From the Department of Biological Chemistry and the
¶ Department of Medicinal Chemistry, Merck Research Laboratories,
West Point, Pennsylvania 19486-0004
Received for publication, November 6, 2002, and in revised form, December 10, 2002
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ABSTRACT |
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Human immunodeficiency virus-type 1 (HIV-1) reverse transcriptase (RT) coordinates DNA polymerization and
ribonuclease H (RNase H) activities using two discrete active sites
embedded within a single heterodimeric polyprotein. We have identified
a novel thiophene diketo acid,
4-[5-(benzoylamino)thien-2-yl]-2,4-dioxobutanoic acid, that
selectively inhibits polymerase-independent RNase H cleavage
(IC50 = 3.2 µM) but has no effect on
DNA polymerization (IC50 > 50 µM). The
activity profile of the diketo acid is shown to be distinct from
previously described compounds, including the polymerase inhibitor
foscarnet and the putative RNase H inhibitor 4-chlorophenylhydrazone.
Both foscarnet and the hydrazone inhibit RNase H cleavage and DNA
polymerization activities of RT, yet neither inhibits the RNase H
activity of RT containing a mutation in the polymerase active site
(D185N) or an isolated HIV-1 RNase H domain chimera containing the
HIV-1 reverse transcriptase
(RT)1 catalyzes the
conversion of single-stranded RNA into double-stranded viral DNA. This
heterodimeric protein is composed of two polypeptide subunits: p66 and
p51. The two catalytic activities of reverse transcriptase are carried out within discrete active sites located in the p66 subunit. The N
terminus of p66 catalyzes RNA- and DNA-dependent DNA
polymerase activity, while the p15 domain at the C terminus of p66
catalyzes ribonuclease H (RNase H) activity. RNase H is required to
cleave the RNA strand of the RNA:DNA heteroduplex intermediates in
reverse transcription. This activity is essential for HIV-1 replication (1, 2, 3), thus RNase H is an attractive target for the development of
new antiretroviral agents.
All RT inhibitors currently licensed for the treatment of HIV-1
infection inhibit the polymerase activity and bind to the polymerase
domain of RT (reviewed in Ref. 4). Although there are several published
examples of compounds that inhibit RNase H activity in vitro
(5-11), it is still unclear whether their affect on RNase H function
is mediated by direct binding to the RNase H domain. In some cases,
these inhibitors have been shown to affect both the polymerase and
RNase H activities of RT (8, 9), which superficially suggests that
these compounds may bind at both active sites. However, several studies
have demonstrated that there is significant interdomain communication
between the polymerase and RNase H domains of RT (e.g. Ref.
12), and potential allosteric effects on RNase H function are usually
not ruled out.
In this report we have employed a series of RT polymerase and RNase H
mutants to characterize the effects of several known inhibitors on each
enzymatic function and to explore the behavior of a novel diketo acid
inhibitor of HIV-1 RNase H:
4-[5-(benzoylamino)thien-2-yl]-2,4-dioxobutanoic acid (Compound I,
Fig. 1). We demonstrate here that
inhibitors that bind to the polymerase domain, such as foscarnet, can
inhibit the RNase H activity in the absence of polymerization. The
phenylhydrazone (Compound II) previously reported to be an RNase H
inhibitor (10, 11) is also shown to work by this mechanism. In
contrast, 4-[5-(benzoylamino)thien-2-yl]-2,4-dioxobutanoic acid
selectively binds to and inhibits the RNase H domain of HIV-1 RT. The
metal-dependent behavior of this novel RNase H inhibitor is
analogous to the mechanism of action proposed for HIV-1 integrase inhibitors of the same structural class (13, 14). These studies demonstrate that inhibitors of RNase H function can be either direct or
allosteric modulators of activity and suggest that prototype structures
that have proven successful in developing integrase inhibitors with
potent antiretroviral activity can be exploited to develop selective
inhibitors of the RNase H activity of HIV-1 RT.
Reagents--
All buffer components were obtained from either
Sigma-Aldrich or Ambion, Inc. unless specified.
4-[5-(Benzoylamino)thien-2-yl]-2,4-dioxobutanoic acid (Compound I)
was synthesized according to published methods (15).
4-Chlorophenylhydrazone of mesoxalic acid (Compound II) was synthesized
according to well established methods (16). 3-{[(4,7-Dichloro-1,3-benzoxazol-2-yl)methyl]amino}-5-ethyl-6-methylpyridin-2-(1H)-one (Compound III) was obtained from the Merck sample collection (17). AZT-TP was purchased from Sierra Bioresearch (Tucson, AZ).
