Phosphorothioate oligonucleotides that bind to the V3-loop and uses thereof

The present invention provides phosphorothioate oligonucleotide moieties comprising a phosphorothioate oligonucleotide comprising the sequence G.sub.m X.sub.n G.sub.p, wherein G is guanosine; X is thymidine, adenosine or cytidine, or a combination thereof; each of m, n and p is independently an integer greater than 2; the phosphorothioate oligonucleotide moiety being capable of binding to a V3 loop of HIV envelope glycoprotein. The invention further provides for a method of inhibiting HIV activity. The invention also provides for a method of inhibiting HIV activity in a subject. The invention further provides for a method of treating an HIV related disorder in a subject. Finally, the invention provides a pharmaceutical composition comprising a phosphorothioate oligonucleotide moiety and a pharmaceutically acceptable carrier.

Throughout this application, various publications are referenced by Arabic 
numerals in brackets. Full citations for these publications may be found 
at the end of the specification immediately preceding the claims. The 
disclosures of these publications are in their entirety hereby 
incorporated by reference into this application to more fully describe the 
state of the art to which this invention pertains. 
BACKGROUND OF THE INVENTION 
Phosphorothioate oligodeoxynucleotides belong to a class of polyanions that 
bind to the third variable domain (V3) of HIV-1 gp120 and inhibit 
infectivity of a wide variety of HIV-1 isolates. This potent V3 binding of 
phosphorothioate oligodeoxynucleotides, which is relatively independent of 
the nucleotide sequence of the oligodeoxynucleotides, decreases with chain 
length (below 18-mers) and is low for 8-mers. However, recent studies have 
observed a nucleotide sequence-dependent augmentation of phosphorothioate 
oligodeoxynucleotide binding to V3 for 8-mers that contain the S-d-G.sub.4 
motif (e.g. SdT.sub.2 G.sub.4 T.sub.2), and have suggested that formation 
of quadruple helical tetraplexes (G-tetrads) is associated with the 
acquisition of V3 binding ability by small phosphorothioate 
oligodeoxynucleotides. We synthesized a series of -SdG.sub.4 -containing 
oligodeoxynucleotides with varying tandem length (including, the 8-mer 
SdT.sub.2 G.sub.4 T.sub.2, the 12-mer SdG.sub.4 T.sub.4 G.sub.4 and the 
28-mer SdG.sub.4 (T.sub.4 G.sub.4).sub.3) and compared them with 
phosphorothioate oligodeoxynucleotides (with similar lengths or related 
sequences) for (1) their inhibition of the binding of mAb 9284, which 
binds to the N-terminal portion of the V3 loop, (2) the values of K.sub.c 
when these compounds are used as competitors of the rgp120-binding of an 
alkylating phosphodiester oligodeoxynucleotide probe, and (3) inhibition 
of HIV-1 infectivity in a cell-cell transmission model. The presence of 
S-dG.sub.4 - motifs and the number of tandem motifs augmented V3-binding 
and anti-HIV-1 infectivity for small (8- or 12-mer oligodeoxynucleotides) 
but did not significantly augment the potency of 28-mers. Whereas 
tetraplex formation of SdT.sub.2 G.sub.4 T.sub.2 may contribute to its V3 
binding, the 12-mer SdG.sub.4 T.sub.4 G.sub.4 does not migrate as a 
tetraplex on non-reducing gels, suggesting that SdG.sub.4 - motifs may 
augment anti-HIV activity of multiple mechanisms. 
The V3 binding data correlates with the relative abilities of these 
oligonucleotides to inhibit HIV-1 after cell free or cell associated 
infection of lymphocytoid H9 cells or of monocytoid U937 cells. In each 
case, the potency of small oligonucleotides (8-12 mers) was enhanced by 
-SdG.sub.4 - motifs, but this augmentation was not observed for longer 
oligonucleotides (e.g. 28-mers; SdG.sub.4 T.sub.4 G.sub.4 is not as potent 
a V3 binder as the 28-mers SdG.sub.4 (T.sub.4 G.sub.4).sub.3 or SdC28 
(IC.sub.50 =20 nM)). In addition, although the phosphodiester 
oligonucleotides with -SdG.sub.4 - motifs may also exist as quadruple 
helices or as hairpins, they did not bind avidly to V3 and did inhibit mAb 
9284 binding. Together, these data indicate that the phosphorothioate 
backbone is essential for V3 binding and that -SdG.sub.4 - motifs augment 
the V3 binding of smaller oligos in the absence of quadruple helix 
formation. However, the V3 binding of the larger oligonucleotides (e.g. 
SdC.sub.28) is not augmented by the presence of this motif. 
Polyanionic compounds interact with the V3 loop of gp120 1-3! and inhibit 
infectivity of multiple pathogenic strains of HIV-1 4-9, 28, 29!. Recent 
data have shown that phosphorothioate oligodeoxynucleotides, which are 
nuclease resistant, inhibit HIV-1 fusion induced by a number of strains, 
at least in part by binding to the V3 loop of gp120, in an interaction 
that depends strongly on the sulfur phosphorothioate backbone, but is 
relatively independent of the nucleotide sequence of the phosphorothioate 
oligodeoxynucleotides 10-13!. The ability of homopolymeric 
phosphorothioate oligodeoxynucleotides to bind in a sequence non-specific 
manner to the V3 loop decreases with chain length below 18-mers and is low 
for 8-mers 13!. However, a more recent study, employing SdT.sub.2 G.sub.4 
T.sub.2, showed that nucleotide sequence-dependent augmentation of 8-mer 
phosphorothioate oligodeoxynucleotides binding to the V3 loop of gp120 
could be mediated by S-dG.sub.4 - motifs 14!. Furthermore, SdT.sub.2 
G.sub.4 T.sub.2 possesses the ability to inhibit HIV-1 infectivity, albeit 
with modest potency (IC.sub.50 =1 .mu.M) 30, 14!. The present invention 
addresses how S-dG.sub.4 - motifs contribute to augmented V3 binding. 
The presence of polydeoxyguanine motifs in 8-mer phosphorothioate 
oligodeoxynucleotides suggested that secondary structure may be involved 
in the augmentation of V3-binding 14!. Phosphodiester 
oligodeoxynucleotides with -dG.sub.4 - motifs are known to form quadruple 
helices which are stabilized by guanosine quartet hydrogen bonding 
(G-tetrads) and metal ions such as Na.sup.30 or K.sup.30 ; structures of 
this type have been extensively characterized by NMR and X-ray 
crystallography 15-17!. SdT.sub.2 G.sub.4 T.sub.2 can exist in solution, 
in fact, as a parallel stranded quadruple helix, and it is in this form 
that it is the most potent as a V3 binder 14!. 
