, by its electron-withdrawing nature,
to enhance the hydrogen bonding potential in Watson-Crick base pairing.
These effects may be responsible for the higher maximum rate of
polymerization and tighter affinity.
In conclusion, our mechanistic studies suggest that (-)-FTC-TP may be
an even better anti-HIV drug than 3TC-TP. The molecular mechanism for
the enhanced potency of (-)-FTC-TP appears to be a function of the
higher efficiency with which it is incorporated into DNA during
RNA-dependent DNA synthesis by HIV-1 RT. Our results agree with an
ongoing clinical trial that compares 3TC and (-)-FTC showing that
(-)-FTC is a very potent and selective anti-HIV agent, with an activity
against HIV-1 ~6- to 10-fold greater than that observed for 3TC
(26)
. This study illustrates that a transient kinetic
approach can provide a detailed mechanistic understanding at a
molecular level as well as provide insight and rationale for
interpreting clinical results.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Although we have used the common
nomenclature in the literature, the most appropriate chemical
nomenclature is
(-)-cis-5-fluoro-1-[2-hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine.
4 3TC generally refers to the unnatural (-)-ß-L
isomer of 2',3'-deoxy-3'-thiacytidine in current use
clinically.
Received for publication January 25, 1999. Accepted for publication without revision May 15, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Biochem. Pharmacol. 50,1043-1051[Medline]
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