[ Protein Purification--
HIV-1 RT mutants D185N (polymerase
domain active site) and D443N (RNase H domain active site) were
generated with the QuikChange site-directed mutagenesis kit
(Stratagene). Full-length wild type and mutant RT proteins were
expressed in E. coli BL21(DE3) cells and purified as
described previously (19) with two variations: a 20-ml Hi-Prep 16/10
Heparin FF column (Amersham Biosciences, Inc.) replaced the
phosphocellulose P-11 resin, and as a final step, an 8 ml
poly(U)-Sepharose 4B column (Amersham Biosciences, Inc.) was added to
eliminate residual E. coli RNase HI. The chimeric isolated
HIV-1 RNase H domain protein containing an RNase H Polymerase-independent Cleavage Assays--
RNase H
activity was measured in 50 mM Tris-HCl, pH 7.8, 1 mM dithiothreitol, 6 mM MgCl2, 80 mM KCl, 0.2% polyethylene glycol 8000, 0.1 mM
EGTA, and 5% dimethyl sulfoxide (Me2SO, Pierce Endogen, Inc.). Compounds were preincubated with 5 nM HIV-1 RT for
10 min at 37 °C, and 40-µl reactions were subsequently initiated
with 50 nM nucleic acid substrate composed of the 32-mer
5'-[32P]tC5U RNA template annealed to the complementary
12-mer DNA oligomer. Reactions proceeded for 20 min and were quenched
with an equal volume of formamide gel-loading buffer. Substrate and
product (25-mer) oligomers were resolved on a 20% denaturing
polyacrylamide sequencing gel containing 8 M urea in Tris
borate-EDTA buffer (Fig. 2A), and results were quantified by
PhosphorImager analysis (Amersham Biosciences, Inc.). Cleavage
reactions with p15-EC were conducted in the same buffer with magnesium
replaced by 2 mM MnCl2. After the 10-min
preincubation of 1 nM p15-EC with each compound of
interest, reactions were initiated with 200 nM substrate
and quenched after 10 min. The two product bands (25-mer and 28-mer) observed with p15-EC reflect the ability of this truncated protein to
carry out multiple independent endonuclease cleavages (Fig. 2B); both bands were summed for quantification. To determine
inhibitory potency (IC50), compounds were titrated with
2-fold dilutions (Fig. 2C).
RNA-dependent DNA Polymerase Assays--
The
heterodimeric nucleic acid substrate used in the HIV-1 RT polymerase
reactions was generated by annealing pD500, the DNA primer, to t500,
the 500 nucleotide RNA template created by in vitro
transcription (22). This primer-template substrate (100 nM
final concentration) was combined with HIV-1 RT enzyme (5 nM) in assay buffer (50 mM Tris-HCl, pH 7.8, 1 mM dithiothreitol, 6 mM MgCl2, 80 mM KCl, 0.2% polyethylene glycol 8000) and immediately mixed with inhibitor or Me2SO (5%) prior to addition of
dNTPs (5 µM dNTPs, 5 µM
[ Isothermal Titration Calorimetry--
Isothermal titration
calorimetry experiments were conducted in a MicroCal VP-ITC
calorimeter. Protein samples were dialyzed in Buffer A (50 mM Tris-HCl, pH 7.0, 5% glycerol). The p15-EC protein was
suspended to 18.6 µM in Buffer A, and 200 µM MnCl2 was added when experiments were
conducted with divalent cation. Ligand solutions were made in the same
buffer as the protein. After p15-EC was added to the 1.4 ml of
calorimeter sample cell and equilibrated at 30 °C with a stirring
speed of 490 rpm, a total of 25 aliquots (5 µl) were injected into
the cell. The heat released with each injection was measured, and heats
of reaction were determined by integration of peak areas. Base-line
correction, peak integration, and binding parameters (stoichiometry,
Kd and Inhibition of Polymerase-independent RNase H Cleavage--
There
are striking structural and functional homologies between HIV-1
integrase and the RNase H domain of HIV-1 RT. Potent and specific
compounds containing a diketo acid motif have been shown to inhibit
HIV-1 integrase in vitro and in cell culture (13). As the
purported mechanism of these inhibitors invokes sequestration of the
active site divalent metals in integrase, it has been hypothesized that
a similar mechanism could be exploited for other phosphotransferase
enzymes. In screening a library of diketo acids we identified
4-[5-(benzoylamino)thien-2-yl]-2,4-dioxobutanoic acid (Compound I) as
a novel inhibitor of the RNase H activity of HIV-1 RT.
