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MOLECULAR PHARMACOLOGY 51:567-575 (1997).
Probing Interactions between Viral DNA and Human Immunodeficiency
Virus Type 1 Integrase Using Dinucleotides
Abhijit
Mazumder,
Hiroyuki
Uchida,
Nouri
Neamati,
Sanjay
Sunder,
Maria
Jaworska-Maslanka,
Eric
Wickstrom,
Fan
Zeng,
Roger A.
Jones,
Robert F.
Mandes,
H. Keith
Chenault, and
Yves
Pommier
Laboratories of
Molecular Pharmacology, Division of Basic Sciences
(A.M., N.N., S.S., Y.P.), and
Experimental Retrovirology Section
(H.U.), Medicine Branch, National Cancer Institute, National Institutes
of Health, Bethesda, Maryland 20892, Department of Pharmacology, Thomas
Jefferson University, Philadelphia, Pennsylvania 19107-5541 (M.J.-M.,
E.W.),
Department of Chemistry, Rutgers State University of New Jersey,
Piscataway, New Jersey 08855-0939 (F.Z., R.A.J.), and
Department of
Chemistry and Biochemistry, University of Delaware, Newark,
Delaware 19716 (R.F.M., H.K.C.)
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Summary |
Retroviral integrases are essential for viral replication and represent
an attractive chemotherapeutic target. In the current study, we
demonstrated the activity of micromolar concentrations of dinucleotides
against human immunodeficiency virus type 1 (HIV-1), HIV type 2 (HIV-2), simian immunodeficiency virus, and feline immunodeficiency
virus integrases. The structure-activity relationship indicates that
5
-phosphorylation enhances potency and that phosphodiester and sugar
modifications affect the inhibition of HIV-1 integrase. Base sequence
selectivity was observed: pAC, pAT, and pCT were the most potent
inhibitors, whereas pAA, pGA, and pGC showed low activity at 100 µM. The inhibition by pAC is consistent with the interaction of the enzyme with the 5 end of the noncleaved strand (5-AC-3). The linear and cyclic dinucleotides released by the 3-processing reaction did not affect enzymatic activity at
physiological concentrations. An increase in the length to
trinucleotides or tetranucleotides enhanced potency by only 2-3-fold,
suggesting that two neighboring bases may be sufficient for significant
interactions. Inhibition of a truncated (50-212) integrase mutant and
global inhibition of all nucleophiles in the 3-processing reaction
suggest that dinucleotides bind in the catalytic core. All of the
active dinucleotides inhibited enzyme/DNA binding in their respective IC50 range. Although the dinucleotides tested showed no
antiviral activity, these observations demonstrate the usefulness of
dinucleotides in elucidating enzyme mechanisms and as potential ligands
for cocrystallization and as lead structures for development of
antivirals.
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Introduction |
Several key enzymes in the
replication cycle of the HIV can be targeted for chemotherapeutic
intervention, most notably, reverse transcriptase and protease (1).
Research is now in progress to develop clinically active agents against
other proteins in the viral life cycle. Toward this goal, several
laboratories have investigated the pharmacological activity of various
drugs as inhibitors of HIV integrase (2-4).
Retroviruses encode the integrase protein at the 3 end of the
pol gene (5). This enzyme, a proteolytic cleavage product of
a gag-pol fusion protein precursor, is contained in the
virus particle and is required for viral replication. It integrates a
double-stranded DNA copy of the RNA genome, synthesized by reverse transcriptase, into a host chromosome. During viral infection, integrase catalyzes the excision of the last two nucleotides from each
3 end of the linear viral DNA, leaving the terminal dinucleotide CA-3-OH at these recessed 3 ends. This activity is referred to as the
3-processing or dinucleotide cleavage. After transport to the nucleus
within the preintegration complex, integrase catalyzes a DNA strand
transfer reaction involving the nucleophilic attack of these ends on
the host chromosome [for recent reviews, see Katz and Skalka (6) and
Rice et al. (7)].
We previously demonstrated that AZT nucleotides inhibit HIV-1 integrase
with an IC50 value of ~100 µM (8) and
recently showed that the gyrB inhibitor, coumermycin A1, and
several mononucleotide analogs that are currently undergoing clinical
trials as inhibitors of HIV-1 replication are more potent inhibitors of
HIV-1 integrase than are AZT nucleotides (2).
Non-nucleotide inhibitors of HIV-1 integrase have also been described
(9-12) that have the potential, as shown by molecular modeling, to
stack their aromatic rings and potentially mimic a dinucleotide (11,
12). Because of the structural similarities between RNase H (from
either Escherichia coli or HIV-1) and retroviral integrase
(from either HIV-1 or ASV) with respect to both the overall topology of
the active sites and location of conserved acidic amino acids (7) and
because of the recent finding that dinucleotides can inhibit RNase H
(13), we theorized that dinucleotides may inhibit integrase. In the
present study, we investigated the effect of dinucleotides
(including those released physiologically during 3-processing) on
enzyme activity and the inhibition of HIV-1 integrase by a wide variety
of dinucleotides having differing base sequences or modifications to
the backbone or sugar moiety. These data can be used to explore the
enzyme mechanisms, develop strategies for the design of
integrase-inhibitor cocrystals, and develop novel integrase
inhibitors.