In the subject invention, synthetic phosphorothioate oligodeoxynucleotides 
of defined sequence and length have been utilized to study whether the 
augmentation of V3-binding which is observed for small 
oligodeoxynucleotides by S-dG.sub.4 - motifs occurs for 
oligodeoxynucleotides with longer sequences as might obtain from a 
potentiation of G-tetrads formation by intramolecular folding. The 
presence of four contiguous guanosine residues contributes to the potency 
of V3 binding and anti-HIV effects for small oligos (8-12-mer 
phosphorothioate oligodeoxynucleotides), however, the presence of four 
contiguous guanosine residues does not improve the potency of 
oligodeoxynucleotides with longer sequences (e.g. 28-mers). The S-dG.sub.4 
- motifs augment V3 binding by at least two mechanisms; whereas the 8-mer 
SdT.sub.2 G.sub.4 T.sub.2 was shown to form tetraplex structures, the 
12-mer SdG.sub.4 T.sub.4 G.sub.4 has augmented potency, independent of 
frank higher order structures such as quadruple helices. 
SUMMARY OF THE INVENTION 
The present invention provides a phosphorothioate oligonucleotide moiety 
comprising a phosphorothioate oligonucleotide comprising the sequence 
G.sub.m X.sub.n G.sub.p, wherein G is guanosine; X is thymidine, 
adenosine, or cytidine, or a combination thereof; each of m, n and p is 
independently an integer greater than 2; said phosphorothioate 
oligonucleotide moiety being capable of binding to a V3 loop of HIV 
envelope glycoprotein. 
The present invention also provides for a method of inhibiting HIV activity 
comprising contacting HIV with an amount of a phosphorothioate 
oligonucleotide moiety described herein in an amount effective to inhibit 
HIV activity. 
The present invention further provides for a method of inhibiting HIV 
activity in a subject which comprises administering to the subject an 
amount of a phosphorothioate oligonucleotide moiety described herein 
effective to inhibit HIV activity. 
The present invention provides for a method of treating an HIV related 
disorder in a subject which comprises administering to the subject an 
amount of a phosphorothioate oligonucleotide moiety described herein 
effective to treat the HIV related disorder. 
The present invention also provides a pharmaceutical composition comprising 
a phosphorothioate oligonucleotide moiety described herein in an amount 
effective to bind a V3 loop of HIV envelope glycoprotein and a 
pharmaceutically acceptable carrier.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention provides a phophorothioate oligonucleotide moiety 
comprising a phosphorothioate oligonucleotide comprising the sequence 
G.sub.m X.sub.n G.sub.p ; wherein G is guanosine; X is thymidine, 
adenosine, or cytidine, or a combination thereof; each of m, n and p is 
independently an integer greater than 2; said phosphorothioate 
oligonucleotide moiety being capable of binding to a V3 loop of HIV 
envelope glycoprotein. 
Throughout this application, the following standard abbreviations are used 
to indicate specific nucleotides: 
##EQU1## 
Examples of phosphorothioate oligonucleotide moieties include, but are not 
limited to, a phosphorothioate oligodeoxynucleotide, a phosphorodithioate, 
a chimeric oligonucleotide, or a phosphorothioate oligonucleotide which is 
further linked to another chemical moiety. 
The term "phosphorothioate oligonucleotide" means an oligonucleotide or 
oligodeoxynucleotide in which a sulfur atom replaces one or more of the 
non-bridging oxygen atoms in one or more phosphodiester linkages, i.e. an 
oligonucleotide or oligodeoxynucleotide having one or more 
phosphorothiodiester linkages. Each phosphorothiodiester linkage can occur 
as either an Rp or Sp diastereomer. A bridging oxygen atom is an oxygen 
atom in a phosphodiester linkage of a nucleic acid which joins phosphorous 
to a sugar. 
One or more of the phosphorothiodiester linkages of the phosphorothioate 
oligonucleotide moiety may be modified by replacing one or both of the two 
bridging oxygen atoms of the linkage with analogues such as --NH, 
--CH.sub.2, or --S. Other oxygen analogues known in the art may also be 
used. 
For the purposes of this invention HIV includes, but is not limited to, 
HIV-1 and HIV-2. 
In an embodiment the V3 loop of HIV envelope glycoprotein is as described 
and depicted in Skinner, et al. "Characteristics of a Neutralizing 
Monoclonal Antibody to the HIV Envelope Glycoprotein" 42!. 
In another embodiment the V3 loop of HIV envelope glycoprotein is the 
portion of gp120 corresponding to C 303 to C 338 (using the standard 
numbering of HIV strain IIIb) or comprising C 302 to C337 (using the 
numbering of HIV strain IIIb by Ratner) 46!. 
In one embodiment the phosphorothioate oligonucleotide moiety comprises a 
phosphorothioate oligonucleotide comprising the sequence G.sub.m X.sub.n 
G.sub.p, wherein X is adenosine or thymidine. 
In another embodiment the phosphorothioate oligonucleotide moiety comprises 
a phosphorothioate oligonucleotide comprising the sequence G.sub.m X.sub.n 
G.sub.p, wherein each of m, n and p is 3 to 10 inclusive. 
In another embodiment the phosphorothioate oligonucleotide moiety comprises 
an oligonucleotide comprising the sequence G.sub.m X.sub.n G.sub.p wherein 
each of m, n and p is 4 and X is thymidine. 
The present invention provides that X may be thymidine, adenosine or 
cytidine, or a combination thereof. Accordingly, X may be any combination 
of adenosine, thymidine or cytidine residues, provided that the total 
number of adenosine, thymidine or cytidine residues present in the 
phosphorothioate oligonucleotide is greater than 2. 
In another embodiment the phosophorothioate oligonucleotide of the 
phosphorothioate oligonucleotide moiety has a length of from about 8 to 
about 100 nucleotide residues. 
A phosphorothioate oligonucleotide may be stereo regular, stereo 
non-regular or stereo random. A stereo regular phosphorothioate 
oligonucleotide is a phosphorothioate oligonucleotide in which all the 
phosphodiester linkages or phosphorothiodiester linkages polarize light in 
the same direction. Each phosphorous in each linkage may be either an Sp 
or Rp diastereomer. Phosphorothioate oligonucleotides which are created in 
an automated synthesizer are stereo random which means that each 
phosphorous atom in the phosphorothioate oligonucleotide has a 50% chance 
of being either an Sp or an Rp diastereomer. 
In a further embodiment, the phosophorothioate oligonucleotide moiety 
comprises a phosphorothioate oligonucleotide linked to a chemical moiety, 
such as a cholesteryl moiety, an intercalating agent, a cross-linker, an 
artificial endonuclease, a lipophilic carrier, a peptide conjugate, or a 
combination thereof. 
In another embodiment, the phosphorothioate oligonucleotide moiety 
comprises a phosphorothioate oligonucleotide conjugated to a sulfated 
carbohydrate, a carbohydrate, or a glycan. 
