HIV integrase inhibitors with nucleobase scaffolds: Discovery of a highly potent anti-HIV agent

Article abstract of DOI:10.1021/jm0508890

HIV integrase is essential for HIV replication. However, there are currently no integrase inhibitors in clinical use for AIDS. We have discovered a conceptually new β-diketo acid that is a powerful inhibitor of both the 3′-processing and strand transfer s

Full text of DOI:10.1021/jm0508890

J. Med. Chem. 2006, 49, 445-447
445
HIV Integrase Inhibitors with Nucleobase
Scaffolds: Discovery of a Highly Potent Anti-HIV
Agent
Vasu Nair,*,§ Guochen Chi,§ Roger Ptak,† and
Nouri Neamati‡
Figure 1. Structures of two of the most active, known â-diketo
compounds.
The Center for Drug DiscoVery and the Department of
Pharmaceutical and Biomedical Sciences, UniVersity of Georgia,
Athens, Georgia 30602, Southern Research Institute, Infectious
Disease Research Department, Frederick, Maryland 21701, and
Department of Pharmaceutical Sciences, UniVersity of Southern
California, Los Angeles, California 90089
Table 1. Anti-HIV Data for Two of the Most Active, Known â-Diketo
Compounds (see Figure 1 for structures)
identification no.
S-1360
L-731988
lit. ref no.
IC50 (µM)
CC50 (µM)
cell line
TIa
25
0.14
110
PBMC
786
19
1.0
b
H-9
b
ReceiVed September 8, 2005
Abstract: HIV integrase is essential for HIV replication. However,
there are currently no integrase inhibitors in clinical use for AIDS. We
have discovered a conceptually new â-diketo acid that is a powerful
inhibitor of both the 3′-processing and strand transfer steps of HIV-1
integrase. The in vitro anti-HIV data of this inhibitor were remarkable
as exemplified by its highly potent antiviral therapeutic efficacy against
HIVTEKI and HIV-1NL4-3 replication in PBMC (TI >4,000 and
>10,000, respectively).
a Therapeutic index. b Not specified.
The enzymes of the pol gene of the human immunodeficiency
virus (HIV) have been identified as important viral targets for
the discovery and development of anti-HIV therapeutic agents.1-5
Drug discovery involving two of the enzymes, HIV reverse
transcriptase (RT) and HIV protease (PR), and subsequent
clinical utilization of some of these compounds for the treatment
of acquired immunodeficiency syndrome (AIDS), have sug-
gested that the methodology of targeting key viral enzymes for
inhibition represents a viable approach in antiviral chemo-
therapy.4-8 However, while the viral targets, HIV RT and HIV
PR, have been successfully investigated for the development
of clinically useful therapeutic agents, research efforts on drug
discovery pertaining to the third enzyme of the pol gene, HIV
integrase, have not resulted in a single FDA-approved drug
where the mechanism of action is inhibition of HIV integrase.9-11
Nevertheless, as integrase has no human counterpart and is
essential for HIV replication, it remains a superbly attractive
target for the discovery of new anti-HIV agents.
HIV-1 integrase is a 32 kDa protein encoded at the 3′-end of
the HIV pol gene.12-14 Incorporation of HIV DNA into host
chromosomal DNA, which is catalyzed by HIV integrase, occurs
by a specifically defined sequence of 3′-processing and strand
transfer reactions.12,13,15,16 Prior to the initiation of integration
in the cytoplasm, there is assembly of viral DNA, previously
produced by reverse transcription, on HIV integrase. Following
assembly, site-specific endonucleatic cleavage of two nucleo-
tides from each 3′-end of double-stranded viral DNA (3′-
processing) produces tailored viral DNA recessed by two
nucleotides and with terminal CAOH-3′. In the next step,
identified as strand transfer, there is staggered nicking of
chromosomal DNA and joining of each 3′-end of the recessed
viral DNA to the 5′-ends of the host DNA. Interestingly, the
strand transfer step, occurring in the nucleus, is partitioned from
Figure 2. Our novel integrase inhibitor, 1, shown in its intramolecularly
hydrogen-bonded form (left), and the preferred conformation of its
physiologically relevant anion (right), depicting the relationship of the
two benzyl groups and of the enolic hydrogen with the keto carbonyl.
3′-processing and is carried out after transport of the processed,
preintegration complex from the cytoplasm into the nucleus.
While a number of structurally diverse compounds have been
reported to be inhibitors of HIV integrase,8-10,17-27 only a few
compounds of one group, the â-diketo acids, represent the most
convincing, biologically validated inhibitors23,24 of this viral
enzyme. However, many of the reported â-diketo acids are
inhibitors of only the strand transfer step. The in vitro anti-
HIV data for perhaps the most active â-diketo compound,25
S-1360 (Figure 1, Table 1), reveal a therapeutic index (CC50/
IC50) of 786. Another well-known â-diketo compound, L-731988,
appears to be somewhat less active than S-1360 (Table 1).
In this report, we disclose for the first time, conceptually new
â-diketo acids with nucleobase scaffolds that are potent inhibi-
tors of both the 3′-processing and strand transfer steps of HIV
integrase. We have discovered that the nucleobase scaffold, the
substituents, and the specific spatial relationship of substituents
in the scaffold, are critical for potent integrase inhibitory activity.
The discovery makes these compounds unique among diketo
acids, not only in terms of integrase activity, but also because
of their remarkably potent anti-HIV activity (discussed below).
Our work involved examples of both pyrimidine and purine
scaffolds. The discovery is best illustrated with one notable
example, in which the structural features are a pyrimidine
nucleobase scaffold that bears three specific substituents in a
defined spatial relationship: hydrophobic benzyl groups at N-1
and N-3 and a diketo acid (enolic form predominant) at C-5
(Figure 2). This compound, 4-(1,3-dibenzyl-1,2,3,4-tetrahydro-
2,4-dioxopyrimidin-5-yl)-2-hydroxy-4-oxo-but-2-enoic acid (1),
was synthesized in our laboratory from readily available ethyl
â-ketobutyrate.
* To whom correspondence should be addressed. Tel.: (706) 542-6293;
Fax: (706) 583-8283, E-mail: vnair@rx.uga.edu.
§ The University of Georgia.
† Southern Research Institute.
‡ University of Southern California.
10.1021/jm0508890 CCC: $33.50 © 2006 American Chemical Society
Published on Web 12/21/2005

