U.S. patent application number 10/775818 was filed with the patent office on 2004-11-18 for inhibition of human teomerase by a g-quadruplex-interaction compound.
Invention is credited to Fedoroff, Oleg Y., Hurley, Laurence H., Kerwin, Sean M., Salazar, Miguel.
Application Number | 20040229894 10/775818 |
Document ID | / |
Family ID | 22114844 |
Filed Date | 2004-11-18 |
United States Patent
Application |
20040229894 |
Kind Code |
A1 |
Kerwin, Sean M. ; et
al. |
November 18, 2004 |
Inhibition of human teomerase by a G-quadruplex-interaction
compound
Abstract
Certain non-nucleoside compounds that will selectively inhibit
telomerase by targeting the nucleic add structures, such as
G-quadruplexes, that may be associated with human telomeres or
telomerase have been identified. Inhibition of human telomerase by
two perylenetetracarboxylic acid diimides and a carbocyanine has
been demonstrated. .sup.1H-NMR studies have evidenced the
stabilization of a G-quadruplex by the perylenetetracarboxylic acid
diimide compounds and provided evidence that these and structurally
related compounds inhibit the telomerase enzyme by a mechanism
consistent with interaction with G-quadruplex structures.
Inventors: |
Kerwin, Sean M.; (Round
Rock, TX) ; Fedoroff, Oleg Y.; (Austin, TX) ;
Salazar, Miguel; (Katy, TX) ; Hurley, Laurence
H.; (Austin, TX) |
Correspondence
Address: |
FULBRIGHT & JAWORSKI L.L.P.
600 CONGRESS AVE.
SUITE 2400
AUSTIN
TX
78701
US
|
Family ID: |
22114844 |
Appl. No.: |
10/775818 |
Filed: |
February 10, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10775818 |
Feb 10, 2004 |
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09730893 |
Dec 5, 2000 |
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6689887 |
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09730893 |
Dec 5, 2000 |
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09244675 |
Feb 4, 1999 |
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6156763 |
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60073629 |
Feb 4, 1998 |
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Current U.S.
Class: |
514/279 ;
546/37 |
Current CPC
Class: |
C12Q 2600/136 20130101;
C12Q 1/68 20130101; C07D 471/06 20130101; C07D 277/64 20130101;
C12Q 1/6886 20130101; C12Q 2600/154 20130101 |
Class at
Publication: |
514/279 ;
546/037 |
International
Class: |
A61K 031/4745; C07D
471/02 |
Claims
What is claimed is:
1. A method of reducing proliferative capacity of a cell comprising
contacting said cell with a compound or a salt thereof or a
stereoisomer of compound I that has the formula: 19where R.sup.1
and R.sup.4 are independently -L-A where L is a linking group
having the formula: 20where n is 1-3; and each R5 is independently
H, Me, OH, or OMe; 21where R5 is as before and Y is O, S, SO,
SO.sub.2, NH, NMe, or NCOMe; 22where R5 and Y are as before and X
is CH.sub.2, O, S, SO, SO.sub.2, NH, NMe, or NCOMe; 23where
R.sup.6, R.sup.7, R.sup.8, and R.sup.9 are independently H, OMe,
OEt, halogen, or Me; and A is a compound of the formula: 24where m
is 0-5 and R6 is halogen, NH.sub.2, NO.sub.2, CN, OMe, SO.sub.2NH2,
amidino, guanidino, or Me; 25where o is 0-1; p is 0-2; q is 1-2
provided that when o+q is 2, in which case a pyrrolidine or pyrrole
ring is indicated, or 3, in which a piperidine or pyridine ring is
indicated; r is 0-3; R.sup.7 is H or Me; R.sup.8 is independently
Me, NO.sub.2, OH, CH.sub.2OH or halogen, and when r is 2-3, two
adjacent R8 substituents are --(CH.dbd.CH).sub.2-- or
--(CH2).sub.4-- to form an annulated six-membered ring; 26where
R.sup.9 is independently H, Me, and when R9 is O; s is 0-1; Z is
CH.sub.2, O, NH, NMe, NEt, N(Me).sub.2, N(Et).sub.2, or
NCO.sub.2Et; 27where Q is N, CH, NMe, or NEt; X is O, S, NH, NMe or
NEt; R.sup.10 and R.sup.11 are independently H, Me,
CH.sub.2CO.sub.2Et, R.sup.10 and R.sup.11 taken together are
--(CH.dbd.CH).sub.2-- or --(CH.sub.2).sub.4--; 28where t is 1-4 4;
u is 0-4, and R12 is independently Me, OH, 29CO.sub.2R.sup.13,
CON(R.sup.13).sub.2, SO.sub.3H, SO.sub.2N(R.sup.13).sub.2, CN,
CH(CO.sub.2R.sup.13).sub.2, CH(CON(R.sup.13).sub.2).sub.2,
N(R.sup.13).sub.2, or N(R.sup.13).sub.3 where R.sup.13 is H, Me,
Et, or CH.sub.2CH.sub.2OH; and R.sup.2, R.sup.2', R.sup.2",
R.sup."; R.sup.3, R.sup.3', R.sup.3", R.sup.3'" are each
independently H, OMe, halogen, or NO.sub.2.
2. The method of claim 1, wherein the cell is a mammalian cell.
3. The method of claim 1, wherein the cell is a human cell.
4. The method of claim 1, wherein the cell is a cancer cell.
5. The method of claim 1, wherein said malignant cell is a breast
cancer cell, a prostate cancer cell, liver cancer cell, a
pancreatic cancer cell, a lung cancer cell, a brain cancer cell, an
ovarian cancer cell, a uterine cancer cell, a testicular cancer
cell, a skin cancer cell, a leukemia cell, a head and neck cancer
cell, an esophageal cancer cell, a stomach cancer cell, a colon
cancer cell, a retinal cancer cell, a bladder cancer cell, an anal
cancer cell and a rectal cancer cell.
6. A method of reducing telomeric extension comprising
administering a compound of claim 1 to a telomerase in the presence
of a telomerase substrate.
7. The method of claim 6, where the telomerase is in a cell.
8. The method of claim 1, wherein said compound further promotes
apoptosis.
9. The method of claim 1, wherein said compound further promotes
apoptosis in a cell.
10. The method of claim 1, wherein the compound is a perylene
compound.
11. The method of claim 1, wherein the compound is
N,N'-bis(2-piperdinoeth- yl)-3,4,9,10-perylenetetracarboxylic acid
diimide.
12. The method of claim 1, wherein the compound is
N,N'-bis(2-dimethylamin- oethyl)-3,4,9,10-perylenetetracarboxylic
acid diimide.
13. A compound of the formula 30where R.sup.1 and R.sup.4 are
independently -L-A where L is a linking group having the formula:
31where n is 1-3; and each R5 is independently H, Me, OH, or OMe;
32where R5 is as before and Y is O, S, SO, SO.sub.2, NH, NMe, or
NCOMe; 33where R5 and Y are as before and X is CH.sub.2, O, S, SO,
SO.sub.2, NH, NMe, or NCOMe; 34where R.sup.6, R.sup.7, R.sup.8, and
R.sup.9 are independently H, OMe, OEt, halogen, or Me; and A is a
compound of the formula: 35where m is 0-5 and R.sup.6 is halogen,
NH.sub.2, NO.sub.2, CN, OMe, SO.sub.2NH.sub.2, amidino, guanidino,
or Me; 36where o is 0-1; p is 0-2; q is 1-2 provided that when o+q
is 2, in which case a pyrrolidine or pyrrole ring is indicated, or
3, in which a piperidine or pyridine ring is indicated; r is 0-3;
R.sup.7 is H or Me; R.sup.8 is independently Me, NO.sub.2, OH,
CH.sub.2OH or halogen, and when r is 2-3, two adjacent R.sup.8
substituents are --(CH.dbd.CH)2- or --(CH2)4- to form an annulated
six-membered ring; 37where R.sup.9 is independently H, Me, and when
R9 is O; s is 0-1; Z is CH.sub.2, O, NH, NMe, NEt, N(Me).sub.2,
N(Et).sub.2, or NCO.sub.2Et; 38where Q is N, CH, NMe, or NEt; X is
O, S, NH, NMe or NEt; R.sup.10 and R.sup.11 are independently H,
Me, CH.sub.2CO.sub.2Et, R.sup.10 and R.sup.11 taken together are
--(CH.dbd.CH).sub.2-- or --(CH.sub.2).sub.4; 39where t is 1-4 4; u
is 0-4, and R.sup.12 is independently Me, OH, 40
41CO.sub.2R.sup.13, CON(R.sup.13).sub.2, SO.sub.3H,
SO.sub.2N(R.sup.13).sub.2, CN, CH(CO.sub.2R.sup.13).sub.2,
CH(CON(R.sup.13).sub.2).sub.2, N(R.sup.13).sub.2, or
N(R.sup.13).sub.3 where R.sup.13 is H, Me, Et, or
CH.sub.2CH.sub.2OH; and R.sup.2, R.sup.2', R.sup.2", R.sup.2";
R.sup.3, R.sup.3', R.sup.3", R.sup.3'" are each independently H,
OMe, halogen, or NO.sub.2.