The present invention further provides that one or both ends of the 
phosphorothioate oligonucleotide of the phosphorothioate oligonucleotide 
moiety may be linked with the following chemical moieties: intercalating 
agents, such as acridine derivatives; cross-linkers, such as psoralen 
derivatives, azidophenacyl, proflavin, and azidoproflavin; artificial 
endonucleases, which comprise those conjugates whose nuclease component is 
able to cleave DNA specifically and nonspecifically, and acquires a 
specificity by covalent linkage to the oligonucleotide portion, such as 
metal complexes EDTA-Fe (II), o-phenanthroline-Cu(I), and 
porphyrin-Fe(II); and lipophilic carriers or peptide conjugates, such as 
long chain alcohols , phosphate esters, amino or mercapto groups, dyes or 
nonradioactive markers and polylysine or other polyamines. 
Furthermore, one or both ends of the phosphorothioate oligonucleotide of 
the phosphorothioate oligonucleotide moiety may be linked with the 
following chemical moieties: intercalating agents, such as 
2-methoxy-6-chloroacridine, methylphosphonates, methylesters, and 
aminoalkyls; alkylating oligonucleotides, such as acetyl; artificial 
endonucleases, such as amino-1-hexanolstaphylococcal nuclease, and 
alkaline phosphatase; peptide conjugates, such as polylysine; and terminal 
transferases. 
Furthermore, the phosphorothioate oligonucleotide of the phosphorothioate 
oligonucleotide moiety may be conjugated to a carbohydrate, sulfated 
carbohydrate, or glycan. Such conjugates may be synthesized so as to 
introduce a desired specificity into the phosphorothioate oligonucleotide 
moiety. 
In addition, the phosphorothioate oligonucleotide of the phosphorothioate 
oligonucleotide moiety may be combined with a cationic lipid. Examples of 
cationic lipids include, but are not limited to, lipofectin, dotma, and 
dogs. 
The phosphorothioate oligonucleotide of the phosphorothioate 
oligonucleotide moiety may have one or more of its sugars modified or 
replaced so as to be ribose, glucose, sucrose, or galactose, or any other 
sugar. Alternatively, the phosphorothioate oligonucleotide may have one or 
more of its sugars substituted or modified in its 2' position, i.e. 
2'allyl or 2'-O-allyl. An example of a 2'-O-allyl sugar is a 
2'-O-methylribonucleotide. Further, the phosphorothioate oligonucleotide 
may have one or more of its sugars substituted or modified to form an 
.alpha.-anomeric sugar. 
Furthermore, the phosphorothioate oligonucleotide of the phosphorothioate 
oligonucleotide moiety may have one or more of its nucleotide bases 
substituted or modified. Apart from the bases of adenine, guanine, 
cytosine, and thymine, other natural bases such as inosine, deoxyinosine, 
and hypoxanthine are acceptable in the phosphorothioate oligonucleotide 
moiety useful in the subject invention. In addition, isosteric purine 
2'deoxy-furanoside analogues, 2'-deoxynebularine or 2'deoxyxanthosine, or 
other purine and pyrimidine analogues may also be used. The guanosine 
bases comprising the phosphorothioate oligonucleotide sequence may not be 
substituted or modified. 
In another embodiment the phosphorothioate oligonucleotide has a length of 
from about 8 to about 100 nucleotide residues. 
The present invention also provides a method of inhibiting HIV activity 
which comprises contacting HIV with an amount of a phosphorothioate 
oligonucleotide moiety described herein in an amount effective to inhibit 
HIV activity. 
As used herein, the phrase "an amount effective to inhibit HIV activity" 
means that amount which is effective to inhibit the HIV activity of HIV in 
a cell. The IC.sub.50 values of the phosphorothioate oligonucleotides of 
the present invention are approximately 10 nanomolar. Accordingly, the 
preferred serum concentration of the phosphorothioate oligonucleotides of 
the present invention is from about 10 micromolar to about 1 nanomolar, 
preferably about 1 micromolar. An effective amount will vary with the 
particular compound in use, the strength of the preparation, the mode of 
administration, and the advancement of the disease condition. Additional 
factors depending on the particular subject being treated that will result 
in a need to adjust dosages, include subject age, weight, gender, diet and 
time of administration. 
The present invention further provides for a method of treating an HIV 
related disorder in a subject which comprises administering to the subject 
an amount of the phosphorothioate oligonucleotide moiety described herein 
effective to treat the HIV related disorder. 
Examples of HIV related disorders include, but are not limited to, AIDS, 
immunodeficiency, central nervous system disease, HIV encephalopathy, 
neuropathy, pneumocystis, Kaposi's sarcoma, carinii pneumonia, 
non-Hodgkin's lymphoma, and Hodgkin's lymphoma. Other HIV related 
disorders are known in the art, and any of these disorders may be treated 
according to the invented method. 
Throughout this application the use of the present invention has been 
described in association with HIV. As stated previously, the invention may 
be used to inhibit HIV-1 and/or HIV-2 activity. The invention may also be 
used to inhibit activity of any virus that uses a cationic moiety to 
mediate fusion with the host membrane. This invention further provides a 
phosphorothioate oligonucleotide moiety comprising a phosphorothioate 
oligonucleotide having the sequence G.sub.m X.sub.n G.sub.p, wherein G is 
guanosine; X is thymidine, adenosine or cytidine, or a combination 
thereof; each of m, n and p is independently an integer greater than 2; 
said phosphorothioate oligonucleotide moiety being capable of binding to a 
surface protein of a virus that uses a cationic moiety to mediate fusion 
with the host membrane. 
The present invention may be used to treat an HIV infection in which the 
infection was acquired by transfusion with blood or blood products, sexual 
contact, a laboratory accident, a needle stick injury (seroconversion), 
trauma, intravenous drug use, transfusion of organs or organ products, or 
any other route of infection. The HIV infection may have been transmitted 
via maternal-fetal transmission. 
The present invention may be used when HIV infection has occurred or when 
there is a chance that HIV infection may have occurred or in instances in 
which HIV infection may occur (ie. needle stick injury). Thus, "inhibiting 
HIV activity in a subject" according to the present invention encompasses 
both prevention and treatment of HIV. 
The present invention also provides a pharmaceutical composition comprising 
a phosphorothioate oligonucleotide moiety described herein in an amount 
effective to bind a V3 loop of HIV envelope glycoprotein and a 
pharmaceutically acceptable carrier. 
As used herein, the phrase "an amount effective to bind to a V3 loop of HIV 
envelope glycoprotein" means that amount which is effective to bind to a 
V3 loop of HIV envelope glycoprotein. An effective amount will vary with 
the particular compound in use, the strength of the preparation, the mode 
of administration, and the advancement of the disease condition. 
Additional factors depending on the particular subject being treated that 
will result in a need to adjust dosages, include subject age, weight, 
gender, diet and time of administration. 