In RNase H cleavage assays, the activity of Compound I against
full-length wild type HIV-1 RT (IC50 = 3.2 µM, Table I) was as potent
as the most active RNase H inhibitors reported previously (8, 23).
Inhibition of RNase H by Compound I was independent of the order of
addition to the assay; reactions initiated by either enzyme or
substrate behaved identically. To further characterize this activity,
Compound I was also evaluated in RNase H cleavage assays using an RT
polymerase active site mutant (D185N) and a chimeric isolated HIV-1
RNase H domain, p15-EC. As expected for a bona fide RNase H
inhibitor, the activity of the diketo acid against p15-EC
(IC50 = 4.7 µM) was comparable with wild type
HIV-1 RT (Table I, Fig. 2) and was only
slightly weaker when assayed against the D185N mutant (IC50 = 8.8 µM). Compound I did not inhibit E. coli
RNase HI activity under similar conditions when tested at
concentrations up to 50 µM. Neither the nucleoside RT
inhibitor (NRTI) AZT-TP nor the non-nucleoside RT inhibitor (NNRTI;
Compound III) inhibited the RNase H activity of the wild type, D185N,
or p15-EC enzymes at concentrations up to 100 µM (Table
I).
The HIV-1 RT inhibitor foscarnet and a previously described RNase H
inhibitor, 4-chlorophenylhydrazone (Compound II), were also effective
inhibitors of the RNase H activity of HIV-1 RT. However, in contrast to
the diketo acid, both foscarnet and the phenylhydrazone inhibited RNase
H cleavage only when the assays were conducted with full-length wild
type RT (IC50 = 1.8 and 6.1 µM, respectively)
and not with either the D185N mutant or p15-EC (IC50 > 100 µM). The lack of activity observed for foscarnet and Compound II with p15-EC cannot be attributed to the unique manganese requirement for this enzyme. Foscarnet, Compound I, and Compound II
were comparably active in RNase H assays using full-length RT when
assayed in either 6 mM MgCl2 (IC50 = 2-6 µM) or in 2 mM MnCl2
(IC50 = 50-100 nM). Although the effect of
foscarnet on RNase H function was initially surprising because this
compound is well known as a pyrophosphate mimetic inhibitor of RT
polymerization, the lack of activity observed with the D185N mutant is
consistent with binding at the polymerase active site.
These results strongly suggest that foscarnet and Compound II are
polymerase inhibitors which indirectly affect RNase H function. Their
affect on RNase H activity may be due to an indirect change in either
RT conformation or nucleic acid positioning. An allosteric effect on
the RNase H function is not unprecedented; several NNRTIs have been
shown to enhance RNase H activity. In particular, Compound III (data
not shown) and nevirapine (12) increase RNase H cleavage rates and
alter the position and number of cleavage products observed in vitro.
Characterization of Allosteric Inhibitors with HIV-1 RT
Polymerase--
To substantiate that the allosteric effect of
foscarnet and Compound II on RNase H function is as a result of binding
to the polymerase domain of RT, the compounds were evaluated in HIV-1 RT polymerization assays using wild type HIV-1 RT and an active site
RNase H mutant (D443N). Wild type HIV-1 RT polymerase activity was
inhibited by foscarnet and Compound II as well as AZT-TP and the NNRTI
Compound III, but not by Compound I (Table
II). The potencies listed are consistent
with previously reported values for foscarnet, AZT-TP, and Compound III
(17, 24). In contrast to the effect of D185N mutation on RNase H
inhibition, polymerase inhibition by foscarnet and Compound II was not
affected by the RNase H active site mutation, D443N.
Under these conditions, Compound II was a moderately potent polymerase
inhibitor (IC50 = 2.3 µM). The negligible
change in potency observed with the RNase H active site D443N mutant
(IC50 = 2.1 µM) is consistent with the
observation that both foscarnet and Compound II are affected by the
D185N mutation and likely bind at or near the polymerase active site.
Although in previously published work Compound II did not inhibit DNA
polymerization (10), the reaction conditions were not optimal for
evaluation; DNA synthesis is not rate-limiting when enzyme is
equivalent or in excess of substrate, thus explaining the apparent
discordance from results presented here.
