Recognition and inhibition of HIV integrase by a novel dinucleotide

Article abstract of DOI:10.1016/S0960-894X(99)00677-0

The viral enzyme, HIV integrase, is involved in the integration of viral DNA into host cell DNA. In the quest for a small nucleotide system with nuclease stability of the internucleotide phosphate bond and critical structural features for recognition and inhibition of HIV-1 integrase, we have discovered a conceptually novel dinucleotide, pIsodApdC, which is a potent inhibitor of this key viral enzyme. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(99)00677-0

Bioorganic & Medicinal Chemistry Letters 10 (2000) 249±251
Recognition and Inhibition of HIV Integrase by a Novel
Dinucleotide
Michael Taktakishvili, a Nouri Neamati, b Yves Pommier b and Vasu Nair a,*
aDepartment of Chemistry, The University of Iowa, Iowa City, IA 52242, USA
bLaboratory of Pharmacology, National Cancer Institute, NIH, Bethesda, MD 20892, USA
Received 26 October 1999; accepted 22 November 1999
AbstractÐThe viral enzyme, HIV integrase, is involved in the integration of viral DNA into host cell DNA. In the quest for a small
nucleotide system with nuclease stability of the internucleotide phosphate bond and critical structural features for recognition and
inhibition of HIV-1 integrase, we have discovered a conceptually novel dinucleotide, pIsodApdC, which is a potent inhibitor of this
key viral enzyme. # 2000 Elsevier Science Ltd. All rights reserved.
The viral enzyme, HIV integrase, is involved in the
integration of viral DNA into host cell DNA, a biolog-
ical process that occurs by a sequence involving DNA
splicing (30-processing) and coupling (integration) reac-
tions.1±4 The enzyme apparently recognizes speci®c
sequences (50-ACTG. . .CAGT-30) in the LTRs of viral
DNA. In the ®rst step which involves 30-processing (or
cleavage step), speci®c endonuclease activity removes
two nucleotides from each end of the double helical
viral DNA producing new 30-hydroxyl ends (CAOH-30).
This truncated viral DNA is coupled in the next steps to
host cell DNA (integration) which includes the DNA
strand transfer reaction. Both the 30-processing and
strand transfer steps involve transesteri®cations. The
integration process is essential for the replication of
HIV. Although studies on the search for clinically useful
anti-integrase agents are relatively recent, the screening
of inhibitors against puri®ed recombinant integrase has
contributed to the identi®cation of some interesting lead
compounds which include small and large nucleotides.5±8
In the quest for a small nucleotide system with nuclease
stability of the internucleotide phosphate bond and
critical structural features for recognition and inhibition
of HIV integrase, we have discovered a novel dinucleo-
tide, pIsodApdC (1), which is a potent inhibitor of this
key viral enzyme.
Compound 1 is an unusual phosphorylated dinucleotide
which is composed of an l-related isodeoxynucleoside9±13
and a d-deoxynucleoside. The stereochemical implica-
tions of such a structure are illustrated in 1 (right).
Dinucleotide 1 and its precursor 2 were synthesized in
several steps from protected (S,S)-isodeoxyadenosine14
in an overall yield of over 10% (Scheme 1). These target
compounds were puri®ed by reversed-phase HPLC on a
C18 column (ethanol/water elution) and initially identi-
®ed by comparison of ion-exchange retention times with
natural dinucleotides (Partisil-10 SAX ion-exchange
HPLC column, phosphate bu€er system, retention
times for 1 and 2: 40 and 70 min, respectively). Their
complete structures were established by multinuclear
NMR data (1H, 13C, 31P NMR spectra and COSY,
HMQC and HMBC data), quantitative UV spectra, CD
spectra, and electrospray (ESI) HRMS data (calculated
for 1: 619.1067 (M±H) , found: 619.1056; calculated for
2: 539.1404 (M±H) , found: 539.1396). The quantitative
UV spectral data (lmax 263 nm, e 19,500 and 19,400)
gave evidence of hypochromicity in these molecules.
This and the CD data suggested the existence of base
stacking interactions. In order for stacking interactions
to occur, the carbohydrate moieties must assume an
orthogonal relationship, which results in an unusual
internucleotide phosphate linkage. The consequences of
this with respect to nuclease stability are discussed
below.
HIV integrase inhibition assays were conducted with
puri®ed recombinant integrase using a 21-mer oligo-
nucleotide substrate as described previously by Pommier
Keywords: antivirals; nucleotides; enzyme inhibitors.
*Corresponding author. Tel.: +1-319-335-1364; fax: +1-319-353-
2621; e-mail: vasu-nair@uiowa.edu
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(99)00677-0