For the purposes of this invention examples of "HIV activity" include, but 
are not limited to, replication, infectivity, cell-cell fusion (syncytia), 
the formation of multinucleated giant cells and the formation of 
heterokaryons. Means of measuring HIV infectivity and/or replication 
include but are not limited to, measuring p24 production, measuring 
reverse transcriptase activity, and/or measuring viral load. 
In any of the methods disclosed herein the phosphorothioate oligonucleotide 
moiety may be administered to the subject in a pharmaceutical composition 
via any known mode of administration. 
Such means of administration are well known to those skilled in the art and 
include, but are not limited to, topical administration, parenteral 
administration, oral administration, or intraperitoneal, intravenous, 
intrathecal, intratracheal, intramuscular, or subcutaneous injection. 
Administration of the phosphorothioate oligonucleotide moiety may be 
effected continuously or intermittently. Furthermore, the phosphorothioate 
oligonucleotide may be administered by itself or in combination with 
cationic lipids or other carriers. 
A pharmaceutical composition comprising a phosphorothioate oligonucleotide 
moiety described herein may include any of the known pharmaceutical 
carriers. Examples of suitable pharmaceutical carriers include any of the 
standard pharmaceutically accepted carriers known to those of ordinary 
skill in the art. Examples of such pharmaceutical carriers include, but 
are not limited to, phosphate buffered saline solution, water, emulsions 
such as oil/water emulsions or a triglyceride emulsion, various types of 
wetting agents, tablets, coated tablets and capsules. A suitable 
pharmaceutically acceptable carrier may be selected taking into account 
the chosen mode of administration. 
An effective amount of a pharmaceutical composition comprising a 
phosphorothioate oligonucleotide moiety is that amount which is effective 
to bring about the desired effect in the subject. Accordingly, an 
effective amount will depend on various factors known to those of ordinary 
skill in the art. Such factors include, but are not limited to, the size 
of the subject and the degree to which the disease from which the subject 
suffers has progressed. The effective amount will also depend on whether 
the phosphorothioate oligonucleotide moiety is going to be administered to 
the subject in a single dosage or periodically over a stretch of time. 
The phosphorothioate oligonucleotide moieties of the present invention may 
be combined with other medicaments commonly used to treat subjects 
suffering from HIV related disorders. Examples of such medicaments 
include, but are not limited to, agents that inhibit CD4-gp120 
interactions, including antibodies to CD4 or gp120, soluble CD4, and 
peptides from CD4 or gp120 that inhibit gp120 activity; protease 
inhibitors such as indinavir sulfate; reverse transcriptase inhibitors 
such as zidovudine, azidothymidine, lamivudine or a combination of reverse 
transcriptase inhibitors; and therapeutic HIV vaccines. 
This invention will be better understood from the Examples in the 
"Experimental Details" Section which follows. However, one skilled in the 
art will readily appreciate that the specific methods and results 
discussed therein are merely illustrative of, and are not intended to, nor 
should they be construed to, limit the invention as described more fully 
in the claims which follow thereafter. 
EXPERIMENTAL DETAILS 
Materials and Methods 
Reagents: 
The rgp120 used was purchased from American BioTechnologies (Cambridge, 
MASS.). The murine IgG1 anti-gp120 mAbs, NEA 9284 18!, NEA 9301 and NEA 
9305 (also known as mAb 0.5 .beta.20!) were purchased (Dupont, Boston, 
Mass.). AZT was obtained from Burroughs Welcome Research Laboratories. 
Oligodeoxynucleotides: 
Oligodeoxynucleotides were synthesized on a DNA Synthesizer by the 
manufacturer's suggested procedures (ABI 380B, Applied Biosystems, Foster 
City, Calif.). After synthesis, the oligodeoxynucleotides were deblocked 
in aqueous ammonia at 60.degree. C. for 8 hrs and HPLC purified over a 
PRP-1 column in a gradient of 0.1M triethylammonium 
bicarbonate/acetonitrile as described previously 19!. After detritylation 
in 3% acetic acid and lyophilization, the oligodeoxynucleotides were 
dissolved in water, and precipitated with a 10-fold excess volume of 2% 
lithium perchlorate/acetone, washed with acetone, and dried in vacuo. The 
oligodeoxynucleotides were then reprecipitated as the sodium salt from 
aqueous ethanol. 
Synthesis of the probe, alkylating oligodeoxynucleotide RCl.sup.32 
PNH-OdT18 
Approximately 6 OD units of OdT.sub.18 were 5' labeled with .sup.32 
P-phosphate by reaction with 5'-polynucleotide kinase 31!. Excess ATP was 
separated from reaction product by Sephadex G25 chromatography in 0.1M 
lithium perchlorate. The oligodeoxynucleotide was then precipitated by 
addition of 2% LiClO.sub.4 /acetone, and dissolved in water at a 
concentration of 200 OD units/.mu.L. The oligodeoxynucleotide was then 
precipitated by addition of 7 .mu.L of an 8% aqueous solution of 
cetyltrimethylammonium bromide solution and dried. To this was added 6 
.mu.g of p-(benzylamino)-N-2-chloroethyl-N-methylamine (ClRNH.sub.2) in 20 
.mu.L of dimethylformamide, followed by 8 .mu.g of dipyridyl disulfide and 
9.5 .mu.g of triphenylphosphine. 50 .mu.L of methanol were added, and the 
oligodeoxynucleotide was precipitated by addition of 2% lithium 
perchlorate/acetone. The product (ClRNH.sup.32 P-OdT.sub.18) was 
redissolved in water, precipitated again as above, and stored in water at 
-80.degree.C. 
Determination of K.sub.c of oligodeoxynucleotides competing for 
ClRNH.sup.32 P-OdT.sub.18 binding to rgp120 
The value of K.sub.d (approximately 1 .mu.M) has been previously determined 
from the concentration dependence of rgp120 binding and alkylation by 
ClRNH.sup.32 P-OdT.sub.18 13!. To 10 .mu.L of a solution of 0.025 mg/mL 
rgp120 (in Tris HCl, 0.1M, pH 7.5) were added ClRNH.sup.32 P-OdT.sub.18 
(in 5 .mu.L Tris HCl, 0.1M, pH 7.5) to give the final concentration of 2 
.mu.M. After incubation of the mixture at 37.degree. C. for 1 hour, 7.5 
.mu.L of a buffer containing 50% glycerol, 0.1M dithiothreitol, 2% SDS and 
0.001% bromophenol blue was added to each sample, and polyacrylamide gel 
electrophoresis performed. The gel was dried and allowed to expose Kodak 
X-ray film for the appropriate times. For quantitation of rgp120-bound 
oligodeoxynucleotides, the developed film was overlayed on the gel, and 
the gel regions were excised and counted in a .beta. counter. To determine 
competition constants (K.sub.c) for phosphorothioate oligodeoxynucleotide 
competitors of ClRNH.sup.32 P-OdT.sub.18 binding to rgp120, the 
appropriate concentration of competitor (1 or 2 AM) was added to the 
reaction mixture. 