446 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2
Letters
Table 3. Antiviral Efficacy of Compound 1 and the Positive Control
Compound, AZT, in the Inhibition of HIV-1TEKI and HIV-1NL4-3
Replication in PBMC
Table 2. IC50 Data for the Inhibition of Wild-Type HIV-1 Integrase by
Compound 1 and Its Closely Related Analogues
3′-processing
(µM)
strand transfer
inhibitors
(µM)
high concn
IC50
CC50
compd
(µM)
HIV-1 isolate
(nM)
(µM)
TI
compound 1 (Figure 1)
3.7 ((1.0)
4.1 ((0.4)
3.9 ((1.1)
10 ((2.0)
0.2 ((0.1)
< 0.6
2 (bis-(o-F-Bn) analogue of 1)
3 (bis-(p-F-Bn) analogue of 1)
4 (1 minus N3-benzyl)
1a
200
200
1
TEKI
NL4-3
TEKI
NL4-3
50
>200
>200
>1
>4000
>10000
>7143
>5556
< 0.7
1a
<20
0.14
0.18
0.5 ((0.2)
AZT
AZT
1
>1
a For compound 1: IC90 3.91 µM (HIV-1TEKI), CC90 > 200 µM. IC90
1.54 µM (HIV-1NL4-3), CC90 > 200 µM.
terminations were performed in triplicate with serial 1/2log10
dilution of the test materials (six to nine concentrations total).
The overall performance of both assays was validated by the
MOI-sensitive positive control compound, AZT, exhibiting the
expected level of antiviral activity.
As summarized in Table 3 (see also Supporting Information),
in vitro anti-HIV studies against HIV-1 isolates in PBMC
showed that compound 1 (highest test concentration ) 200 µM)
was extremely active with IC50 values in the nM range and with
antiviral efficacy data (therapeutic indices, TI ) CC50/IC50) of
> 4000 (HIV-1TEKI) and >10000 (HIV-1NL4-3). The IC90 data
for 1, which were in the low µM range, were also compelling
(Table 3). Cell viability data showed only mild cellular
cytotoxicity at higher test concentrations; however, a CC50
(>200 µM) was not reached. The control compound, AZT
(highest test concentration ) 1 µM), gave therapeutic indices
of > 7143 (HIV-1TEKI) and >5556 (HIV-1NL4-3). Also, in
comparison, the antiviral efficacy data of 1 in PBMC (HIV-
1NL4-3) were well over 1 order of magnitude greater than those
for the most active known â-diketo integrase inhibitor, S-1360
in PBMC (see Table 1).25
Figure 3. Docking of HIV integrase inhibitor (1) with HIV-1 integrase
core structure (PDB code 1BL3). The DDE catalytic triad (Asp64,
Asp116, and Glu152) is shown in blue-green, the Mg2+ as a red sphere,
other H-bonding residues (Asn155 and Glu92) in orange, the lysine
residue with NH3+ (electrostatic interaction) in red. Involvement of the
uracil ring of inhibitor in active site binding is clearly apparent.
To obtain further Supporting Information for the aforemen-
tioned PBMC data and the anti-HIV-1 integrase data, evaluations
were performed for compound 1 against HIV-1NL4-3 in a MAGI-
X4 assay. This assay is designed to detect compounds that block
HIV-1 replication via targets in the viral life cycle up to and
including Tat transactivation (e.g., virus attachment/fusion/entry,
uncoating, reverse transcription, nuclear import, integration, LTR
transactivation). Inhibitors of viral targets after LTR transacti-
vation (e.g., Rev, virus egress/packaging/release, protease) do
not score well in this assay.
Integrase inhibitor, 1, and the known HIV replication inhibi-
tors, AZT (reverse transcriptase inhibitor), T-20 (fusion inhibi-
tor), and dextran sulfate (entry inhibitor) all exhibited anti-HIV
activity in the MAGI-X4 assay against HIV-1NL4-3 in HeLa-
CD4-LTR-â-gal cells. The protease inhibitor, saquinavir was