250
M. Taktakishvili et al. / Bioorg. Med. Chem. Lett. 10 (2000) 249±251
and co-workers.15 The data are summarized in Table 1.
Compound 1 was found to have strong inhibitory
activity against recombinant wild type HIV integrase in
reproducible assays (IC50 19 mM for 30-processing and
25 mM for strand transfer). The activity is much greater
than for dideoxynucleoside monophosphates5,6 and is
close to the activity of the corresponding ``natural
dinucleotide'' pdApdC.16 The signi®cant anti-integrase
activity of dinucleotide 1 suggests some sequence selec-
tivity, which is consistent with the catalytic mechanism
of 30-processing. Molecular recognition by the integrase
of the ultimate and penultimate bases (50-AC-30) at the
50-end of the minus strand of non-cleaved viral DNA
may result in stable complex formation before the
strand transfer reaction.17 Thus, the potent inhibitor
activity of 1 may re¯ect the anity that HIV integrase
has for this dinucleotide sequence and other data16 also
suggest that two neighboring bases may ful®ll a sub-
stantial part of the essential interaction requirements
when integrase recognizes its viral DNA substrate. The
terminal 50-phosphate appears to be essential for activ-
ity as the precursor of 2, i.e. the dinucleotide that is
devoid of the 50-phosphate group, is much less potent.
Compounds 1 and 2 inhibited all enzymatic activities of
integrase and DNA-integrase cross-linking in the same
range as indicated in Table 1 when Mn+2 or Mg+2 was
used as a cofactor (data not shown). This suggests that
compound 1 binds to the catalytic core of integrase and
the inhibition of integrase by 1 is metal-independent.
Another remarkable aspect of compounds 1 and 2 is
that the internucleotide bond exhibits resistance to
cleavage by mammalian 50- and 30-exonucleases (phos-
phodiesterases (PDE)). For example, for compound 2,
cleavage of the internucleotide phosphate bond is
approximately 33% of that for the natural dinucleotide,
dApdC, with PDE I and approximately 20% with PDE
II. The results are graphically represented in Figures 1
and 2. The resistance to internucleotide phosphate
bond cleavage is not associated with chemical alteration
of the phosphate bond, as is commonly found with
nuclease-resistant compounds, but with the structural
distortion of this phosphate bond as illustrated in 1.
Further studies of conceptually-new, nuclease-resistant
dinucleotides as inhibitors of HIV integrase are in
progress.
Table 1. Anti-integrase inhibition data for 1 and 2 and some selected
compounds
Compound
30-processing
Strand transfer
(mM)
(mM)
PIsodApdC 1
IsodApdC 2
(S,S)-IsoddAMP
L-ddCMP5
19
200
>200
50
>110
6
25
200
>200
45
140
3
AZTMP6
pdApdC13
Scheme 1.

M. Taktakishvili et al. / Bioorg. Med. Chem. Lett. 10 (2000) 249±251
251
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Figure 1. Hydrolysis of isodApdC in comparison with the natural
dinucleotide, dApdC, with PDE I from bovine intestinal mucosa.
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Antiviral Evaluation; Kinchington, D., Schinazi, R., Eds.;
Humana: Totowa, 1998. Integrase Assays. In brief, IN was
preincubated at a ®nal concentration of 200 nM with the
inhibitor in reaction bu€er (50 mM NaCl, 1 mM HEPES, (pH
7.5), 50 mM EDTA, 50 mM dithiothreitol, 10% glycerol (wt/
vol), 7.5 mM MnCl2, 0.1 mg of bovine serum albumin per ml,
10 mM 2-mercaptoethanol, 10% DMSO, and 25 mM MOPS
(morpholinepropanesulfonic acid) (pH 7.2) at 30 ꢀC for
30 min. Then, the 50-end 32P-labeled linear oligonucleotide
substrate (20 nM) was added, and incubation was continued
for 1 h. Reactions were quenched by the addition of an equal
volume (16 mL) of loading dye (98% deionized formamide,
10 mM EDTA, 0.025% xylene cyanol, 0.025% bromophenol
blue). An aliquot (5 mL) 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 PhosphorImager cassette, and analyzed with a Molecular
Dynamics PhosphorImager (Molecular Dynamics, Sunnyvale,
CA). Percent inhibition was calculated using the following
equation: inhibition=100Â[1 (D C)/(N C)], where C, N,
and D are the fractions of 21-mer substrate converted to 19-
mer (30-processing product) or strand transfer products for
DNA alone, DNA plus IN, and IN plus drug, respectively.
The 50% inhibitory concentrations (IC50) were determined by
plotting the log of drug concentration versus percent inhibi-
tion and identifying the concentration which produced an
inhibition of 50%.
Figure 2. PDE II from bovine spleen.
Acknowledgments
We thank Dr. Suresh Pal for help with the PDE studies,
Dr. Lynn Teesch for the ESI HRMS data, and Mr.
Guisen Zhao for the preparation of isodeoxyadenosine.
We gratefully acknowledge support of this research by
the National Institutes of Health (AI 43181).
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