The value of Kc could be calculated from Equation 1 24! 
EQU K.sub.c =K.sub.d .times.C/L.sub.1 .times.1/R.sub.o /R.sub.c)-1!Equation 1 
where K.sub.c and K.sub.d are as previously described C=competitor!, 
L.sub.1 =ClNH.sup.32 P-OdT.sub.18 ! (=2 .mu.M), and R.sub.o /R.sub.c =the 
number of bound counts in the absence and presence of competitor, 
respectively. The determination of K.sub.c via Equation 1 is a variation 
of the method of Cheng and Prusoff 32!. Equation 1 is valid only if the 
alkylating oligodeoxynucleotide or competitor are used at saturating 
concentrations, if not, the actual value of K.sub.c may be lower than the 
measured value. 
ELISA binding assays 
The adherence of rgp120 to the U bottoms of untreated polystyrene plates 
(Corning, Corning N.Y.) was achieved by incubation of 50 .mu.L of a rgp120 
solution (1 .mu.g/mL) in phosphate buffered saline (PBS) for 2 h at room 
temperature. Control wells were treated with PBS without added protein. 
Wells were washed three times with PBS containing 0.05% Tween-20 
(PBS-Tween) followed by blocking with 0.15 mL of 1% BSA at 4.degree. C. 
overnight. The wells were then washed three times in PBS-Tween before 50 
.mu.L of solutions containing the phosphorothioate or phosphodiester 
oligodeoxynucleotides, at the given concentrations, plus the mAb were 
added to control and protein-coated wells and allowed to react for 30 min 
at room temperature. Control solutions contained 1% BSA in water or in 
PBS. To detect bound ligands, 50 .mu.L of the anti-gp120 mAb in 1% BSA 
were added to the wells and reacted for 90 min at 20.degree. C. The wells 
were washed four times before the addition of 50 .mu.L of 1:1000 affinity 
purified goat anti-mouse IgG conjugated to alkaline phosphatase (Bio-rad 
Laboratories, Richmond, Calif.) in 1% BSA. After 60 min, wells were washed 
four times before addition of 50 .mu.L substrate solution 
(p-nitrophenylphosphate). The enzyme reaction was allowed to progress at 
20.degree. C. for 20-60 min before absorption at 405 nm was measured in an 
ELISA plate analyzer (VMAX, Molecular Devices Corporation, Palo Alto, 
Calif.). Assays were performed in triplicate. Standard deviations (SD) 
were obtained by the STDEV formula (Excel, Microsoft, Redmond, Wash.). The 
standard deviations of the subtracted means (A-B) obtained from the 
formula S.D. (A-B)=SQR(SD(A).sup.2 =SD(B).sup.2). 
Viral Replication Assays 
Antiviral effects of oligodeoxynucleotides were evaluated in tissue 
culture. H9 cells chronically infected with HIV-1 LAI strain were 
maintained as previously described 33!. Cells and supernatant were used 
to transmit virus to fresh lymphocytoid H9 cells or monocytoid U937 cells. 
5.times.10.sup.6 recipient cells in 1 ml of RPMI 10% FCS were incubated 
with 1 ml of a dilution of chronically infected cells containing 10.sup.4 
viable cells (1:100 dilution), with gentle mixing. After 2 hrs at 
37.degree. C., the cells were diluted to 15 ml of RPMI FCS and 100 .mu.l 
samples (containing 3.times.10.sup.4 cells) were added to wells of a 96 
well plate containing an equal volume of media plus oligodeoxynucleotides 
at 2.times. final concentration. Supernatants were harvested and frozen 
twice per week, and at each time half the volume was replaced with fresh 
drug containing medium. After 14 days, supernatants were quantitatively 
assayed for RT transcriptase (RT) activity as described 33!. RT values 
from the day in which peak RT activity was reached in the absence of drug 
were used to calculate the IC.sub.50 values of the oligodeoxynucleotides 
and AZT. 
Results 
Comparison of the ability of phosphorothioate and phosphodiester 
oligodeoxynucleotides to inhibit the binding of mAb 9284 to rgp120 
The effect of the S-dG.sub.4 -motif on phosphorothioate 
oligodeoxynucleotides binding to gp120 was studied by measuring the effect 
of these compounds on the binding of the two anti-gp120 V3 mAbs, 9284 and 
9305, to rgp120 in a solid phase ELISA assay. It has previously been shown 
that SdC.sub.28 (but not OdC.sub.28) specifically inhibits the gp120 
binding of mAb 9284 13!. The 9284 mAb recognizes the N-terminal portion 
of the V3 loop of rgp120 18!. However, SdC.sub.28 does not inhibit 
binding of mAb 9305 13!, which recognizes a contiguous and overlapping 
site C-terminal to 9284 on the V3-loop 18, 20!. Because SdT.sub.2 G.sub.4 
T.sub.2 has recently been shown to bind V3 14!, the effect of SdT.sub.2 
G.sub.4 T.sub.2 on mAb 9284 binding was studied first. Similar to the 
published reports of its effect on the binding of anti-V3 antiserum 14!, 
SdT.sub.2 G.sub.4 T.sub.2 inhibits mAb 9284 binding to the V3 loop 
(IC.sub.50 =4.2 .mu.M) (FIG. 1A). However, even at 10 .mu.M (at which 
SdT.sub.2 G.sub.4 T.sub.2 inhibits 70% of 9284 binding), neither OdT.sub.2 
G.sub.4 T.sub.2 nor Sd(TG).sub.4 inhibit mAb 9284 binding (FIG. 1B). These 
data are consistent with published reports that both the phosphorothioate 
backbone and the presence of an -SdG.sub.4 - motif are required for V3 
loop binding (FIG. 1B)14!. Moreover, in addition to confirming the 
V3-specific binding of SdT.sub.2 G.sub.4 T.sub.2, the finding that 
SdT.sub.2 G.sub.4 T.sub.2 does not inhibit mAb 9305 binding to the V3 loop 
(FIG. 1B) extends the understanding of its binding domain by mapping 
binding to the N-terminal half of the V3 loop. This behavior is similar to 
that of larger SdC-homopolymers, such as SdC.sub.28 as well as sulfated 
polysaccharides 3, 13!. However, although, the -SdG.sub.4 - motif confers 
on SdT.sub.2 G.sub.4 T.sub.2 a sequence-dependent augmented potency for 
V3-binding relative to oligodeoxynucleotides of comparable size and base 
composition, the binding of SdT.sub.2 G.sub.4 T.sub.2 to gp120 V3 is 
substantially weaker than that of SdC.sub.28 (FIG. 1A, Table 1) 13!. 
To address the possibility that tandem S-dG.sub.4 - motifs may further 
facilitate binding to the V3 loop of gp120, the effect of SdG.sub.4 
T.sub.4 G.sub.4 on mAb anti-gp120 V3 binding to rgp120 was studied. 