14. A method of reducing proliferative capacity of a cell
comprising contacting said cell with a compound having the formula
II or a salt thereof or a stereoisomer of said compound: 42where C
is --CH.dbd.CH--, --(CH.dbd.CH).sub.2--, --(CH.dbd.CH).sub.3--,
p-phenylene, o-phenylene, p-phenylene-CH.dbd.CH--, or
o-phenylene-CH.dbd.CH--; B is O, S, or NR, and R is r Me or Et.
15. The method of claim 14, wherein the cell is a mammalian
cell.
16. The method of claim 14, wherein the cell is a human cell.
17. The method of claim 14, wherein the cell is a cancer cell.
18. The method of claim 14, wherein said cancer cell is a breast
cancer cell, a prostate cancer cell, liver cancer cell, a
pancreatic cancer cell, a lung cancer cell, a brain cancer cell, an
ovarian cancer cell, a uterine cancer cell, a testicular cancer
cell, a skin cancer cell, a leukemia cell, a head and neck cancer
cell, an esophageal cancer cell, a stomach cancer cell, a colon
cancer cell, a retinal cancer cell, a bladder cancer cell, an anal
cancer cell and a rectal cancer cell.
19. A method of reducing telomeric extension comprising
administering a compound of claim 14, to a telomerase in the
presence of a telomerase substrate.
20. The method of claim 19, where telomerase is in a cell.
21. The method of claim 14, wherein said compound further promotes
apoptosis in a cell.
22. The method of claim 14, wherein the compound is a
carbocyanine.
23. The method of claim 22, wherein the carbocyanine is
3,3'-diethyloxadicarbocyanine (DODC).
24. A method for identifying a candidate compound that inhibits
telomerase activity, comprising the steps: a) obtaining the
three-dimensional structure of a selected compound; and b)
determining the complementarity of the compound to telomere DNA
G-quadruplex wherein a compound that exhibits at least 75% of the
favourable intermolecular interaction energy of the perylene
diimide 2-d(TTAGGG).sub.4 complex structure is indicated to inhibit
telomerase activity.
25. A method of identifying a telomerase inhibitor comprising: a)
contacting a compound with DNA G-quadruplex; and b) determining the
melting point of the DNA G-quadruplex wherein a compound exhibiting
an increase in melting point of said quadruplex, relative to
unbound DNA G-quadruplex, is indicated to inhibit telomerase
activity.
26. A method of identifying a telomerase inhibitor comprising the
steps: a) preparing a DNA G-quadruplex/dye complex wherein the dye
is bound with the G-quadruplex; b) contacting said complex with a
candidate compound; and c) determining displacement of said dye in
the complex by said candidate, wherein displacement of the dye
identifies the candidate as a telomerase inhibitor.
27. A method of identifying a telomerase inhibitor comprising: a)
contacting a candidate compound to be identified as a telomerase
inhibitor with DNA G-quadruplex; and b) determining the
fluorescence or UV/VIS spectrum of the compound wherein an increase
or decrease of the UV/VIS absorption or fluorescence emission
intensity of said compound relative to the UV/VIS absorption or
fluorescence emission intensity in the absence of DNA-G-quadruplex
indicates telomerase inhibitory activity of the compound.
28. A compound of the formula: 43in which C is --CH.dbd.CH--,
--(CH.dbd.CH).sub.2--, --(CH.dbd.CH).sub.3--, p-phenylene,
o-phenylene, p-phenylene-CH.dbd.CH--, or o-phenylene-CH.dbd.CH--; B
is O, S, or NR, and R is Me or Et.
29. The method of claim 1, wherein the mitotic division of a cell
is inhibited.
30. The method of claim 14, wherein the mitotic division of a cell
is inhibited.
31. A compound of claim 28, having the structure: 44
32. The method of claim 14, having the structure: 45
33. A compound of claim 13, having the formula: 46
34. The method of claim 1, having the formula: 47
35. The compound of claim 13, having the formula: 48
36. The method of claim 1, having the structure: 49
Description
[0001] The present application claims the priority of co-pending
U.S. Provisional Patent Application Ser. No. 60/073,629, filed Feb.
4, 1998, the entire disclosure of which is incorporated herein by
reference without disclaimer. The government may own rights in the
present invention pursuant to contract number U19CA-67760-O.sub.2,
and contract number NCDDG, CA67760 from the National Cancer
Institute, and contract number CA49751 and contract number CA77000
from the National Institutes of Health.
BACKGROUND OF THE INVENTION
[0002] I. Field of the Invention
[0003] This invention relates to the field of cancer therapy. The
invention also relates to screening methods for identifying
pharmacologically active compounds that may be useful for treating
proliferative diseases. More particularly, the inventors have
identified non-nucleoside molecule compounds that interact with
specific DNA structures and which inhibit human telomerase.
[0004] II. Description of Related Art
[0005] Cancer, which is a cell proliferative disorder, is one of
the leading causes of disease, being responsible for 526,000 deaths
in the United States each year (Boring et al., 1993). For example,
breast cancer is the most common form of malignant disease among
women in Western countries and, in the United States, is the most
common cause of death among women between 40 and 55 years of age
(Forrest, 1990). The incidence of breast cancer is increasing,
especially in older women, but the cause of this increase is
unknown. Malignant melanoma is another form of cancer whose
incidence is increasing at a frightening rate, at least sixfold in
the United States since 1945, and is the single most deadly of all
skin diseases (Fitzpatrick, 1986).
[0006] One of the devastating aspects of cancer is the propensity
of cells from malignant neoplasms which disseminate from their
primary site to distant organs and develop into metastatic cancers.
Animal tests indicate that about 0.01% of circulating cancer cells
from solid tumors establish successful metastatic colonies (Fidler,
1993). Despite advances in surgical treatment of primary neoplasms
and aggressive therapies, most cancer patients die as a result of
metastatic disease. Hence, there is a need for new and more
efficacious cures for cancer.
[0007] The ends of chromosomes have specialized sequences, termed
telomeres, comprising tandem repeats of simple DNA sequences. Human
telomeres consist of the sequence 5'-TTAGGG (Blackburn, 1991;
Blackburn et al., 1995). Telomeres have several functions apart
from protecting the ends of chromosomes, the most important of
which appear to be associated with senescence, replication, and the
cell cycle clock (Counter et al., 1992). Progressive rounds of cell
division result in a shortening of the telomeres by some 50-200
nucleotides per round. Almost all tumor cells have shortened
telomeres, which are maintained at a constant length (Allshire et
al., 1988; Harley et al., 1990; Harley et al., 1994) and are
associated with chromosome instability and cell
immortalization.
[0008] The enzyme telomerase adds the telomeric repeat sequences
onto telomere ends, ensuring the net maintenance of telomere length
in tumor cells commensurate with successive rounds of cell
division. Telomerase is a DNA polymerase with an endogenous RNA
template (Feng et al., 1995), on which the nascent telomeric
repeats are synthesized. A significant recent finding has been that
approximately 85-90% of all human cancers are positive for
telomerase, both in cultured tumor cells and primary tumor tissue,
whereas most somatic cells appear to lack detectable levels of
telomerase (Kim et al., 1994; Hiyama et al., 1995a). This finding
has been extended to a wide range of human tumors (see, for
example, references Broccoli, 1994 and Hiyama et al., 1995b) and is
likely to be of use in diagnosis.
[0009] Human telomerase has been proposed as a novel and
potentially highly selective target for antitumor drug design (Feng
et al., 1995; Rhyu et al., 1995; Parkinson, 1996). Studies with
antisense constructs against telomerase RNA in HeLa cells show that
telomere shortening is produced, together with the death of these
otherwise immortal cells (Feng et al., 1995). Sequence-specific
peptide-nucleic acids directed against telomerase RNA have also
been found to exert an inhibitory effect on the enzyme (Norton et
al., 1996).
[0010] Among chemical agents, 2,6-diamido-anthraquinones have been
reported as DNA-interactive agents (Collier and Neidle, 1988; 1992;
Agbandje et al., 1992). These compounds have been shown to act as
selective DNA triplex interactive compounds (Fox et al., 1995; Haq
et al., 1996), with reduced affinity for duplex DNA and only
moderate conventional cytotoxicity in a range of tumor cell lines.