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Materials and Methods |
Dinucleotides and analogs.
All dinucleotides and
trinucleotides were purchased from Midland Certified Reagent (Midland,
TX) with the exception of the following. The 2,5-oligoadenylates were
a gift from Dr. Robert J. Suhadolnik (Temple University, Philadelphia,
PA). Nonphosphorylated dinucleotides incorporating modified nucleoside
analogs were obtained from Dr. Jean-Pierre Sommadossi (University of
Alabama-Birmingham, Birmingham, Alabama). The PNA dinucleotide was
supplied by Dr. Peter Nielsen (University of Copenhagen, Copenhagen,
Denmark). The cyclic dinucleotides were from the laboratories of Dr.
Roger Jones and Dr. H. Keith Chenault.
The methylphosphonate dCpdA and phosphorothioates dCpdA and dCpdT were
synthesized by phosphoramidite coupling, separated into their protected
Rp and Sp diastereomers by normal-phase HPLC, chemically
phosphorylated, deprotected, and purified by reverse-phase HPLC to
homogeneous peaks. Each diastereomer displayed the expected mass
spectra and 31P NMR peaks.
Preparation of radiolabeled DNA substrates.
The following
oligodeoxynucleotides were HPLC purified by and purchased from Midland
Certified Reagent: AE117, 5-ACTGCTAGAGATTTTCCACAC-3; AE118,
5-GTGTGGAAAATCTCTAGCAGT-3; AE157, 5-GAAAGCGACCGCGCC-3; AE146,
5-GGACGCCATAGCCCCGGCGCGGTCGCTTTC-3; AE156,
5-GTGTGGAAAATCTCTAGCAGGGGCTATGGCGTCC-3; AE118S,
5-GTGTGGAAAATCTCTAGCA-3; and RM22M,
5-TACTGCTAGAGATTTTCCACAC-3. AE117, AE118, and the first 19 nucleotides of AE156 correspond to the U5 end of the HIV-1 LTR.
To analyze the extents of 3-processing and strand transfer using 5
end-labeled substrates, AE118 was 5 end labeled using T4
polynucleotide kinase (GIBCO BRL, Baltimore, MD) and
[
-32P]ATP (DuPont-New England Nuclear). The kinase
was heat inactivated, and AE117 was added to the same final
concentration. The mixture was heated at 95°, allowed to cool slowly
to room temperature, and run on a G-25 Sephadex quick-spin column
(Boehringer-Mannheim Biochemicals, Indianapolis, IN) to separate
annealed double-stranded oligonucleotide (Fig. 1C) from
unincorporated label.
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Fig. 1.
Inhibition of HIV-1 integrase by CA dinucleotides.
A, Oligonucleotide used in the 3-processing and strand transfer
reactions and corresponding to the last 21 bp of the U5 end of the
HIV-1 LTR. Arrow, dinucleotide cleavage site in the
3-processing reaction. B, The two catalytic activities of HIV-1
integrase: 3-processing (3-P) and strand transfer
(S. T.). C, PhosphorImager picture of a representative
experiment with CA, pCA, and CAr. The DNA substrate and
3-processing product are shown as 21 and 19 mer, respectively.
STP, strand transfer products. Lane 1,
DNA alone. Lanes 2 and 18, DNA plus integrase without
dinucleotide. Lanes 3-7, DNA plus integrase in the
presence of pCA at 7.5, 15, 30, 100, and 132 µM,
respectively. Lanes 8-11, DNA plus integrase in the
presence of CA at 15, 30, 100, and 132 µM, respectively. Lanes 12-17, DNA plus integrase in the presence of
ribo-CA at 7.5, 15, 30, 70, 100, and 132 µM,
respectively. D, Quantification of C.  , CAr;  , pCA;
 , CA.
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To analyze the choice of nucleophile for the 3-processing reaction
(14, 15), AE118 was 3 end-labeled using
[
-32P]cordycepin triphosphate (DuPont-New England
Nuclear) and terminal transferase (Boehringer-Mannheim Biochemicals).
The transferase was heat inactivated, and RM22M was added to the same
final concentration. The mixture was heated at 95°, allowed to cool
slowly to room temperature, and run on a G-25 spin column as before. To
analyze the extent of strand transfer using the "precleaved"
substrate, AE118S was 5 end-labeled, annealed to AE117, and column
purified as described above. To determine the extent of 30-mer target
strand generation during disintegration (the reverse of the strand
transfer reaction) (16), AE157 was 5 end labeled; annealed to AE156, AE146, and AE117; annealed; and column purified as described above.