Similar to other phosphorothioate oligodeoxynucleotides, SdG.sub.4 T.sub.4 
G.sub.4 inhibits the binding of the anti-gp120 V3 mAb 9284 but not mAb 
9305 (FIG. 1A). In addition, the corresponding phosphodiester 
oligodeoxynucleotide, OdG.sub.4 T.sub.4 G.sub.4, does not inhibit mAb 9284 
binding at the concentrations tested (10 .mu.M, FIG. 1B). SdG.sub.4 
T.sub.4 G.sub.4 blocks mAb 9284 binding to the V3 loop with IC.sub.50 
=0.16 .mu.M; thus, its activity is higher than the 8-mer SdT.sub.2 G.sub.4 
T.sub.2 (IC.sub.50 =4.2 .mu.M (FIG. 1A, Table 1). Its activity is slightly 
lower than SdC.sub.28 (IC.sub.50 =0.044 .mu.M), which is an 
oligodeoxynucleotide of substantially longer length (28-mer vs 12-mer) 
(FIG. 1A, Table 1). The corresponding value of IC.sub.50 for SdT.sub.19 is 
0.570 .mu.M (FIG. 1A). These data suggested that the tandem -S-dG.sub.4 - 
repeats in SdG.sub.4 T.sub.4 G.sub.4 increase the ability of the 
phosphorothioate oligodeoxynucleotides to bind to the V3 loop of gp120. 
The next series of experiments addressed whether an additional tandem 
repeat, provided by appending -SdT.sub.4 G.sub.4 T.sub.4 G.sub.4 - to 
SdG.sub.4 T.sub.4 G.sub.4, would confer increased V3 binding on the new 
oligodeoxynucleotide, SdG.sub.4 (T.sub.4 G.sub.4).sub.3. SdG.sub.4 
(T.sub.4 G.sub.4).sub.3 binds to rgp120 and inhibits mAb 9284 binding with 
IC.sub.50 =0.042 .mu.M (FIG. 1A, Table 1). It is thus more potent on a 
molar basis than SdG.sub.4 T.sub.4 G.sub.4, but its almost identical in 
potency to SdC.sub.28, which does not contain S-dG.sub.4 - motifs. 
Determination of the value of K.sub.c for competition by phosphorothioate 
oligodeoxynucleotides of the binding of RCLNH.sup.32 P-OdT18 to rgp120 
The nature of the interaction S-dG.sub.4 - motif containing 
oligodeoxynucleotides with gp120 was probed by measuring the K.sub.c of 
competition for a probe, alkylating phosphodiester oligodeoxynucleotide. 
An 18-mer phosphodiester homopolymer of thymidine which has been 5'-end 
modified with an alkylating moiety and .sup.32 p was employed to produce 
the probe RClNH.sup.32 P-OdT.sub.18. (FIG. 2). The use of this probe 
permits the evaluation of the ability of other phosphodiester and 
phosphorothioate oligodeoxynucleotides to inhibit probe binding and 
subsequent alkylation of gp120 by the radiolabeled probe 
oligodeoxynucleotide. 
Subsequent to alkylation, the .sup.32 P-labeled alkylating 
oligodeoxynucleotide-protein complex was visualized autoradiographically 
after denaturing gel electrophoresis 13!. 
Phosphorothioate and phosphodiester oligodeoxynucleotides were studied as 
competitors of probe oligodeoxynucleotides binding to rgp120. As shown in 
FIG. 2, in the presence of a competitor of probe oligodeoxynucleotide 
binding, the intensity of the gel band, which represents rgp120 modified 
by the probe oligodeoxynucleotide decreased (FIG. 2, compare lane 9 
(control) with lanes 6 (SdG.sub.4 T.sub.4 G.sub.4), lane 7 (SdT.sub.18) 
and lane 8 (SdC.sub.18)). In the presence of phosphorothioate 
oligodeoxynucleotides, ClRNH.sup.32 P-OdT.sup.18 binding to rgp120 was 
competed to a greater extent than in the presence of phosphodiester 
oligodeoxynucleotides (FIG. 2, compare lanes 6(SdG.sub.4 T.sub.4 G.sub.4) 
and 2(SdT.sub.2 G.sub.4 T.sub.2), with lane 5(OdG.sub.4 T.sub.4 G.sub.4) 
Determination of the approximate values of K.sub.c of phosphorothioate 
oligodeoxynucleotide binding to rgp120 was accomplished by use of Equation 
1; these are shown in Table 1. An error of 15% is assumed due to the 
presence of non-specific background on the X-ray film. The value of 
K.sub.c for SdC.sub.18 as determined by Equation 1 is 0.020 .mu.M. The 
value of K.sub.c for SdC.sub.18, as determined via the Cheng-Prusoff 
equation 13! is 0.022 .mu.M. Thus, under the conditions used in these 
experiments, the two methods of determination of K.sub.c give 
approximately equivalent results. 
It is apparent that there is a substantial diminution in the value of 
K.sub.c for SdG.sub.4 T.sub.4 G.sub.4 relative to SdT.sub.2 G.sub.4 
T.sub.2. Further, the value of K.sub.c for the 12-mer SdG.sub.4 T.sub.4 
G.sub.4 is similar for that determined for SdC.sub.28 or SdG.sub.4 
(T.sub.4 G.sub.4).sub.3. In each case, the magnitude of K.sub.c for each 
compound correlates with their ability to block mAb 9284 binding to the V3 
loop of rgp120 in the solid phase ELISA binding assay (Table 1). 
Effect of phosphorothioate and phosphodiester oligodeoxynucleotides on 
HIV-1 infectivity 
To determine if the V3-loop binding correlates with an antiviral effect on 
HIV-1 infection, the ability of these oligodeoxynucleotides to limit HIV-1 
infection in both the lymphocytoid cell line, H9 (FIG. 3A and 3B) and the 
monocytoid cell line, U937, (FIG. 3C and 3D), was examined. Infection of 
these cell lines was initiated by addition of a small inoculum of 
chronically infected H9 cells, followed by culture in the presence of 
different concentrations of oligodeoxynucleotides. Interference with viral 
replication in this assay is a stringent test of the ability of a compound 
to affect new rounds of both cell free and cell-cell viral infection. Cell 
viability in the absence of infection was not affected by 
oligodeoxynucleotides at the concentrations used (data not shown). FIGS. 
3A and 3B show that SdT.sub.2 G.sub.4 T.sub.2 inhibits HIV-1 infectivity 
in H9 cells with IC.sub.50 of 4.0 .mu.M, which is similar to the results 
of published studies 14!. In contrast, SdG.sub.4 T.sub.4 G.sub.4 inhibits 
HIV-1 more potently, with IC.sub.50 of 0.55 .mu.M (FIGS. 3A and 3B). 