Unlike foscarnet and the hydrazone, the diketo acid did not inhibit
RNA-dependent DNA polymerization of wild type or D443N RT
(IC50 > 50 µM). Although all three compounds
had activity in RNase H cleavage assays using wild type RT, these
results suggest that only Compound I is a selective inhibitor of HIV-1
RNase H catalysis.
Metal-dependent Interactions Observed by Isothermal
Titration Calorimetry--
Isothermal titration calorimetry
experiments were conducted with the isolated RNase H domain hybrid
protein (p15-EC) to characterize the interaction between the diketo
acid (Compound I) and the RNase H domain of HIV-1 RT. Although no
binding was observed either in the absence of metal or in the presence
of Mg2+ (Fig. 3B),
a robust signal corresponding to the heat released was detected in the
presence of MnCl2 (Fig. 3, A and B).
The divalent cation specificity observed for Compound I binding to
p15-EC is consistent with the strict manganese requirement for p15-EC
catalytic activity and supports the hypothesis that Compound I might
interact with metal ions in the RNase H active site. As expected, no
change in the heat of reaction was detected when either foscarnet or Compound II were titrated into buffer containing p15-EC (data not
shown).
For Compound I, the fitted parameters (Fig. 3B,
insert) derived from the ITC studies suggest that there is
one binding site (n = 0.9 ± 0.2) on the RNase H
domain for the diketo acid with an exothermic reaction upon association
as shown by the negative Gibbs free energy and enthalpy changes. In
addition, the dissociation constant calculated from the ITC analysis
(8.9 ± 3.5 µM) is consistent with the
IC50 determined in enzymatic activity assays (3.2 µM, Table I); the slight difference between the values
may be due to a shift in solvent proton concentration (pH 7.0 versus pH 7.8, respectively). As the ITC studies were
performed in the absence of nucleic acid substrate, the observation
that binding is metal-dependent and that the calculated
Kd is comparable with the observed IC50
suggests nucleic acid is not required for either binding of the
inhibitor or the active site metals.
In summary, we have demonstrated that compounds from the diketo acid
structural class can inhibit HIV-1 RNase H catalysis in
vitro. Compound I is distinct from previously described RNase H
inhibitors, and it selectively inhibits RNase H cleavage without affecting RNA-dependent DNA polymerization. Isothermal
titration calorimetry studies confirm a direct interaction with the
isolated RNase H domain and demonstrate a requirement for divalent
cation. The suggestion that the thiophene diketo acid may interact with metals in the RNase H active site is consistent with the metal sequestration mechanism by which similar compounds affect HIV-1 integrase activity. Compound I also inhibits HIV-1 integrase in vitro (1.9 µM)2; however,
the activity relationship for RNase H inhibition is clearly distinct
from integrase, as previously described integrase inhibitors with the
diketo acid pharmacophore have no effect on RNase H activity (Refs. 13
and 14 and data not shown). Although Compound I does not inhibit HIV-1
replication in cell culture, it provides an important proof of concept
for direct inhibitors of the RNase H activity of HIV-1 RT. This work
also outlines methods to distinguish indirect affects on RNase H
function by compounds that bind to the polymerase domain and to
identify selective RNase H inhibitors that may ultimately lead to the
development of novel chemotherapeutics for HIV-1 infection.
-C helix from Escherichia coli RNase HI,
suggesting these compounds affect RNase H indirectly. In contrast, the
diketo acid inhibits the RNase H activity of the isolated RNase H
domain as well as full-length RT, and inhibition is not affected by the
polymerase active site mutation. In isothermal titration calorimetry
studies using the isolated RNase H domain, binding of the diketo acid
is independent of nucleic acid but strictly requires Mn2+
implying a direct interaction between the inhibitor and the RNase H
active site. These studies demonstrate that inhibition of HIV-1 RNase H
may occur by either direct or indirect mechanisms, and they provide a
framework for identifying novel agents such as 4-[5-(benzoylamino)thien- 2-yl]-2,4-dioxobutanoic acid that
specifically targets RNase H.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Chemical structures of the five compounds
used in this study.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-32P]ATP (Amersham Biosciences, Inc.) was used to
5'-end-label RNA substrates in standard T4 polynucleotide kinase
reactions (Roche Molecular Biochemicals). The 32-mer
PAGE-purified RNA oligonucleotide (tC5U:
5'-CCCCCUCUCAAAAACAGGAGCAGAAAGACAAG-3') (18) was purchased from
Dharmacon Research, Inc. The 12-mer DNA oligonucleotide (p12: 5'-GTCTTTCTGCTC-3') was purchased from Integrated DNA
Technologies, Inc.