Integrase proteins.
Purified recombinant wild-type HIV-1
integrase (14) and deletion mutant IN50-212 (17) were
generous gifts of Drs. R. Craigie and A. Engelman (Laboratory of
Molecular Biology, National Institute of Diabetes and Digestive and
Kidney Diseases, National Institutes of Health, Bethesda, MD). Dr.
Craigie also provided the expression system for the wild-type HIV-1
integrase. A plasmid encoding the HIV-2 integrase was generously
provided by Dr. R. H. A. Plasterk (Netherlands Cancer Institute,
Amsterdam, The Netherlands). Purified recombinant wild-type FIV and SIV
integrases were generous gifts of Drs. S. Chow (University of
California, Los Angeles) and R. Craigie (National Institute of Diabetes
and Digestive and Kidney Diseases), respectively.
3-Processing, strand transfer, and disintegration assays.
Integrase was preincubated at a final concentration of 200 nM (for HIV-1 and HIV-2) or 600 nM (for FIV and
SIV) with inhibitor in reaction buffer [50 mM NaCl, 1 mM HEPES, pH 7.5, 50 µM EDTA, 50 µM dithiothreitol, 10% glycerol (w/v), 7.5 mM MnCl2, 0.1 µg/ml bovine serum albumin, 10 mM 2-mercaptoethanol, 10% dimethylsulfoxide, and 25 mM 3-(N-morpholino)propanesulfonic acid, pH
7.2] at 30° for 30 min. Preincubation for 30 min of the enzyme with
inhibitor was performed to optimize the inhibitory activity in the
3-processing reaction (18). Then, 20 nM of the 5 end
32P-labeled linear oligonucleotide substrate was added, and
incubation was continued for an additional hour. The final reaction
volume was 16 µl.
Disintegration reactions (16) were performed as above except that the Y
oligonucleotide (i.e., the branched substrate in which the U5 end was
"integrated" into target DNA) was used.
Electrophoresis and quantification.
Reactions were quenched
by the addition of an equal volume (16 µl) of Maxam-Gilbert loading
dye (98% deionized formamide, 10 mM EDTA, 0.025% xylene
cyanol, 0.025% bromphenol blue). An aliquot (5 µl) was
electrophoresed on a denaturing 20% polyacrylamide gel (0.09 M Tris-borate, pH 8.3, 2 mM EDTA, 20%
acrylamide, 8 M urea). Gels were dried, exposed in a
Molecular Dynamics PhosphorImager cassette (Sunnyvale, CA), and
analyzed using a Molecular Dynamics PhosphorImager. Percent inhibition
was calculated using the equation 100 × [1 
(D C)/(N C)], where C, N, and D are the fractions of 21-mer
substrate converted to 19-mer (3-processing product) or strand
transfer products for DNA alone, DNA plus integrase, and integrase plus
drug, respectively. The IC50 value was determined by
plotting the drug concentration versus percent inhibition and determining the concentration that produced 50% inhibition.
UV cross-linking experiments.
We used the method of
Yoshinaga et al. (19). Briefly, integrase was incubated with
substrate in reaction buffer as described above for 5 min at 30°.
Reactions were then irradiated with an UV transilluminator (254-nm
wavelength) from 3 cm above (2.4 mW/cm2) at room
temperature for 10 min. An equal volume (16 µl) of 2× sodium dodecyl
sulfate-polyacrylamide gel electrophoresis buffer (100 mM
Tris, pH 6.8, 4% 2-mercaptoethanol, 4% sodium dodecyl sulfate, 0.2%
bromphenol blue, 20% glycerol) was added to each reaction, and the
reaction was heated at 95° for 3 min before loading of a 20-µl
aliquot onto a 12% sodium dodecyl sulfate-polyacrylamide gel. The gel
was run at 120 V for 1.5 hr, dried, and exposed in a PhosphorImager
cassette. For inhibition of DNA binding experiments, integrase (200 nM) was preincubated with the dinucleotide (at the
indicated concentration) for 30 min at 30° before the subsequent addition of the radiolabeled viral DNA substrate (20 nM).
HIV cytopathic effect assay.
HIV cytopathic effect assays
were performed essentially as previously described (20). ATH8 cells
were used as the host cell line and were grown in cell culture in the
absence or presence of 300 median tissue culture infective dose of
HIV-1LAI strain and in the absence or presence of
dinucleotides.
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Results |
Inhibition of HIV-1 integrase by dinucleotides and
trinucleotides.
The first dinucleotide tested was 5-CA as an
inhibitor of HIV-1 integrase because this sequence is conserved at the
3 end of all retroviral LTR sequences and immediately 5 from the
dinucleotide cleavage site (Fig. 1A) (5) and is critical for optimum
enzymatic activity (21, 22). To be as concise as possible, from this point on, the nonphosphorylated CA dinucleotide will be represented as
CA and its 5-phosphorylated version will be represented as pCA. Fig.