A carbocyanine dye, 3,3'-diethyloxadicarbocyanine (DODC,), has been
reported to bind dimeric hairpin G-quadruplex structures (Chen et
al., 1996).
[0011] This invention describes a novel class of non-nucleoside
molecules that are telomerase inhibitors. These compounds have
demonstrated their ability to interact with telomeres which form
structures called the G-quadruplex structures. As telomeres are
involved in controlling the cell cycle, cell replication and aging,
these inhibitors of telomerase prevent uncontrolled cell growth and
the immortality of tumor cells.
SUMMARY OF THE INVENTION
[0012] The present invention has demonstrated for the first time
that a non-nucleoside, small molecule can target the G-quadruplex
structure and can act as a telomerase inhibitor. Accordingly,
methods have been developed that identify these classes of
compounds and several inhibitors identified.
[0013] Compounds such as those described here, which interact
selectively with G-quadruplex structures and inhibit telomerase,
are expected to be useful as inhibitors of the proliferation of
cells that require telomerase to maintain telomere length for
continued growth. The invention thus relates to novel methods for
identifying compounds that will be useful in this regard, and also
includes new classes of telomerase inhibitors. In this regard,
several perylene compounds and carbocyanines have been shown to
interact with G-quadruplex structures. Since telomerase appears to
be found almost exclusively in tumor cells, these agents are
contemplated to be useful as antitumor agents.
[0014] In one aspect of the invention, compounds that act as
telomerase inhibitors have been identified. It has been found that
compounds that bind to the human G-quadruplex structure inhibit the
human telomerase. The identification of such G-quadruplex
interactive agents is an efficient approach for identifying human
telomerase inhibitors. Methods for identifying these G-quadruplex
interactive agents include identifying compounds whose
three-dimensional structure is complementary to that of the
G-quadruplex structure. Another method for identifying G-quadruplex
interactive compounds is to identify compounds that interact with
G-quadruplexes using such methods as dye displacement or melting
points of G-quadruplex/compound hybrids.
[0015] More particularly, candidate compounds that inhibit
telomerase activity are identified by first obtaining the
three-dimensional structure of a compound that might interact with
the G-quadruplex.selected compound. The complementarity of the
compound to human telomere DNA G-quadruplex is then determined. If
there is a high degree of complementarity, telomerase inhibition
activity is indicated.
[0016] Alternatively, one can contact a telomerase inhibitor
candidate compound with human DNA G-quadruplex; and then determine
the melting point of the human DNA G-quadruplex. The inventors have
found that an increase in melting point of the quadruplex indicates
telomerase inhibitory activity of the compound.
[0017] Additionally, telomerase inhibitors may be identified by
first preparing a DNA G-quadruplex/dye complex with a dye
intercalated into the G-quadruplex; then contacting complex with a
telomerase inhibitor candidate. Displacement of the dye in the
complex identifies the candidate as a telomerase inhibitor.
[0018] Yet another aspect of this invention is to provide
non-nucleoside inhibitors of telomerase. Using the disclosed
screening methods, compounds have been identified that bind to
human G-quadruplex structures. The invention includes perylene
compounds, exemplified by
N,N'-bis(2-dimethylaminoethyl)-3,4,9,10-perylenetetracarboxylic
acid diimide that are useful telomerase inhibitors. Novel compounds
such as N,N'-bis(2-piperdinoethyl)-3,4,9,10-perylenetetracarboxylic
acid diimide are also within the scope of the invention.
[0019] A preferred G-quadruplex structure is formed from the
sequence d(AGGGTTAGGGTTAGGGTTAGGG) or the sequences
d(TTAGGG).sub.4, d(TAAGGGT).sub.4, or d(TTAGGGTT).sub.4 either
alone or in the presence of a G-quadruplex interactive perylene
diimide of general structure I. The structures were determined by
NMR spectroscopy. Alternatively, one may determine the
three-dimensional structure of potential G-quadruplex interactive
agents by x-ray diffraction or molecular mechanics calculations.
Preferred programs for determining the degree of complementarity
between the potential G-quadruplex interactive agent and these
G-quadruplex structures include DOCK, autoDOCK, AMBER and SYBYL.
The preferred methods for generating orientations between the
potential G-quadruplex interactive agents and these G-quadruplex
structures are manual and using the DOCK or autoDOCK programs. The
cutoff for determining the likelihood that the orientation of the
potential G-quadruplex interactive agent and the G-quadruplex
structure have sufficient chemical interaction to form a complex is
roughly 75% of the favorable intermolecular interaction energy,
calculated with the above programs, of the perylene diimide
2-d(TTAGGG).sub.4 complex structure as determined by NMR
spectroscopy.
[0020] Preferred G-quadruplex structures are those formed by the
sequences d(TTAGGG).sub.4, d(AATGGGT).sub.4 and d(TTAGGGTT).sub.4.
Several methods of determining the interaction of potential
G-quadruplex interactive agents with these structures include
UV/VIS spectroscopy, in which the changes in the UV/VIS spectrum of
the potential agent under more than a 10% change at the wavelength
due solely to the ligand and which undergoes the most change, upon
addition of an excess of the G-quadruplex structure; UV
spectroscopy, in which the melting temperature of the G-quadruplex
structure as determined by a hyperchromicity transition at a given
temperature range is increased by >5.degree. C. upon addition of
an excess of the agent; UV/VIS spectroscopy in which addition of a
potential G-quadruplex interactive agent to a complex of a
G-quadruplex-interactive perylene diimide and a G-quadruplex
produces a >25% change in the absorption of due to the
G-quadruplex-interactive perylene diimine-G-quadruplex complex;
UV/VIS spectroscopy in which addition of a potential G-quadruplex
interactive agent to a complex of a G-quadruplex-interactive
carbocyanine and a G-quadruplex produces a >25% change in the
absorption of due to the G-quadruplex-interactive
carbocyanine-G-quadruplex complex; NMR spectroscopy in which the
melting temperature of the G-quadruplex as determined by the
disappearance of the imino proton signals of the G-quadruplex is
increase by >5.degree. C. in the presence of one- to
two-equivalents of the agent; NMR spectroscopy in which the
interaction of the agent with the G-quadruplex structure is
determined by the shift of at least one of the imino protons of the
G-quadruplex by >0.01 ppm upon addition of one- to
two-equivalents of the agent; fluorescence spectroscopy in which
the fluorescence emission spectrum of the agent undergoes a shift
of >5 nm and/or a change in intensity of >25% upon the
addition of an excess of the G-quadruplex structure; fluorescence
spectroscopy in which the fluorescence emission spectrum of a
G-quadruplex-interactive perylene diimide-G-quadruplex complex
undergoes a >25% change upon the addition of an excess of the
agent; or fluorescence spectroscopy in which the fluorescence
emission spectrum of a G-quadruplex-interactive
carbocyanine-G-quadruplex complex undergoes a >25% change upon
the addition of an excess of the agent.
[0021] The preferred embodiments of the invention as it related to
one class of G-quadruplex interactive telomerase inhibitors are
compounds of the structure I in which 1
[0022] in which R.sup.1 and R.sup.4 are independently taken from
the set of sub-structures given by the formula -L-A in which L is a
linking group taken from the set consisting of: 2
[0023] where n is 1, 2, or 3; and each R5 is independently taken
from the set H, Me, OH, or OMe; 3
[0024] where R5 is as before and Y is taken from the set O, S, SO,
SO2, NH, NMe, NCOMe; 4
[0025] where R5 and Y are as before and X is taken from the set
CH2, O, S, SO, SO2, NH, NMe, NCOMe; 5
[0026] where R6, R7, R8, and R9 are independently taken from the
set consisting of H, OMe, OEt, halo, or Me;
[0027] or a bond;
[0028] and A is taken from the set consisting of: 6
[0029] where m is 0-5 and each R6 is taken from the set consisting
of halo, NH2, NO2, CN, OMe, SO2NH2, amidino, guanidino, or Me;
7
[0030] where o is 0 or 1; p is 0, 1, or 2; q is 1 or 2such that o+q
is either 2, in which case a pyrrolidine or pyrrole ring is
indicated, or 3, in which a piperidine or pyridine ring is
indicated; r is 0, 1, 2, or 3; R7 is H or Me; each R8 is
independently taken from the set consisting of Me, NO2, OH, CH2OH,
halo, or when r is 2 or 3, two adjacent R8 substituents may be
together taken as --(CH.dbd.CH)2- or --(CH2)4- such as to form an
annulated six-membered ring; 8
[0031] where each R9 is independently taken from the set consisting
of H, Me, or both R9 can taken together be .dbd.O; s is equal to 0
or 1; and Z is taken from the set consisting of CH2, 0, NH, NMe,
NEt, N(Me).sub.2, N(Et)2, or NCO2Et; 9
[0032] where Q is either N, CH, NMe, or NEt; X is either O, S, NH,
NMe or NEt; R10 and R11 are independently taken from the set
consisting of H, Me, CH2CO2Et, or R10 and R11 taken together
consist of --(CH.dbd.CH)2- or --(CH2)4-; 10
[0033] where t is equal to 1, 2, 3, or 4; u is equal to 0, 1, 2, 3,
or 4, and each R12 is individually taken from the set consisting of
Me, or OH; 11
[0034] OH, CO2R.sup.13, CON(R.sup.13).sub.2, SO3H,
SO2N(R.sup.13).sub.2, CN, CH(CO2R.sup.13).sub.2,
CH(CON(R.sup.13).sub.2).sub.2, N(R.sup.13).sub.2, or
N(R.sup.13).sub.3 where R13 is either H, Me, Et, or
CH2CH.sub.2OH;
[0035] R2, R2', R2", R2"; R3, R3', R3", R3'" are each independently
taken from the set H, OMe, halo, or NO2.