-C helix substrate-binding loop derived from E. coli RNase HI (20,
21) (p15-EC) was overexpressed from a synthetic codon-optimized gene encoding the HIV-1 NL4-3 sequence, and this protein was purified by
methods described previously (21).
-33P]dATP) to initiate the reactions (50 µl total
volume). After 1-h incubation at 37 °C, reactions were quenched by
EDTA, spotted onto DE81 filter plates (Whatman), and washed (twice with
250 µl of phosphate-buffered saline, once with 250 µl of water, and once with 50 µl of ethanol). After drying for 15 min, 50 µl of scintillation fluid was added prior to quantification via Topcount (Packard).
H°) were performed using the
ORIGIN analysis software. Gibbs free energy (
G° =
RT ln(1/Kd)) and entropy of binding (T
S° =
H°
G°) were calculated from measured experimental values.
Samples were run in triplicate and results were averaged to obtain
binding parameters.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
Inhibition of HIV-1 RNase H cleavage
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Fig. 2.
Titrations of compounds in the RNase H gel
cleavage assay. Reactions were carried out as described under
"Experimental Procedures." A, 50 nM
5'-[32P]tC5U/p12 substrate initiated reactions with 10 nM wild type HIV-1 RT in 6 mM
MgCl2. One cleavage product (25-mer) was resolved.
B, 200 nM 5'-[32P]tC5U/p12
substrate initiated reactions with 1 nM p15-EC in 2 mM MnCl2. Two cleavage products (25-mer and
28-mer) were resolved. Compounds were titrated with 2-fold dilutions
starting at a concentration of 20 µM for Compound I and
100 µM for Compound II. AZT-TP was used as a negative
control with both enzymes, and rates from reactions conducted with no
compound (5% Me2SO alone) were used as the base
line for calculating percent inhibition. C, plot of percent
inhibition relative to Me2SO controls. Compound I
(squares) and Compound II (circles) are titrated
against wild type HIV-1 RT (open) and p15-EC
(closed).
Inhibition of HIV-1 RNA-dependent DNA polymerization
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Fig. 3.
Isothermal titration calorimetry with the
isolated RNase H domain protein, p15-EC. A, raw power
traces of heat released per unit time for titration of 1.54 mM Compound I into buffer (50 mM Tris-HCl, pH
7.0, 200 µM MnCl2, 5% glycerol) containing
18.6 µM p15-EC. B, heats of reaction (kcal/mol
ligand) for Compound I (black squares) with least squares
fit (solid line) based on integrated areas of raw power
traces shown above. Compound II and foscarnet heats of reaction are
below the limit of ITC detection and superimposable over the control
titrations of Compound I into buffer with either no metal (open
triangles) or Mg2+ (black circles).
Thermodynamic parameters for Compound I bound to p15-EC are given in
the tabular inset, and values represent the average of
experiments conducted in triplicate.
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ACKNOWLEDGEMENTS |
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We are grateful to Steve Young for the synthesis of Compound II, Paul Darke for the use of the VP-ITC instrument, and we appreciate the assistance of Susan Barr during preparation of this manuscript.
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FOOTNOTES |
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* 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.
§ To whom correspondence should be addressed: Dept. of Biological Chemistry, Merck and Co., Inc., WP42-300, P. O. Box 4, West Point, PA 19486-0004. E-mail: cathryn_shawreid@merck.com.
Present address: Dept. of Molecular and Cell Biology,
University of Connecticut, Storrs, CT 06269-3125.
Published, JBC Papers in Press, December 11, 2002, DOI 10.1074/jbc.C200621200
2 D. Hazuda, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: RT, reverse transcriptase; HIV-1, human immunodeficiency virus-type 1; RNase H, ribonuclease H; ITC, isothermal titration calorimetry; AZT-TP, 3'-azido-3'-deoxthymidine triphosphate; NRTI, nucleoside reverse transcriptase inhibitor; NNRTI, non-nucleoside reverse transcriptase inhibitor; Me2SO, dimethyl sulfoxide.
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REFERENCES |
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