1A shows the oligonucleotide used in the 3-processing and strand
transfer reactions (Fig. 1B). Fig. 1, C and D, shows that CA was only
minimally active against strand transfer (IC50 ~ 60 µM) and even less active against 3-processing (<50%
inhibition at 132 µM). Similar results were obtained for
a CT dinucleotide (data not shown). Our previous observation that
modification of the deoxyribose of a physiological deoxynucleotide can
confer inhibitory activity against integrase (2) led us to test whether incorporation of modified nucleoside analogs (e.g.,
3-azido-3-deoxythymidine or 2,3-dideoxyinosine) into a dinucleotide
would result in increased potency. No increase in potency was observed
(data not shown).
Our previous observation that 5-phosphorylation of nucleoside analogs
conferred activity against integrase (2, 8) led us to test the
phosphorylated version of CA and other dinucleotides. 5-Phosphorylation of CA (generating pCA) enhanced the inhibitory activity (Fig. 1, C and D). This pCA dinucleotide also inhibited strand
transfer using a precleaved oligonucleotide (which mimics the product
of the 3-processing reaction) consisting of sequences from the U3 end
of the LTR in the same concentration range (data not shown).
Interestingly, when the nonphosphorylated CA dinucleotide in which a
ribose was substituted for the deoxyribose was tested, significant
inhibition of both strand transfer and 3-processing was observed at
100 µM (Fig. 1, C and D). Therefore, substitution of the
deoxyribose by a ribose also enhanced the potency of the nonphosphorylated CA dinucleotide.
We then tested all 16 possible sequence combinations of
5-phosphorylated deoxydinucleotides against HIV-1 integrase to
determine whether base sequence selectivity existed (Table
1). The inhibitory activity observed with pAC, pAT, and
pCT is in contrast with the low level of activity observed with the pTN
family (N = A, C, T, or G).
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TABLE 1
Inhibition of HIV-1 integrase by 5-phosphorylated deoxydinucleotides
For each dinucleotide combination, the first line represents the
IC50 for 3-processing and the lower line the
IC50 for strand transfer (in micromolar).
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The correlation between potency and oligonucleotide length was tested
further using several trinucleotides and tetranucleotides. In addition,
the effect of 5-phosphorylation on trinucleotide potency was examined
(Table 2). The nonphosphorylated trinucleotides CAG and
CTA inhibited 3-processing at ~60 µM (Table 2),
whereas nonphosphorylated dinucleotides were inactive in this range
(Fig. 1). The phosphorylated CTA trinucleotide showed a further
enhancement of potency by ~2-3-fold (Table 2), which is consistent
with results obtained from dinucleotides. Two 5-phosphorylated
tetranucleotides corresponding to the HIV LTR end were also tested
(Table 2), and they were found to not be better inhibitors than the
corresponding trinucleotides or dinucleotides (Tables 1 and 2).
Changes in the DNA backbone.
Substitution of the
phosphodiester backbone of pCA by an anionic phosphorothiodiester did
not abolish potency (Fig. 2). For example, both the Rp
(lanes 8-12) and Sp (lanes 13-17) diastereomers of pCA inhibited strand transfer, although at higher concentrations than did pCA (lanes 3-7).
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Fig. 2.
Effect of phosphodiester substitution on the
inhibition of 3-processing and strand transfer by the
5-phosphorylated deoxyribodinucleotides. Darker (A) and lighter (B)
exposures of the gel are shown for inhibition of the strand transfer
and 3-processing activities, respectively. Dinucleotide concentrations
in micromolar are indicated above each lane.
STP, DNA strand transfer products. 19
(mer), 3-processing product. 21 (mer), DNA substrate.
Lane 1, DNA alone. Lanes 2, 18, and
30, DNA plus integrase (IN) without
dinucleotide. Lanes 3-7, DNA plus integrase in the
presence of pCA, which has a phosphodiester backbone
(PD). Lanes 8-12, DNA plus integrase in
the presence of pCA, which has a phosphorothiodiester backbone (Rp
diastereomer) (PS Rp). Lanes 13-17, DNA
plus integrase in the presence of pCA, which has a phosphorothiodiester
backbone (Sp diastereomer) (PS Sp). Lanes
19-21, DNA plus integrase in the presence of pCA, which
has a methylphosphonodiester backbone (Rp diastereomer) (MP
Rp). Lanes 22-24, DNA plus integrase in the
presence of pCA, which has methylphosphonodiester backbone (Sp
diastereomer) (MP Sp). Lanes 25-29, DNA
plus integrase in the presence of CA, which has a PNA backbone
(PNA). C, Quantification of A and B.