[0036] In addition, this invention includes the development of
other G-quadruplex interactive telomerase inhibitors compounds
derived from structure I, having the following structures: 12
[0037] The preferred embodiment of the invention as it relates to
another class of G-quadruplex interactive telomerase inhibitors are
compounds of the general structure II: 13
[0038] in which C is either a bond, --CH.dbd.CH--, --(CH.dbd.CH)2-,
--(CH.dbd.CH)3-, p-phenylene, o-phenylene, p-phenylene-CH.dbd.CH--,
or o-phenylene-CH.dbd.CH--; B is O, S, or NR, and R is either Me or
Et.
[0039] In addition, this invention includes the development of
another G-quadruplex interactive telomerase inhibitor compound
derived from structure II, having the following structure: 14
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0041] FIG. 1. Effect of increasing concentrations of
N,N'-bis(2-dimethylaminoethyl)-3,4,9,10-perylenetetracarboxylic
acid diimide on inhibition of telomerase catalyzed extension of an
18-mer primer d[TTAGGG].sub.3 (1 .mu.M). Elongated primer was
labeled with 1.5 .mu.M of [.alpha.-.sup.32P]-dGTP (800 Ci
mmol.sup.-1, 10 mCi ml.sup.-1) with 1 mM DATP and dTTP using a
standard telomerase assay. Lanes 1-5 are 0, 10, 50, and 100 .mu.M
of N,N'-bis(2-dimethylaminoethyl)-3,4,9,10-peryl-
enetetracarboxylic acid diimide.
[0042] FIG. 2. Changes in the UV/VIS absorbance spectrum of
N,N'-bis(2-dimethylaminoethyl)-3,4,9,10-perylenetetracarboxylic
acid diimide upon addition of increasing amount of
[d(TTAGGGT)].sub.4, an oligodeoxyribonucleotide which adopts a
G-quadruplex structure.
[0043] FIG. 3. Titration of [d(TTAGGG)].sub.4 with
N,N'-bis(2-piperdinoeth- yl)-3,4,9,10-perylenetetracarboxylic acid
diimide. Imino proton region of the 500-MHz .sup.1H NMR is shown
with increasing amounts of added ligand. The resonances labeled
G4*, G5*, and G6* represent resonances of final 2:1
ligand/G-quadruplex complexes.
[0044] FIG. 4. NMR-based model of
[d(TTAGGG)].sub.4--N,N'-bis(2-piperdinoe-
thyl)-3,4,9,10-perylenetetracarboxylic acid diimide complex. The
ligand is stacked under the G6 guanine tetrad with positively
charged side chains located in the grooves.
[0045] FIG. 5. Shows the design of an intramolecular quadruplex DNA
that contains the human telomere repeats.
[0046] FIG. 6. Photocleavage of G4A DNA by TMPyP.sup.4 in K.sup.+
buffer.
[0047] FIG. 7. Model depicting G-quadruplex structure blocking
primer extension by DNA polymerase.
[0048] FIG. 8. Primer extension of PQ sequence in the presence of
compounds. Lane 1: water control; Lane 2: 50 mM K+; Lanes 3-6;
QQ23; Lanes 7-10; QQ30; Lanes 11-14; QQ31; Lanes 15-18; APPER
(Bis[1-(2-aminoethyl)piperdine]-3,4,9,10-perylenetetracarboxylic
diimide); Lanes 3,7,11, and 15; 0.5 .mu.M; Lanes 4,6,12, and 16; 1
.mu.M; Lanes 5,9,13 and 17; 10 .mu.M; Lanes 6,10, 14 and 18; 50
.mu.M.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0049] I. The Present Invention
[0050] A structure-based approach to discovering non-nucleoside
compounds that will selectively inhibit human telomerase by
targeting the nucleic acid structures, such as G-quadruplexes, that
may be associated with human telomeres or telomerase has been
utilized. Inhibition of human telomerase by a 2,6-diamido
anthraquinone has been successfully demonstrated. .sup.1H-NMR has
demonstrated the stabilization of a G-quadruplex by this compound
and evidence has been provided that this compound inhibits the
telomerase enzyme by a mechanism consistent with interaction with
G-quadruplex structures. The present work shows that
non-nucleoside, small molecules can interact with G-quadruplexes
and inhibit telomerase.
[0051] Using the methods described, it was found that compounds
that bind to the human G-quadruplex structure inhibit the human
telomerase. The identification of such G-quadruplex interactive
agents is a novel and efficient approach for identifying human
telomerase inhibitors.
[0052] It is envisioned that the telomerase inhibitors will provide
therapy for tumors and cancers including skin cancers, connective
tissue cancers, adipose cancers, breast cancers, lung cancers,
stomach cancers, pancreatic cancers, ovarian cancers, cervical
cancers, uterine cancers, anogenital cancers, kidney cancers,
bladder cancers, colon cancers, prostate cancers, central nervous
system (CNS) cancers, retinal cancer, blood, lymphoid cancers and
the like.
[0053] II. Telomerase
[0054] Telomerase is a ribonucleoprotein enzyme that synthesizes
one strand of the telomeric DNA using as a template a sequence
contained within the RNA component of the enzyme. The ends of
chromosomes have specialized sequences, termed telomeres,
comprising tandem repeats of simple DNA sequences which in humans
is 5'-TTAGGG (Blackburn, 1991; Blackburn et al., 1995). Apart from
protecting ends of chromosomes telomeres have several other
functions, the most important of which appear to be associated with
replication, regulating the cell cycle clock and ageing (Counter et
al., 1992). Progressive rounds of cell division shorten telomeres
by 50-200 nucleotides per round. Almost all tumor cells have
shortened telomeres, which are maintained at a constant length
(Allshire et al., 1988; Harley et al., 1990; Harley et al., 1994)
and are associated with chromosome instability and cell
immortalization.
[0055] With regard to human cells and tissues telomerase activity
has been identified in immortal cell lines and in ovarian carcinoma
but has not been detected at biologically significant levels (that
are required to maintain telomere length over many cell divisions)
in mortal cell strains or in normal non-germline tissues (Counter
et al., 1992; Counter et al, 1994). These observations suggest
telomerase activity is directly involved in telomere maintenance,
linking this enzyme to cell immortality.
[0056] As described above, the immortalization of cells involves
the activation of telomerase. More specifically, the connection
between telomerase activity and the ability of many tumor cell
lines, including skin, connective tissue, adipose, breast, lung,
stomach, pancreas, ovary, cervix, uterus, kidney, bladder, colon,
prostate, central nervous system (CNS), retina and blood tumor cell
lines, to remain immortal has been demonstrated by analysis of
telomerase activity (Kim, et al., 1994). This analysis,
supplemented by data that indicates that the shortening of telomere
length can provide the signal for replicative senescence in normal
cells, see PCT Application No. 93/23572, incorporated herein by
reference, demonstrates that inhibition of telomerase activity can
be an effective anti-cancer therapy. Thus, telomerase activity can
prevent the onset of otherwise normal replicative senescence by
preventing the normal reduction of telomere length and the
concurrent cessation of cell replication that occurs in normal
somatic cells after many cell divisions. In cancer cells, where the
malignant phenotype is due to loss of cell cycle or growth controls
or other genetic damage, an absence of telomerase activity permits
the loss of telomeric DNA during cell division, resulting in
chromosomal rearrangements and aberrations that lead ultimately to
cell death. However, in cancer cells having telomerase activity,
telomeric DNA is not lost during cell division, thereby allowing
the cancer cells to become immortal, leading to a terminal
prognosis for the patient.
[0057] Methods for detecting telomerase activity, as well as for
identifying compounds that regulate or affect telomerase activity,
together with methods for therapy and diagnosis of cellular
senescence and immortalization by controlling telomere length and
telomerase activity, have also been described elsewhere.