inactive. In addition, compound 1 did not display any cellular
cytotoxicity up to the highest concentrations (200 µM) tested
in this assay.
In summary, a conceptually new â-diketo acid discovered
by us is a powerful inhibitor of both steps of wild-type HIV-1
integrase and exhibits remarkably potent in vitro anti-HIV
activity, making it the most active inhibitor of HIV replication
of this class of biologically validated integrase inhibitors. The
discovery of this remarkably active molecule, representative of
a unique set of diketo acids bearing nucleobase scaffolds, has
uncovered a whole new direction in the chemistry and biology
of integrase inhibitors and their potential therapeutic applica-
tions.
Integrase inhibition studies were conducted with recombinant
wild-type HIV-1 integrase and a 21-mer oligonucleotide sub-
strate, following a previously described procedure.28 Compound
1 and its analogues, 2 and 3, showed strong inhibition of both
3′-processing and strand transfer steps of HIV-1 integrase (Table
2). The observed integrase inhibition is unusual, as the reported
â-diketo compounds are commonly inhibitors of only the strand
transfer step (e.g., S-1360). Although the same active site
residues appear to be involved in 3′-processing and strand
transfer, it is not clear whether the mechanism of inhibition of
HIV integrase by 1 is the same for 3′-processing in the
cytoplasm and strand transfer in the nucleus, i.e., interaction
with the DDE motif (Asp64, Asp116, and Glu152) and other
proximal amino acid residues and possible sequestration of
critical metal cofactors in the catalytic site (Figure 3). Participa-
tion of the uracil amide carbonyl (4-position) in the binding of
this inhibitor to the active site is suggested by our docking
experiments. Some support for the contribution of the uracil
ring in the inhibition of both steps of HIV integrase comes from
the inhibition data for L-708906 that lacks the nucleobase
scaffold [IC50 for L-708906 >1000 µM (3′-processing) and 0.48
µM ( 0.08 µM (strand transfer), TI ) 16, MT-4 cells].27
Compound 1 and the positive control compound, AZT, were
tested in a PBMC cell-based, microtiter anti-HIV assay against
the clinical isolate, HIV-1TEKI (NSI phenotype) and HIV-1NL4-3
(SI phenotype), and in a MAGI-X4 assay against HIV-1NL4-3
performed with HeLa-CD4-LTR-â-gal cells. All antiviral de-
Acknowledgment. This project was supported by Grant No.
RO1 AI 43181 (to V.N.) from the National Institutes of Health
(NIAID). Its contents are solely the responsibility of the authors
and do not necessarily represent the official views of the NIH.

Letters
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2 447
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Frederick, MD, under NIAID Contract NO1-AI-25478 (to R.P.).
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of AIDS, for his support and facilitation of the anti-HIV studies.
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Supporting Information Available: Synthetic methods and
analytical data for 1 and its precursors, and for compounds 2-4;
procedures for HIV integrase assays and HIV antiviral assays,
representative graphical anti-HIV data and standard deviation data.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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