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The importance of the ionic character of the dinucleotide backbone in
conferring potency was examined by its replacement with neutral,
hydrophobic internucleotidic linkages. We examined replacement of the
phosphodiester backbone in the pCA dinucleotide with a methylphosphonodiester linkage to preserve the basic phosphorus-linked backbone. Both the Rp and Sp diastereomers of the
methylphosphonodiester pCA were inactive for both 3-processing and
strand transfer at 500 µM (Fig. 2, lanes
19-24), suggesting that the ionic character of the backbone is
important. More radical alterations were tested by using a CA
dinucleotide analogue with a PNA backbone (23) in the CA dinucleotide.
Like the phosphodiester version, the CA with the PNA backbone exhibited
IC50 values for both 3-processing and strand transfer at
~100 µM (Fig. 2, lanes 25-29). We conclude that the anionic character of the backbone, although important for
activity (Fig. 2, compare lanes 3-7 with lanes
19-24), can be substituted by an amide backbone, which may
exhibit a partial dipole character, simulating the anionic character of
the phosphodiester.
The results of sugar and backbone substitution on the CA dinucleotide
are summarized in Fig. 3. Modification of both the sugar and the internucleotidic linkage had a significant impact on potency. Taken together with the data in Table 1, interactions between HIV-1
integrase and the base, sugar, and phosphodiester linkages all appear
to play critical roles in potency (and, presumably, binding affinity).
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Fig. 3.
Activities and structures of the CA dinucleotides
and PNA analog with the sugar and backbone modifications used in this
study. Micromolar IC50 values for each activity are shown
above each structure.
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Other backbone modifications that retained the anionic character of the
backbone but altered its conformation were also tested. For example,
nine available cyclic dinucleotides were assayed. Such cyclic
dinucleotides can be generated by HIV-1 integrase during the
3-processing reaction in vitro by the attack of the 3-hydroxyl of the viral DNA on the scissile bond (14, 15). Cyclic
dinucleotides also exhibited a sequence-dependent activity, although
they were active at >100 µM (Fig. 4). For
example, the cyclic CC dinucleotide (lanes 25-27) was the
most potent of the nine tested, whereas the cyclic CT dinucleotide was
inactive at 1 mM (lanes 6-8). Interestingly, a
cyclic dinucleotide with abasic sites in place of the bases (DD) also
exhibited inhibitory activity (lanes 13-15). These results
are in contrast to the significant inhibition of both 3-processing and
strand transfer obtained with the linear, 5-phosphorylated
dinucleotides (Table 2). We conclude that the cyclic structure reduces
potency relative to the linear structure. In addition,
2-5-oligoadenylates, recently shown to be active against HIV-1
replication due to inhibition of reverse transcriptase (24), were found
to be inactive at 300 µM (data not shown). These results
are not unexpected given the lack of inhibition of integrase observed
with the pAA (Table 1). Therefore, the 2-5 phosphodiester backbone
does not seem to enhance potency relative to the 3-5 linkage.
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Fig. 4.
Effects of cyclic dinucleotides on HIV-1 integrase
activity. Darker (top) and lighter
(bottom) exposures of the gel are shown for inhibition
of the strand transfer and 3-processing activities, respectively.
STP, DNA strand transfer products. 19
(mer), 3-processing product. 21 (mer), DNA substrate.
Dinucleotide concentrations are indicated above each
lane. Lane 1, DNA alone. Lanes 2 and 12, DNA plus integrase without dinucleotide. Lanes
3-5, DNA plus integrase in the presence of cyclic AC.
Lanes 6-8, DNA plus integrase in the presence of cyclic
CT. Lanes 9-11, DNA plus integrase in the presence of
cyclic GC. Lanes 13-15, DNA plus integrase in the
presence of cyclic DD (where D is a tetrahydrofuran moiety representing
a synthetic version of an abasic site). Lanes 16-18, DNA plus integrase in the presence of cyclic AG. Lanes
19-21, DNA plus integrase in the presence of cyclic TT.
Lanes 22-24, DNA plus integrase in the presence of
cyclic GG. Lanes 25-27, DNA plus integrase in the
presence of cyclic CC. Lanes 28-30, DNA plus integrase
in the presence of cyclic AA. Micromolar IC50 values for
each activity are shown below each dinucleotide.
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Site and mechanism of action.
The binding site of these
deoxyribodinucleotides was first examined by testing the dinucleotide
pCT with an integrase deletion mutant containing only amino acids
50-212 (catalytic core of integrase). Because deletion mutants of
integrase are inactive in both 3-processing and strand transfer (17)
but can catalyze the disintegration reaction (16), the branched Y
oligonucleotide was used as the substrate. Our finding that the
integrase deletion mutant missing both the amino-terminal zinc finger
and the carboxyl-terminal DNA-binding domains could be inhibited by pCT
(Fig. 5A) suggests that dinucleotides bind in the
catalytic core.