[0058] A. G-Quadruplex Structures
[0059] Human telomeres form structures known as G-quadruplexes.
Human telomeres contain numerous repeats of the sequence TTAGGG,
exhibiting an enhancement of G and T residues and a paucity of A
residues. Intramolecular G-quadruplex DNA may be designed by
generating a sequence of human telomere repeats (FIG. 5). The G
tetrad consists of four G bases hydrogen bonded in Hoogsteen
fashion symmetrically disposed about a central axis, as shown in
FIG. 5.
[0060] G-rich DNA is known to assume highly stable structures
formed by Hoogsteen base pairs between guanine residues
(Williamson, 1994; Nadel et al., 1995). These structures, known as
G-quadruplexes, are stabilized in the presence of K.sup.+ and may
have biological roles that are yet to be determined (Henderson et
al., 1987; Hardin et al., 1997; Williamson et al., 1989). One
particular region of the genome where these structures may play a
significant biological role is at the ends of chromosomes where
G-rich DNA is normally found (e.g., TTAGGG and TTGGGG tandem
repeats in human cells and ciliate Tetrahymena, respectively)
(Henderson et al., 1987; Blackburn and Greider, 1995; Sundquist and
Heaphy, 1993). In addition, a number of genes containing G-rich DNA
have been identified recently, and it has been proposed that the
G-rich regions within these genes may regulate gene expression by
forming G-quadruplex structures (Sen and Gilbert, 1988;
Hommond-Kosack et al., 1993; Murchie and Lilley, 1992; Simonsson et
al., 1998). One potential biologically relevant role of
G-quadruplex DNA is as a barrier to DNA synthesis (Howell et al.,
1996). This barrier has been thoroughly investigated and has been
found to be K.sup.+ dependent (Woodword et al., 1994). This
observation strongly suggests that the formation of G-quadruplex
species is responsible for the observed effect on DNA synthesis
(Weitzmann et al., 1996).
[0061] The inventors have shown that the 2,6-iamidoanthraquinone
BSU-1051 modulates human telomerase activity by a mechanism that is
dependent on the elongation of the telomeric primer d(TTAGGG).sub.3
to a length that is then capable of forming an intramolecular
G-quadruplex structure (Sun et al., 1997). The inventors have also
shown that BSU-1051, by virtue of its interaction with G-quadruplex
DNA, enhances the block of DNA synthesis by the G-quadruplex
structure in the presence of K.sup.+.
[0062] B. Methods for Identifying G-quadruplex Interactive
Agents
[0063] Several methods for identifying classes of G-quadruplex
interactive agents may be employed. One method involves identifying
compounds whose three-dimensional structure is complementary to
that of the G-quadruplex structure. G-quadruplex structure is
understood to mean at least in one sense the structure of the
G-quadruplex that is formed by the single-stranded DNA
corresponding to at least four repeats of the telomeric sequence.
In humans, the telomeric sequence is d(TTAGGG). Thus, the
G-quadruplex structure of interest for the identification of human
telomerase inhibitors may be any sequence of the form
{d([N.sub.1]TAGGG[N.sub.2])}.sub.4 where [N.sub.1] is zero to two
bases corresponding to the human telomeric sequence; for example,
[N.sub.1] may equal G, GG, or may be absent; where [N.sub.2] is
zero to three bases corresponding to the human telomeric sequence;
for example, [N.sub.2] can equal T, TT, TTA or it may be
absent.
[0064] Alternatively, G-quadruplex structure is understood to mean
the fold-over or intramolecular G-quadruplex formed from at least
four repeats of the G-triad of telomeric sequence. Thus, the
G-quadruplex structure of interest for the identification of human
telomerase inhibitors may be any sequence of the form
d([N.sub.3][TTAGGG].sub.3[N.su- b.2]) where [N.sub.2] is as defined
above and [N3] is three G's preceded by zero to three nucleotides
corresponding to the human telomeric sequence. These structures may
be determined by a variety of techniques including molecular
mechanics calculations, molecular dynamics calculations,
constrained molecular dynamics calculations in which the
constraints are determined by NMR spectroscopy, distance geometry
in which the distance matrix is partially determined by NMR
spectroscopy, x-ray diffraction, or neutron diffraction techniques.
In the case of all these techniques, the structure can be
determined in the presence or absence of any ligands known to
interact with G-quadruplex structures, including but not limited to
potassium and other metal ions, 2,6-diamidoanthraquinones, perylene
diimides, or carbocyanines.
[0065] Complementary is understood to mean the existence of a
chemical attraction between the G-quadruplex interactive agent and
the G-quadruplex. The chemical interaction may be due to one or a
variety of favorable interactions, including ionic, ion-dipole,
dipole-dipole, van der Waals, charge-transfer, and hydrophobic
interactions. Each of these type of interactions, alone or
together, may be determined by existing computer programs using as
inputs the structure of the compound, the structure of the
G-quadruplex, and the relative orientation of the two. Such
computer programs include but are not limited to AMBER, CHARMM,
MM2, SYBYL, CHEMX, MACROMODEL, GRID, and BioSym. Such programs are
contemplated as being useful for the determination of the chemical
interaction between two molecules, either isolated, or surrounded
by solvent molecules, such as water molecules, or using
calculational techniques that approximate the effect of solvating
the interacting molecules. The relative orientation of the two can
be determined manually, by visual inspection, or by using other
computer programs which generate a large number of possible
orientations.
[0066] Examples of computer programs include but are not limited to
DOCK and AutoDOCK. Each orientation can be tested for its degree of
complementarity using the computer programs. An advantage of this
method is that it does not require availability of physical samples
of the compounds, only that their three-dimensional structure is
known. It thus can be used to design novel compounds that possess
the desired ability to inhibit telomerase.
[0067] Alternatively, this method may be used as a screening method
for identifying telomerase inhibitors from a collection of
compounds that are available, provided that the structure of these
compounds is known. If only the two-dimensional structure is known,
the corresponding three-dimensional structure can be obtained using
existing computer programs. Such computer programs include but are
not limited to CONCORD, CHEM3D, and MM2.
[0068] Another method for identifying G-quadruplex interactive
compounds that may inhibit telomerase involves use of techniques
such as UV/VIS spectroscopy, polarimetry, CD or ORD spectroscopy,
IR or Raman spectroscopy, NMR spectroscopy, fluorescence
spectroscopy, HPLC, gel electrophoresis, capillary gel
electrophoresis, dialysis, refractometry, conductometry, atomic
force microscopy, polarography, dielectometry, calorimetry,
solubility, EPR or mass spectroscopy. The application of these
methods can be direct, in which the G-quadruplex interactive
compound's interaction with the G-quadruplex is measured directly,
or it can be indirect, in which a particular G-quadruplex
interactive agent having a useful spectroscopic property is used as
a probe for the ability of other compounds to bind to the
G-quadruplex; for example, by displacement or by fluorescence
quenching.
[0069] III. Telomerase Inhibitors
[0070] The identification of compounds that inhibit telomerase
activity provides important benefits to efforts at treating human
disease. Compounds that inhibit telomerase activity can be used to
treat cancer, as cancer cells express telomerase activity and
normal human somatic cells do not express telomerase activity at
biologically relevant levels (i.e. at levels sufficient to maintain
telomere length over many cell divisions). Unfortunately, few such
compounds have been identified and characterized. Hence, there
remains a need for compounds that act as telomerase inhibitors and
for compositions and methods for treating cancer and other diseases
in telomerase activity is present abnormally. The present invention
meets these and other needs.
[0071] Once a compound has been identified as being a G-quadruplex
interactive agent, confirmatory evidence for the ability of said
compound to inhibit telomerase may be obtained using a standard
primer extension assay that does not use a PCR.TM.-based
amplification of the telomerase primer extension products such as
described in Sun et al., 1997. The identified inhibitors may be
used therapeutically to interfere with the function of telomerase
and thus to treat cancers.
[0072] Using the screening methods described above, compounds have
been identified that bind to human G-quadruplex structures and have
been shown to inhibit human telomerase. One group of compounds is
represented by general structure I: 15
[0073] in which R.sup.1 and R.sup.4 are represented by L-A where L
is a linking group which may be any of a group of substituted
(X)methylene, (X)dimethylene, (X)trimethylene, (X)dimethyleneamine,
(X)dimethyleneoxy, (X)dimethyleneaminodimethylene,
(X)-dimethyleneoxydimethylene, (X)-p-phenylene, (X)-m-phenylene,
(X)-o-phenylene, or an unsubstituted covalent bond;
[0074] A is a group that interacts with the grooves of the
G-quadruplex structure, examples being a substituted carbocyclic
ring, a (substituted) heterocyclic ring, an hydroxyl, a carboxylic
acid, a carboxylic acid ester, a carboxamide, a sulfonamide, a
sulfonic acid, a nitrile, a malonate diester, a malonate diamide, a
disubstituted amine, a quartemized nitrogen-containing
heterocyclce, or a quaternary amine.