Similarly, SdT.sub.2 G.sub.4 T.sub.2 inhibits HIV-1 infectivity in U937 
cells with IC.sub.50 of 1.8 .mu.M, whereas, SdG.sub.4 T.sub.4 G.sub.4 
inhibits HIV-1 infection in U937 cells more potently (3-9 fold), with 
IC.sub.50 of 0.55 .mu.M (FIGS. 3C and 3D). These effects correlate with 
the increased potency of SdG.sub.4 T.sub.4 G.sub.4 for binding rgp120 in 
both the solid phase and solution competition assays (FIGS. 1A, 1B and 2, 
Table 1). Furthermore, SdG.sub.4 (T.sub.4 G.sub.4).sub.3 inhibits HIV-1 
infection in H9 cells with IC.sub.50 of 0.01 .mu.M, which is more potent 
(3-9 fold) than the smaller oligodeoxynucleotides, but not significantly 
different from SdC.sub.28 (IC.sub.50 =0.01 .mu.M) (Table 1). Taken 
together, these data suggest a relationship between the affinity of gp120 
V3-loop binding and the ability of the oligodoexynucleotides to inhibit 
the infectivity of HIV-1 in tissue culture. Therefore, similar to the in 
vitro binding and inhibition of HIV-1 infectivity, the augmented potency 
of S-dG.sub.4 -containing oligodoexynucleotides is particularly evident 
for the 8-mer SdT.sub.2 G.sub.4 T.sub.2 and the 12-mer SdG.sub.4 T.sub.4 
G.sub.4 compounds, but is lost for longer sequences (e.g. 28-mers). 
Solution structure of phosphorothioate oligodeoxynucleotides 
The next series of experiments addressed the state of the phosphorothioate 
oligodoexynucleotides in solution. The solution states of these 
oligodeoxynucleotides were studied by examining their migration in non-SDS 
containing polyacrylamide gels after staining with 0.25 mM monobromobimane 
(Calbiochem, San Diego, CALIF.) and visualization by fluorescent 
transillumination. As a positive control for tetraplex formation SdG.sub.6 
T.sub.9 which exists in solution as quadruple helix (similar to SdT.sub.2 
G.sub.4 T.sub.2) was studied. Although SdG.sub.6 T.sub.9 is a 15-mer (FIG. 
2, lane 4) it migrates on a native polyacrylamide gel much slower than 
SdC.sub.28 (FIG. 2, lane 5), however, treatment of SdG.sub.6 T.sub.9 with 
10M urea/formamide dissociates it into rapidly mobile monomers (FIG. 2, 
lane 2). 
Surprisingly, SdG.sub.4 T.sub.4 G.sub.4 that displayed potent V3 binding 
relative to other 12-mers, migrates at approximately the rate expected for 
a monomer (FIG. 2; compare lanes 1 and 5). Moreover, SdG.sub.4 (T.sub.4 
G.sub.4).sub.3, that displayed V3 binding similar to homopolymeric 
oligodeoxynucleotides, migrates predominately at the rate expected for a 
dimer. Although the 12-mer and 28-mer oligodeoxynucleotides containing the 
-SdG.sub.4 - motif did not migrate with the characteristic retardation of 
tetraplexes, it was of interest that the migration of the 12-mer SdG.sub.4 
T.sub.4 G.sub.4 is somewhat slower than that of SdC.sub.19 (FIG. 2; 
compare lanes 2 and 3). Moreover, the migration of the 28-mer SdG.sub.4 
(T.sub.4 G.sub.4).sub.3 is also slightly slower than that of SdC.sub.28 
(FIG. 2; compare lanes 1 and 3). This slow migration is still observed 
even after treatment of the oligodeoxynucleotides with 10M urea/formamide 
(FIG. 2; compare lanes 2 SdC.sub.28 ! and 5 SdG.sub.4 (T.sub.4 
G.sub.4).sub.3 !). The reason for the relatively slow migration of 
phosphorothioate oligodeoxynucleotides containing the -SdG.sub.4 -motif is 
obscure. 
Taken together, these data suggested that the tandem -SdG.sub.4 -repeats in 
SdG.sub.4 T.sub.4 G.sub.4, in the absence of quadruple helix formation, 
increase the ability of the phosphorothioate oligodeoxynucleotides to bind 
to the V3 loop of gp120. Moreover, despite the presence of higher order 
structure in SdG.sub.4 (T.sub.4 G.sub.4).sub.3, our data indicate that the 
augmentation of phosphorothioate oligodeoxynucleotides binding to gp120 V3 
by -SdG.sub.4 - motifs, while pronounced for smaller oligodeoxynucleotides 
(8- or 12-mers), is not significant for a phosphorothioate 
oligodeoxynucleotide of longer length (28-mer ). 
Discussion 
In both solid phase ELISA assay and solution competition assay, the 
phosphorothioate oligodeoxynucleotides SdT.sub.2 G.sub.4 T.sub.2 and 
SdG.sub.4 T.sub.4 G.sub.4 demonstrate sequence-dependent augmentation of 
binding to the V3 loop of gp120 relative to other phosphorothioate 
oligodeoxynucleotides of similar length and base composition. The V3-loop 
binding data correlates with the relative abilities of these 
oligodeoxynucleotides to inhibit HIV-1 after cell-free or cell-associated 
infection of lymphocytoid H9 cells or of monocytoid U937 cells. Moreover, 
in each case, the potency of small oligodeoxynucleotides (8-12 mers) was 
enhanced by -SdG.sub.4 - motifs, but this augmentation was not observed 
for longer oligodeoxynucleotides (e.g. 28-mers; SdG.sub.4 (T.sub.4 
G.sub.4).sub.3 or SdC.sub.28 displayed similar V3 binding and inhibition 
of HIV-1 infectivity). Finally, examination of the solution structures of 
these oligodeoxynucleotides, studied by SDS gel electrophoresis, showed 
that -S-dG.sub.4 - motifs augment the V3 binding and anti-HIV-1 effects of 
the 12-mer SdG.sub.4 T.sub.4 G.sub.4 oligodeoxynucleotide in the absence 
of guadruple helix formation. 
In both solid phase ELISA assay and solution competition assay, the 
phosphorothioate oligodeoxynucleotides SdT.sub.2 G.sub.4 T.sub.2 and 
SdG.sub.4 T.sub.4 G.sub.4 demonstrate sequence-dependent augmentation of 
binding to the V3 loop of gp120 relative to other oligodeoxynucleotides of 
similar length and base composition. In addition, the use of conventional 
polyacrylamide gels enables relatively facile determination of the nature 
of molecules formed by oligodeoxynucleotides folding 27!. However, 
whereas SdT.sub.2 G.sub.4 T.sub.2 exists predominantly as a quadruple 
helix in polyacrylamide gels, SdG.sub.4 T.sub.4 G.sub.4 has the 
appropriate gel mobility of a monomer. These data suggest that 
augmentation of V3 binding efficiency and the anti-HIV activity of these 
G-rich compounds are not solely dependent on the formation of higher order 
complexes 25, 26!. Rather, the presence of four contiguous guanosine 
residues alone is sufficient to increase activity. The ability of 
oligodeoxynucleotides with four contiguous guanosine residues to 
non-specifically bind with high affinity to proteins, such as basic 
fibroblast growth factor, has previously been noted 34!. 