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Fig. 5.
Analysis of the dinucleotide binding site and
effect on choice of nucleophile in the 3-processing reaction. A,
Inhibition of disintegration catalyzed by the integrase catalytic
domain (IN50-212) in the presence of pCT. Lane
1, DNA alone. Lane 2, DNA plus integrase.
Lanes 3-6, DNA plus integrase in the presence of the indicated concentration of pCA. 15mer, DNA substrate.
30mer, DNA product. B, Inhibition of the 3-processing
reaction using a 3 end-labeled substrate. G, Products
of the glycerolysis reactions. C, Products of the
circular nucleotide formation reactions. L, Products of
the hydrolysis reactions. Lane 1, DNA alone. Lane 2, DNA plus wild-type integrase. Lanes 3-4,
5-6, and 7-8, DNA plus integrase in the
presence of the indicated micromolar concentrations of pCA,
CAr, and pAC, respectively.
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The second question we asked was whether dinucleotides affected the
choice of nucleophile in the 3-processing reaction. An oligonucleotide
substrate labeled at the 3 end was used for these experiments (14,
15). As seen in Fig. 5B, all of the dinucleotides tested inhibited
glycerolysis, hydrolysis, and circular nucleotide formation to the same
extent. Thus, dinucleotides exert a global inhibition by blocking the
use of the three nucleophiles (glycerol, water, or the hydroxyl group
of the viral DNA terminus) in the 3-processing reaction. These results
are consistent with the binding of dinucleotides to the central core
domain of the HIV-1 integrase.
We next wanted to determine whether dinucleotides inhibited binding of
the substrate DNA, which presumably interacts with the catalytic acidic
acid residues Asp64, Asp116, and Glu152 of the core enzyme domain (6,
25). Dinucleotides inhibited binding of the enzyme to its substrate DNA
(Fig. 6). For example, pTA showed strong inhibition of
binding only at 132 µM (lane 16), whereas pAC
(lane 13) and pGT (lane 23) exhibited the same
level of inhibition at 3.2 and 8 µM, respectively. Thus,
IC50 values for DNA binding inhibition correlated with
IC50 values for inhibition of HIV-1 integrase catalytic
activities (Table 1). These results suggest that dinucleotides affect
the binding site of the enzyme for its DNA substrate.
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Fig. 6.
Inhibition of the DNA-binding activities of HIV-1
integrase in the presence of dinucleotides. Inset,
PhosphorImager picture showing UV cross-linking of wild-type integrase
with duplex U5 oligonucleotide substrates in the presence of increasing
concentrations of dinucleotides. Right, migrations of
proteins of known molecular mass. DNA, DNA alone.
IN, DNA plus integrase without dinucleotide. pCA, DNA plus integrase in the presence of 100, 50, 20, and 8 µM pCA, respectively. rCA, DNA plus
integrase in the presence of 132, 60, 30, and 15 µM rCA,
respectively. pAC, DNA plus integrase in the presence of
50, 20, 8, 3.2, and 1.3 µM pAC. pTA , DNA
plus integrase in the presence of 132, 60, 30, 15, and 7.5 µM pTA, respectively. pGT, DNA plus
integrase in the presence of 50, 20, 8, and 3.2 µM pGT,
respectively.
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Inhibition of related lentiviral integrases.
The pCA
dinucleotide was tested for inhibition of the related retroviral
integrases from HIV-2, SIV, and FIV. As seen in Fig. 7,
when HIV-1 DNA was used in all reactions, pCA inhibited both 3-processing and strand transfer catalyzed by HIV-1 integrase in the
expected concentration range. The IC50 values were 88 and 15 µM for 3-processing and strand transfer,
respectively. Similarly, HIV-2 integrase was inhibited with
IC50 values of 125 and 10 µM for
3-processing and strand transfer, respectively. As in the case of
HIV-1 integrase, pCA was ~12-fold more selective in inhibiting the
strand transfer reaction than the 3-processing reaction with HIV-2
integrase. Although strand transfer catalyzed by FIV integrase was
inhibited in the same concentration range (IC50 = 46 µM) as that for the HIV-1 and -2 integrases,
3-processing catalyzed by this integrase was inhibited with an
IC50 value of ~1000 µM. The differential
inhibition of the two activities catalyzed by FIV and SIV integrases
was ~20-40-fold. Therefore, the pCA dinucleotide appeared to inhibit
HIV-1 and -2 integrases more efficiently than the FIV and SIV
integrases.
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Fig. 7.
Inhibition of the related retroviral integrases
from HIV-1, HIV-2, FIV, and SIV by the pCA dinucleotide.
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Discussion |
Sequence selectivity in the inhibition of HIV-1 integrase.