[0075] R2, R2', R2", R2'", R3, R3', R", R'" are independently
hydrogen, alkyl, halo, amino, nitro, hydroxy, alkoxy, alkylamino,
dialkylamino, aryl, or cyano.
[0076] Another group of compounds suitable as telomerase inhibitors
is shown by the general structure II in which B is O, S, or NR; C
is an unsaturated linking group, 1 to 3 (substituted) ethylene
groups, a substituted or unsubstituted carbocyclic group, or a
heterocyclic group; and R is lower alkyl. 16
[0077] These compounds may interact specifically with G-quadruplex
structures as compared to other nucleic acid structures such as
double-stranded DNA, single-stranded DNA, and RNA structures. The
degree of selectivity in the interaction of these compounds with
G-quadruplex structures versus other nucleic acid structures is
given by the ratio of the affinity of these compounds for
G-qudruplex structures to the affinity for the other nucleic acid
structures. In particular, the ability of these compounds to
distinguish between G-quadruplex structures and double-stranded DNA
may be an important criterion. Compounds with general ability to
bind double-stranded DNA are known to inhibit or alter a variety of
DNA-associated enzymes or proteins, including but not limited to:
histone binding, topoisomerase I, topoisomerase II,
DNA-polymerases, RNA-polymerases, DNA repair enzymes, cytosine
methyltransferase, and transcription factor binding. In order to
discover compounds that are able to selectively inhibit telomerase
and other G-qudruplex-associated enzymes and proteins, one would
want to select compounds that have a high ratio of affinities for
G-qudruplex structures versus double-stranded DNA. These selective
G-quadruplex-binding compounds can be identified by selecting those
G-quadruplex binding compounds that display weak or no ability for
binding to double-stranded DNA, as determined by UV/VIS
spectroscopy, polarimetry, CD or ORD spectroscopy, IR or Raman
spectroscopy, NMR spectroscopy, fluorescence spectroscopy, HPLC,
gel electrophoresis, capillary gel electrophoresis, dialysis,
refractometry, conductometry, atomic force microscopy,
polarography, dielectometry, calorimetry, solubility, EPR and mass
spectroscopy.
[0078] In addition to the thermodynamic considerations of
G-quadruplex binding by these compounds, the kinetics of the
interaction between these compounds and G-quadruplexes is also
considered to be important. The relative rates of the association
and dissociation of these compounds with G-quadruplex structures
can affect their biological properties. In particular, those
compounds with slow dissociation rates may be more effective in
inhibiting telomerase and other G-quadruplex-associated enzymes and
proteins than hose with identical G-quadruplex binding ability, but
whose dissociation rates are faster. For a given compound, the
overall equilibrium binding affinity (binding constant) to
G-quadruplex structures is a ratio of the association rate and the
dissociation rate. The dissociation rate of a complex consisting of
a G-quadruplex structure and a G-quadruplex interactive compound
can be determined by a variety of methods. In one example, the
dissociation of the complex can be determined
spectrophotometrically upon the addition of a detergent, such as
SDS. Alternatively, the dissociation rate can be determined in a
T-jump study, in which the temperature of the complex is quickly
raised to a point at which the complex dissociates and this process
is monitored spectrophotometrically. In another example the
dissociation rate for a G-quadruplex-compound complex can be
determined by monitoring by a variety of techniques the
dissociation of a complex in which one partner, either the
G-quadruplex or the compound, is tethered, either covalently or
non-covalently, to an immobile phase, and a solution is passed over
this immobilized complex. The dissociation rate for a
G-quadruplex-compound complex also can be determined indirectly, by
measuring both the equilibrium binding constant and the association
rate. For example, if two compounds have similar equilibrium
G-quadruplex binding constants, but one has a slower association
rate, then that same compound must also have a proportionately slow
dissociation rate.
[0079] Using the above techniques, one can select from those
G-quadruplex interactive agents identified, those that have
additional desired properties of selective G-quadruplex interaction
when compared to other nucleic acids structures, such as
double-stranded DNA, and/or slow kinetics of association with
G-quadruplex structures.
[0080] Agents capable of inhibiting telomerase activity in tumor
cells offer therapeutic benefits with respect to a wide variety of
cancers and other conditions (for example, fungal infections) in
which immortalized cells telomerase activity are a factor in
disease progression or in which inhibition of telomerase activity
is desired for treatment purposes. The telomerase inhibitors of the
invention can also be used to inhibit telomerase activity in germ
line cells, which may be useful for contraceptive purposes.
[0081] IV. Pharmaceutical Formulations and Administration
[0082] The invention further comprises the therapeutic treatment of
cancer by the administration of an effective dose of one or more
inhibitors of telomerase. Where clinical applications are
contemplated, it will be necessary to prepare pharmaceutical
compositions of drugs in a form appropriate for the intended
application. Generally, this will entail preparing compositions
that are essentially free of pyrogens, as well as other impurities
that could be harmful to humans or animals.
[0083] The phrase "pharmaceutically or pharmacologically
acceptable" refer to molecular entities and compositions that do
not produce adverse, allergic, or other untoward reactions when
administered to an animal or a human. As used herein,
"pharmaceutically acceptable carrier" includes any and all
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents and the like. The
use of such media and agents for pharmaceutically active substances
is well know in the art. Supplementary active ingredients also can
be incorporated into the compositions.
[0084] The active compositions of the present invention may include
classic pharmaceutical preparations. Administration of these
compositions according to the present invention will be via any
common route so long as the target tissue is available via that
route. This includes oral, nasal, buccal, rectal, vaginal or
topical. Alternatively, administration may be by orthotopic,
intradermal, subcutaneous, intramuscular, intraperitoneal or
intravenous injection. Such compositions would normally be
administered as pharmaceutically acceptable compositions, described
supra. A preferred route is direct intra-tumoral injection,
injection into the tumor vasculature or local or regional
administration relative to the tumor site.
[0085] The active compounds may also be administered parenterally
or intraperitoneally.
[0086] Solutions of the active compounds as free base or
pharmacologically acceptable salts can be prepared in water
suitably mixed with a surfactant, such as hydroxypropylcellulose.
Dispersions can also be prepared in glycerol, liquid polyethylene
glycols, and mixtures thereof and in oils. Under ordinary
conditions of storage and use, these preparations contain a
preservative to prevent the growth of microorganisms.
[0087] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions and sterile powders for
the extemporaneous preparation of sterile injectable solutions or
dispersions. In all cases the form must be sterile and must be
fluid to the extent that easy syringability exists. It must be
stable under the conditions of manufacture and storage and must be
preserved against the contaminating action of microorganisms, such
as bacteria and fungi. The carrier can be a solvent or dispersion
medium containing, for example, water, ethanol, polyol (for
example, glycerol, propylene glycol, and liquid polyethylene
glycol, and the like), suitable mixtures thereof, and vegetable
oils. The proper fluidity can be maintained, for example, by the
use of a coating, such as lecithin, by the maintenance of the
required particle size in the case of dispersion and by the use of
surfactants. The prevention of the action of microorganisms can be
brought about by various antibacterial an antifingal agents, for
example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal,
and the like. In many cases, it will be preferable to include
isotonic agents, for example, sugars or sodium chloride. Prolonged
absorption of the injectable compositions can be brought about by
the use in the compositions of agents delaying absorption, for
example, aluminum monostearate and gelatin.
[0088] Sterile injectable solutions are prepared by incorporating
the active compounds in the required amount in the appropriate
solvent with various of the other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and freeze-drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0089] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents and the like. The use of such media and agents for
pharmaceutical active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
active ingredient, its use in the therapeutic compositions is
contemplated. Supplementary active ingredients can also be
incorporated into the compositions.
[0090] For oral administration the compounds developed in the
present invention may be incorporated with excipients and used in
the form of non-ingestible mouthwashes and dentifrices. A mouthwash
may be prepared incorporating the active ingredient in the required
amount in an appropriate solvent, such as a sodium borate solution
(Dobell's Solution). Alternatively, the active ingredient may be
incorporated into an antiseptic wash containing sodium borate,
glycerin and potassium bicarbonate. The active ingredient may also
be dispersed in dentifrices, including: gels, pastes, powders and
slurries. The active ingredient may be added in a therapeutically
effective amount to a paste dentifrice that may include water,
binders, abrasives, flavoring agents, foaming agents, and
humectants.