The binding of deoxyguanosine containing phosphodiester 
oligodeoxynucleotides to the V3 loop is also of interest relative to 
whether the V3 loop has specific target structures on cells and if so, may 
provide insight into the chemical nature of the V3-target structure 
interaction. Several observations suggest that the V3 loop may have a 
molecular ligand or target. First, it is notable that although variable in 
primary sequence, the V3 loop of gp120 of all pathogenic strains of HIV-1 
is positively charged and known to interact with sulfated polysaccharides 
4-9!. On the V3 loop of the HIV-1 .sub.111B gp120 that binds mAb 9284, 
there are three arginine and one lysine residues in a 12 amino-acid 
epitope 18! indicating that the N-terminal region of the V3 loop that 
binds mAb 9284 contains a dense region of positive charges. These 
properties suggest that the molecular target(s) for V3 may be negatively 
charged moieties. 
In this regard, the V3 has been shown to interact with cell surface 
sulfated proteoglycans (heparan sulfate 40!), sulfated glycolipids (3' 
sulfogalactosyl ceramide 39!). In addition, V3 interacts with 
proteinaceous species on U937 and Molt-4 cells 41!, which might contain 
carbohydrate moieties, (for example sialyl-groups), since lectins are 
known to have clusters of positively charged amino acids at their 
carbohydrate-binding regions 38!. A separate line of inquiry has observed 
interactions between V3 and a cell surface protease, cathepsin G and has 
suggested that cathepsin G cleaves the V3 loop 35, 36!. The fact that V3 
may interact with a variety of target structures is also supported by the 
finding that the V3 plays a role in restricting viral trophism for 
macrophages/neural cells or T cells 37, 22, 21! which may depend on 
interactions with cell-type specific cell surface molecules. Furthermore, 
it is probably the positively charged V3 region that is primarily 
responsible for the binding of phosphorothioate oligodeoxynucleotides. 
The possibility exists that one or more ligands for the V3 loop will be 
identified and the phosphorothioate oligonucleotides described are 
expected to inhibit the interaction of the V3 loop with such structures. 
For example, recently a seven transmembrane domain protein, with homology 
to chemokine receptors, has been identified, termed fusin, that functions 
as a co-factor for human CD4 in allowing HIV-1 infection of mouse cells 
44!. Since several members of the C--C and C-X-C chemokine receptor 
family have negatively charged motifs (characterized by E-rich stretches) 
in the ligand binding domains, it is possible that fusin and other related 
members of this family may bind the positively charged V3-loop of gp120. 
Thus, the phosphorothioate oligonucleotides described herein bind to the 
V3 loop and inhibit the interaction of the V3 loop with such co-receptors 
such as chemokine receptors. 
Polyanions appear to have advantages over mAbs in interacting with the V3 
loop, because these compounds bind to and inhibit the infectivity of a 
diverse range of viral isolates. In this regard, phosphorothioate 
oligodeoxynucleotides thus share several potentially important anti-HIV 
properties with sulfated polysaccharides, but may have some advantages. 
Oligodeoxynucleotides are discrete compounds and SdC.sub.28 binds more 
avidly to gp120 than sulfated polysaccharides. Moreover, the potential 
utility of phosphorothioate oligodeoxynucleotides as therapeutic anti-HIV 
agents is further supported by their ability to inhibit HIV-1 infection in 
both lymphocytoid and monocytoid cell lines and by the fact that compounds 
such as SdG.sub.4 T.sub.4 G.sub.4 and SdG.sub.4 (T.sub.4 G.sub.4).sub.3 
are not toxic to human cell lines in vitro. 
These data are also interesting from the point of view of drug design. To 
the extent that small oligodeoxynucleotides are easier to manufacture, the 
12-mer SdG.sub.4 T.sub.4 T.sub.4 may be an important lead compound. On the 
other hand, the longer oligodeoxynucleotides represent a distinct class of 
lead compounds because they bind V3 potently. Since the binding of 28-mer 
is sequence insensitive to the best of our knowledge, it is possible that 
28-mer sequences can be identified that confer other critical properties, 
such as augmentation of bioavailability. Furthermore, in addition to 
binding V3, phosphorothioate oligodeoxynucleotides bind to the gp120 
binding site of CD4 23!, and may contribute to inhibition of infectivity, 
since, it is known that mAb binding to the V3 loop does not inhibit 
gp120-CD4 binding 23!. Thus, it is a goal of future research to determine 
if phosphorothioate oligodeoxynucleotides have synergistic effects on 
inhibiting HIV infectivity by inhibiting both the V3 function and 
gp120-CD4 binding. Together, these observations suggest that clinical 
therapeutic trials of 12-mer or 28-mer phosphorothioate 
oligodeoxynucleotides in patients at high risk for HIV-1 seroconversion 
(e.g., needle stick injuries) might well be worthwhile. 
TABLE 1 
______________________________________ 
Binding to gp120 V3 and inhibition of HIV-1 
infectivity by phosphorothioate (PS) and 
phosphodiester (PO) oligodeoxynucleotides 
V3 gp120 
Binding Binding 
IC.sub.50 
K.sub.d (nM) 
H9 U937 
solid solution 
HIV inf. 
HIV inf. 
Abbrev. OdX/PdX phase phase IC.sub.50 (nM) 
IC.sub.50 (nM) 
______________________________________ 
OdT.sub.2 G.sub.4 T.sub.2 
PO &gt;10,000 760,000 
N.D. N.D. 
SdT.sub.2 G.sub.4 T.sub.2 
PS 4,200 1,540 4,000 1,800 
Od(TG).sub.4 
PO &gt;10,000 10,200 N.D. N.D. 
Sd(TG).sub.4 
PS &gt;10,000 900 &gt;10,000 
&gt;10,000 
OdG.sub.4 T.sub.4 G.sub.4 
PO &gt;10,000 4,650 N.D. N.D. 
SdG.sub.4 T.sub.4 G.sub.4 
PS 160 50 550 550 
SdG.sub.4 (T.sub.4 G.sub.4).sub.3 
PS 42 N.D. 10 N.D. 
SdC.sub.28 
PS N.D. *23 10 N.D. 
______________________________________ 
*This data point was published previously in Stein, C. A., et al., 
Antisense Res. and Dev. 3: 19-31 (1993). 
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46. Ratner, et al., Nature (1985) 313: 277--284. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 1 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
GAGGGGAACAGTTCGTCCATGGC23 
__________________________________________________________________________