In the present study, several dinucleotides were found to be potent
inhibitors of both 3-processing and strand transfer catalyzed by HIV-1
integrase. Some sequence selectivity was evident, with pAC, pAT, and
pCT being the most potent and pAA, pGA, and pGC showing weak activity
at 100 µM. The lack of a more unambiguous sequence
selectivity was consistent with the nonspecific DNA binding capability
of integrase (26) and with interactions with other parts of the
dinucleotide (e.g, the phosphodiester backbone or the sugar moiety)
being critical (Figs. 3 and 4).
The sequence selectivity observed in the inhibition by dinucleotides
is, however, consistent with the catalytic mechanism for 3-processing.
For example, before the reaction, integrase presumably binds near the
scissile bond (i.e., the bond between the A and G nucleotides on the
plus strand) (Fig. 1A). The dinucleotides immediately upstream of the
scissile bond are a 5-CA-3 and 5-TG-3 on the plus and minus strand,
respectively (Fig. 1A). Binding of integrase to these dinucleotides in
the recognition sequence should therefore not inhibit the subsequent
endonuclease reaction or catalysis would not occur. Accordingly,
neither of these dinucleotides exhibited potency against 3-processing
at <100 µM (Table 1). These dinucleotides could
therefore provide a binding site on the viral DNA substrate, which
would allow the 3-processing reaction to proceed unhindered. These
results are consistent with earlier reports that mutation of the
5-GCA-3 trinucleotide at the-processing site on the scissile strand
resulted in inhibition of the 3-processing reaction (21, 27, 28),
further supporting the contention that the residues immediately
upstream of the dinucleotide cleavage site in the 3-processing
reaction may provide a critical recognition/binding site for the
integrase. The relative lack of potency observed with the 5-GT-3
dinucleotide (Table 1) suggests that this reaction product (Fig. 1C),
which results from hydrolysis of the scissile phosphodiester bond in
the 3-processing reaction, may not serve to regulate this reaction.
The most potent dinucleotide that exhibited IC50 values of
5 µM was pAC. This finding is consistent with the
observation that interactions between integrase and the ultimate and
penultimate bases at the 5 end of the noncleaved strand (5-AC-3)
(Fig. 1A) could be the basis for stable complex formation before the strand transfer reaction (29). Therefore, the potent inhibitory activity of pAC is presumably a manifestation of the binding affinity that integrase may have for this dinucleotide sequence.
Effects of 5-phosphorylation and trinucleotides.
5-Phosphorylation of both dinucleotides and trinucleotides enhanced
potency compared with the corresponding nonphosphorylated form, which
is consistent with the earlier reported effects of 5-phosphorylation
on mononucleosides (8) and with the nonspecific binding of integrase to
and inhibition by nucleic acids (26) and polyanions (30). Inhibition
with 5-phosphorylated dinucleotides was observed at 10-20-fold lower
concentrations than with mononucleotides such as AZT (8) and D4T
monophosphates (2). However, lengthening of the oligonucleotide further
did not increase potency as significantly. These data suggest that two
neighboring bases may be sufficient to provide the essential
interactions when integrase recognizes its viral DNA substrate.
Interaction of HIV-1 integrase with the phosphodiester
backbone.
Previous reports using duplex oligonucleotide substrates
containing ethylated phosphodiester (31) or methylphosphonodiester (32)
backbones have shown that these substitutions dramatically lower the
extent of both 3-processing and strand transfer reactions. These data
suggested the importance of interactions with the phosphodiester backbone. This hypothesis was probed by investigating the effect of
neutralizing the negative charge in the backbone of various CA
dinucleotides and examining the resulting extent of inhibition. Elimination of the anionic character of the backbone by substitution with a methylphosphonodiester backbone was sufficient to abolish potency. Therefore, the negative charge in these dinucleotides may
serve to position the inhibitor for critical interactions with the
divalent metal ion (29) or basic amino acids in the active site (33) so
that high affinity binding can occur.
Two predictions would be consistent with this hypothesis. First, an
increase in anionic character could serve to optimize interactions
within the active site. This prediction is supported by the observation
that inhibition with 5-phosphorylated dinucleotides was observed at
10-20-fold lower concentrations than with nonphosphorylated dinucleotides. The same trend was observed with mononucleotides of AZT
(8) and D4T (2). Second, correct positioning of the 5-phosphorylated
dinucleotide may require proximity of the anionic charge from both the
phosphodiester backbone and the 5-phosphate to appropriate counter
ions. This prediction is supported by the observation that the cyclic
structure abolished potency relative to the linear structure. In this
case, either the decrease in anionic charge from three (for the linear,
phosphorylated dinucleotide) to two (for the cyclic dinucleotide) or
the movement of the 5-phosphate might be responsible for the lack of
inhibition. The lack of inhibition by, and presumably low affinity for,
the cyclic dinucleotides may also be a manifestation of the requirement
for product dissociation after the 3-processing reaction (if the viral
DNA hydroxyl has been used as a nucleophile in this endonuclease
reaction to generate a cyclic dinucleotide) and before the strand
transfer reaction. The low affinity for the cyclic dinucleotides also
suggests that these compounds, like their linear counterparts, do not
inhibit the enzymatic activities of integrase through a feedback loop.