[0091] The compositions of the present invention may be formulated
in a neutral or salt form. Pharmaceutically-acceptable salts
include the acid addition salts (formed with the free amino groups
of the protein) and which are formed with inorganic acids such as,
for example, hydrochloric or phosphoric acids, or such organic
acids as acetic, oxalic, tartaric, mandelic, and the like. Salts
formed with the free carboxyl groups can also be derived from
inorganic bases such as, for example, sodium, potassium, ammonium,
calcium, or ferric hydroxides, and such organic bases as
isopropylamine, trimethylamine, histidine, procaine and the
like.
[0092] Upon formulation, solutions will be administered in a manner
compatible with the dosage formulation and in such amount as is
therapeutically effective. The formulations are easily administered
in a variety of dosage forms such as injectable solutions, drug
release capsules and the like. For parenteral administration in an
aqueous solution, for example, the solution should be suitably
buffered if necessary and the liquid diluent first rendered
isotonic with sufficient saline or glucose. These particular
aqueous solutions are especially suitable for intravenous,
intramuscular, subcutaneous and intraperitoneal administration. In
this connection, sterile aqueous media which can be employed will
be known to those of skill in the art in light of the present
disclosure. For example, one dosage could be dissolved in 1 ml of
isotonic NaCl solution and either added to 1000 ml of
hypodermoclysis fluid or injected at the proposed site of infusion,
(see for example, "Remington's Pharmaceutical Sciences" 15th
Edition, pages 1035-1038 and 1570-1580). Some variation in dosage
will necessarily occur depending on the condition of the subject
being treated. The person responsible for administration will, in
any event, determine the appropriate dose for the individual
subject. Moreover, for human administration, preparations should
meet sterility, pyrogenicity, general safety and purity standards
as required by FDA Office of Biologics standards.
[0093] Because telomerase is active only in tumor, germline, and
certain stem cells of the hematopoietic system, other normal cells
are not affected by telomerase inhibition therapy Steps also can be
taken to avoid contact of telomerase inhibitor with germline or
stem cells, although this may not be essential. For instance,
because germline cells express telomerase activity, inhibition
telomerase may negatively impact spermatogenesis and sperm
viability, suggesting that telomerase inhibitors may be effective
contraceptives or sterilization agents. This contraceptive effect
may not be desired, however, by a patient receiving a telomerase
inhibitor of the invention for treatment of cancer. In such cases,
one can deliver a telomerase inhibitor of the invention in a manner
that ensures the inhibitor will only be produced during the period
of therapy, such that the negative impact on germline cells is only
transient.
[0094] V. Therapies
[0095] One of the major challenges in oncology today is the
effective treatment of a given tumor. Tumors are often resistant to
traditional therapies. Thus, a great deal of effort is being
directed at finding efficous treatment of cancer. One way of
achieving this is by combining new drugs with the traditional
therapies and is discussed below. In the context of the present
invention, it is contemplated that therapies directed against
telomerase could be used in conjunction with surgery, chemotherapy,
radiothearpy and indeed gene therapeutic intervention. It also may
prove effective to combine telomerase targeted chemotherapy with
antisense or immunotherapies directed toward tumor markers or other
oncogenes or oncoproteins.
[0096] "Effective amounts" are those amounts of a candidate
substance effective to reproducibly decrease expression of
telomerase in an assay in comparison to levels in untreated cells.
An "effective amount" also is defined as an amount that will
decrease, reduce, inhibit or otherwise abrogate the growth of a
cancer cell.
[0097] It is envisioned that the telomerase inhibitors will provide
therapy for a wide variety of tumors and cancers including skin
cancers, connective tissue cancers, adipose cancers, breast
cancers, lung cancers, stomach cancers, pancreatic cancers, ovarian
cancers, cervical cancers, uterine cancers, anogenital cancers,
kidney cancers, bladder cancers, colon cancers, prostate cancers,
central nervous system (CNS) cancers, retinal cancer, blood and
lymphoid cancers.
[0098] A. Combination Therapies
[0099] To kill cells, inhibit cell growth, inhibit metastasis,
inhibit angiogenesis or otherwise reverse or reduce the malignant
phenotype of tumor cells, the methods of standard therapy discussed
above are generally insufficient as tumors are often resistant to
several of these agents. Often combining a host of different
treatment methods prove most effective in cancer therapy. Further,
several AIDS afflicted patients have a higher risk of developing
cancers. Combination therapy in these cases is required to treat
AIDS as well as the cancer. Using the methods and compounds
developed in the present invention, one would generally contact a
"target" cell with a telomerase inhibitor and at least one other
agent. These compositions would be provided in a combined amount
effective to kill or inhibit proliferation of the cell. This
process may involve contacting the cells with the telomerase based
therapy and the other agent(s) or factor(s) at the same time. This
may also be achieved by contacting the cell with a single
composition or pharmacological formulation that includes both
agents, or by contacting the cell with two distinct compositions or
formulations, at the same time, wherein one composition includes
the telomerase based therapy and the other includes the agent.
[0100] Alternatively, the telomerase inhibitor-based treatment may
precede or follow the other agent treatment by intervals ranging
from min to wk. In embodiments where the other agent and
telomerase-based therapy are applied separately to the cell, one
would generally ensure that a significant period of time did not
expire between the time of each delivery, such that the agent and
telomerase-based treatment would still be able to exert an
advantageously combined effect on the cell. In such instances, it
is contemplated that one would contact the cell with both
modalities within about 12-24 h of each other and, more preferably,
within about 6-12 h of each other, with a delay time of only about
12 h being most preferred. In some situations, it may be desirable
to extend the time period for treatment significantly, however,
where several days (2, 3, 4, 5, 6 or 7) to several wk (1, 2, 3, 4,
5, 6, 7 or 8) lapse between the respective administrations.
[0101] It also is conceivable that more than one administration of
either telomerase-based treatment or the other agent will be
desired. Various combinations may be employed, where
telomerase-basedtreatment is "A" and the other agent is "B", as
exemplified below:
1 A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B A/A/B/B
A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A A/A/A/B B/A/A/A
A/B/A/A A/A/B/A A/B/B/B B/A/B/B B/B/A/B
[0102] Other combinations are also contemplated. Again, to achieve
cell killing, both agents are delivered to a cell in a combined
amount effective to kill the cell.
[0103] The invention also encompasses the use of a combination of
one or more DNA damaging agents, whether chemotherapeutic compounds
or radiotherapeutics as described in the section below, together
with the telomerase inhibitor. The invention also contemplates the
use of the telomerase inhibitors in combination with surgical
removal of tumors to treat any remaining neoplastic or metastasized
cells. Further, immunotherapy may be directed at tumor antigen
markers that are found on the surface of tumor cells. The invention
also contemplates the use of telomerase inhibitors in combination
with gene therapy, directed toward a variety of oncogenes, such as,
tumor markers, cell cycle controlling genes, described below.
[0104] The other agent may be prepared and used as a combined
therapeutic composition, or kit, by combining it with the
telomerase-inhibitor based treatment, as described above. The
skilled artisan is directed to "Remington's Pharmaceutical
Sciences" 15th Edition, chapter 33, in particular pages 624-652.
Some variation in dosage will necessarily occur depending on the
condition of the subject being treated. The person responsible for
administration will, in any event, determine the appropriate dose
for the individual subject. Moreover, for human administration,
preparations should meet sterility, pyrogenicity, general safety
and purity standards as required by FDA Office of Biologics
standards.
[0105] It is proposed that the regional delivery of non-nucleoside
inhibitors of telomerase to patients with tumors will be a very
efficient method for delivering a therapeutically effective
chemical to counteract the clinical disease. Similarly, other
chemotherapeutics, radiotherapeutics, gene therapeutic agents may
be directed to a particular, affected region of the subjects body.
Alternatively, systemic delivery of telomerase based treatment
and/or the agent may be appropriate in certain circumstances, for
example, where extensive metastasis has occurred.
[0106] It also should be pointed out that any of the standard or
other therapies may prove useful by themselves in treating a
cancer. In this regard, reference to chemotherapeutics and
non-telomerase inhibitor-based treatment in combination should also
be read as a contemplation that these approaches may be employed
separately.
[0107] When such combination therapy is employed for the treatment
of a tumor, the cytotoxic agent may be administered at a dosage
known in the art to be effective for treating the tumor. However,
the G-quadruplex interaction compounds may produce an additive or
synergistic effect with a cytotoxic agent against a particular
tumor. Thus, when such combination antitumor therapy is used, the
dosage of G-quadruplex interaction compounds administered may be
less than that administered when the cytotoxic agent is used alone.
Similarly, for patients afflicted by AIDS, AZT/protease inhibitors
will be used with G-quadruplex interaction compounds, or other
herein mentioned therapeutic agent(s). Again the dosage of
G-quadruplex interaction compounds or other conjunctively utilized
agent, may be altered to suit the AIDS treatment.