Interaction of HIV-1 integrase with the sugar moiety.
Previous
reports with mononucleotides have suggested that critical interactions
between integrase and the sugar moeity may contribute to the binding
affinity of and inhibition by these compounds (2, 33). For example,
deoxyribose modifications such as substitution of a
-L-enantiomer for its -D-counterpart, unsaturation between the 2 and 3 carbons, or substitution by an azido
or a fluoro substituent at the 3 position conferred inhibitory
activity against HIV-1 integrase compared with the unsubstituted
nucleotide (2). Furthermore, oxidation of ATP, generating the
2,3-dialdehyde, resulted in a nucleotide analog that was >30-fold as
potent as an integrase inhibitor than ATP (33).
In this study, the possible effect of sugar pucker in integrase
inhibition was examined by substitution of the deoxyribose (C2-endo) by a ribose (C3-endo) in a CA
dinucleotide. Substitution of the deoxyribose enhanced potency of the
nonphosphorylated CA dinucleotide to approximately the same extent as
did 5-phosphorylation. These data also suggest that interactions with
the sugar may play as critical a role as those with the
5-phosphomonoester.
Dinucleotides as pharmacological agents.
In addition to being
10-100-fold more potent than their corresponding mononucleotides,
5-phosphorylated dinucleotides also exhibit potency in the same range
as other recently described integrase inhibitors (10, 12, 34).
Furthermore, cellular uptake of 5-phosphorylated dinucleotides may not
require specialized delivery vehicles. Other anionic mononucleotides
and oligonucleotides have been found to cross the cell membrane and act
as inhibitors of intracellular targets. For example,
phosphonylmethoxypropyladenine (35) and a guanosine quartet-forming
oligonucleotide (36), both of which are currently under
preclinical investigation as anti-HIV agents, have been shown to
accumulate inside cells. None of the dinucleotides tested in that
study, however, exhibited inhibition of HIV replication or cytotoxicity
at concentrations of 30 µM.
We investigated whether some of the most potent dinucleotides in this
report had antiviral activity by using the cell protection assay
against the cytopathic effects of HIV-1LAI strain. None of
the dinucleotides tested [pCA, rCA, CA, pAC, cDD, cCC, pCA(PS), and
pCA(MP); see Figs. 3 and 4 for structures] had significant antiviral
activity. They also exhibited no cytotoxicity by themselves at
concentrations of 200 µM. At this time, it is not clear
whether this lack of activity/cytotoxicity is due to cellular uptake, stability, or both. These data suggest that additional modifications may be required to achieve antiviral effects. Along these lines, it is
interesting to note that a guanosine quartet oligonucleotide with
potent antiviral activity is also among the most potent known inhibitors of HIV integrases (37). Another application of dinucleotides involves the possible use of these water-soluble inhibitors as lead
compounds for further antiviral development if cocrystals could be
obtained.
| |
Acknowledgments |
We thank Drs. Robert Craigie and Alan Engelman (Laboratory of
Molecular Biology, National Institute of Diabetes and Digestive and
Kidney Diseases) for generously providing us with purified HIV-1
integrase; Dr. Kurt Kohn for his support during the course of these
experiments; and Drs. Jean-Pierre Sommadossi (University of
Alabama-Birmingham), Peter Nielsen (University of Copenhagen, Copenhagen, Denmark), and Robert J. Suhadolnik (Temple University, Philadelphia, Pennsylvania) for providing nonphosphorylated
dinucleotides containing modified nucleoside analogs, the PNA
dinucleotide, and 2,5-oligoadenylates, respectively.
| |
Footnotes |
Received November 8, 1996; Accepted January 13, 1997
This project was supported by a grant from the National
Institutes of Health Intramural AIDS Targeted Antiviral Program and by
the Delaware Research Foundation (H.K.C).
Send reprint requests to: Yves G. Pommier, M.D., Ph.D., Lab
of Molecular Pharmacology, Division of Basic Sciences, NCI/NIH, Bldg.
37, Room 5C25, Bethesda, MD
20892-4255.
| |
Abbreviations |
HIV, human immunodeficiency virus;
HIV-1, human immunodeficiency virus type 1;
HIV-2, human immunodeficiency
virus type 2;
LTR, long terminal repeat;
STP, strand transfer products;
SIV, simian immunodeficiency virus;
FIV, feline immunodeficiency virus;
PNA, peptide-nucleic acid;
AZT, 3-azido-2,3-dideoxythymidine;
HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;
HPLC, high
performance liquid chromatography;
Rp and Sp, chiral phosphorus with an
R or S configuration, respectively.
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Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics
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Summary
Introduction
Summary