[0108] Preferably, the patient is treated with G-quadruplex
interaction compounds for about 1 to 14 days, preferably 4 to 14
days, prior to the beginning of therapy with a cytotoxic agent, and
thereafter, on a daily basis during the course of such therapy.
Daily treatment with the telomerase inhibitor can be continued for
a period of, for example, 1 to 365 days after the last dose of the
cytotoxic agent is administered. This invention encompasses the use
of telomerase inhibitors-based cancer therapy for a wide variety of
tumors and cancers affecting skin, connective tissues, adipose,
breast, lung, stomach, pancreas, ovary, cervix, uterus, kidney,
bladder, colon, prostate, anogenital, central nervous system (CNS),
retina and blood and lymph.
[0109] B. Standard Therapies
[0110] Described herein are the therapies used as standard or
traditional methods for treatment of cancers. The section on
chemotherapy describes the use of non-nucleoside telomerase
inhibitors as chemotherapeutic agents in addition to several other
well known chemotherapeuticagents. As detailed in the section
above, all the methods described below can be used in combination
with the telomerase inhibitors developed in the present
invention.
[0111] a. Surgery: Surgical treatment for removal of the cancerous
growth is generally a standard procedure for the treatment of
tumors and cancers. This attempts to remove the entire cancerous
growth. However, surgery is generally combined with chemotherapy
and/or radiotherapy to ensure the destruction of any remaining
neoplastic or malignant cells.
[0112] b. Chemotherapy: A variety of chemical compounds, also
described as "chemotherapeutic agents", function to induce DNA
damage, are used to treat tumors. Chemotherapeutic agents
contemplated to be of use, include, adriamycin, 5-fluorouracil
(5FU), etoposide (VP-16), camptothecin, actinomycin-D, mitomycin,
cisplatin (CDDP), hydrogen peroxide, carboplatin, procarbazine,
mechlorethamine, cyclophosphamide, ifosfamide, melphalan,
chlorambucil, bisulfan, nitrosurea, dactinomycin, daunorubicin,
doxorubicin, bleomycin, plicomycin, tamoxifen, taxol,
transplatinum, vincristin, vinblastin and methotrexate to mention a
few.
[0113] Agents that damage DNA include compounds that interfere with
DNA replication, mitosis and chromosomal segregation. Such
chemotherapeutic compounds include adriamycin, also known as
doxorubicin, etoposide, verapamil, podophyllotoxin, and the like.
Widely used in a clinical setting for the treatment of neoplasms,
these compounds are administered through bolus injections
intravenously at doses ranging from 25-75 mg/m.sup.2 at 21 day
intervals for adriamycin, to 35-50 mg/m.sup.2 for etoposide
intravenously or double the intravenous dose orally.
[0114] Agents that disrupt the synthesis and fidelity of nucleic
acid precursors and subunits also lead to DNA damage. A number of
such agents have been developed, particularly useful are agents
that have undergone extensive testing and are readily available.
5-fluorouracil (5-FU), is one such agent that is preferentially
used by neoplastic tissue, making it particularly useful for
targeting neoplastic cells. Thus, although quite toxic, 5-FU, is
applicable with a wide range of carriers, including topical and
even intravenous administrations with doses ranging from 3 to 15
mg/kg/day.
[0115] Agents that directly cross-link nucleic acids, specifically
DNA, are envisaged to facilitate DNA damage leading to a useful
antineoplastic treatment. For example, cisplatin, and other DNA
alkylating agents may be used. Cisplatin has been widely used to
treat cancer, with efficacious doses used in clinical applications
of 20 mg/m.sup.2 for 5 days every three wk for a total of three
courses. Cisplatin is not absorbed orally and must therefore be
delivered via injection intravenously, subcutaneously,
intratumorally or intraperitoneally.
[0116] The non-nucleoside G-quadruplex inhibitor compounds
developed in this invention are chemotherapeutic agents that are
cytotoxic and inihibit telomerase function which is critical to
cell replication and maintenance of tumor cell immortality. These
compounds also indirectly inhibit DNA polymerases by their strong
interactions with the G-quadruplex structures (FIG. 7).
[0117] c. Radiotherapy: Radiotherapeutic agents and factors include
radiation and waves that induce DNA damage for example,
y-irradiation, X-rays, UV-irradiation, microwaves, electronic
emissions, radioisotopes, and the like. Therapy may be achieved by
irradiating the localized tumor site with the above described forms
of radiation's. It is most likely that all of these factors effect
a broad range of damage DNA, on the precursors of DNA, the
replication and repair of DNA, and the assembly and maintenance of
chromosomes.
[0118] Dosage ranges for X-rays range from daily doses of 50 to 200
roentgens for prolonged periods of time (3 to 4 wk), to single
doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes
vary widely, and depend on the half-life of the isotope, the
strength and type of radiation emitted, and the uptake by the
neoplastic cells.
[0119] d. Gene Therapy: Gene therapy based treatments targeted
towards oncogenes such as p53, p16, p21, Rb, APC, DCC, NF-1, NF-2,
BCRA2, p16, FHIT, WT-1, MEN-I, MEN-II, BRCA 1, VHL, FCC, MCC, ras,
myc, neu, raf erb, src, fins, jun, trk; ret, gsp, hst, bcl and abl,
which are often mutated versions of their normal cellular
counterparts in cancerous tissues.
[0120] VI. Screening for Anti-Telomere and Anti-Cancer Activity
[0121] In particular embodiments, one may test the inhibitors by
measuring their ability to inhibit growth of cancer cells, to
induce cytotoxic events in cancer cells, to induce apoptosis of the
cancer cells, to reduce tumor burden and to inhibit metastases. For
example, one can measure cell growth according to the MTT assay. A
significant inhibition in growth is represented by decreases of at
least about 30%40% as compared to uninhibited, and most preferably,
of at least about 50%, with more significant decreases also being
possible. Growth assays as measured by the MTT assay are well known
in the art. Other assays to measure cell death, apoptosis are well
known in the art, for example, Mosmann et al., 1983; Rubinstein et
al., 1990.
[0122] Quantitative in vitro testing of the anti-tumor agents
identified herein is not a requirement of the invention as it is
generally envisioned that the agents will often be selected on the
basis of their known properties or by structural and/or functional
comparison to those agents already demonstrated to be effective.
Therefore, the effective amounts will often be those amounts
proposed to be safe for administration to animals in another
context.
VII. EXAMPLES
[0123] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example 1
Telomerase Inhibition by
N,N'-bis(2-dimethylaminoethyl)-3,4,9,10-perylenet- etracarboxylic
acid diimide
[0124] Selection of
N,N'-bis(2-dimethylaminoethyl)-3,4,9,10-perylenetetrac- arboxylic
acid diimide as a potential telomerase inhibitor was determined by
Method (B) as described in Example 2. 17
[0125] The relative inhibition of telomerase by
N,N'-bis(2-dimethylaminoet- hyl)-3,4,9,10-perylenetetracarboxylic
acid diimide was determined in a standard primer extension assay
that does not use a PCR.TM.-based amplification of the telomerase
primer extension products. Briefly, the 18-mer telomeric primer
d[TTAGGG].sub.3 (1 .mu.M) without or with
N,N'-bis(2-dimethylaminoethyl)-3,4,9,10-perylenetetracarboxylic
acid diimide was elongated with telomerase in the presence of 1.5
.mu.M of [.alpha.-.sup.32P]-dGTP (800 Ci mmol.sup.-1, 10 mCi
ml.sup.-1) with 1 mM dATP and 1 mM dTTP. The extension products
were isolated and visualized by autoradiography after denaturing
gel electrophoresis.
[0126] The IC.sub.50 was determined to be 50 .mu.M, and at 100
.mu.M of
N,N'-bis(2-dimethylaminoethyl)-3,4,9,10-perylenetetracarboxylic
acid diimide there is an almost complete inhibition of telomerase
activity.
N,N'-bis(2-piperdinoethyl)-3,4,9,10-perylenetetracarboxylic acid
diimide and 3 showed similar behavior. 18
Example 2
Screening Assays for Telomerase Inhibitors
[0127] Compounds that inhibit telomerase are potential drugs for
the treatment of cancer. The method selects compounds based upon
ability to interact with the human DNA-G-quadruplex. Several
procedures for detecting this interaction include:
[0128] (A) A three dimensional structure of a candidate compound
will be analyzed to determine their degree of complementarity to
the three-dimensional structure of human telomeric DNA
G-quadruplex. The NMR solution structure of
d(AGGGTTAGGGTTAGGGTTAGGG) [pdb entry 143d] and its corresponding
molecular surface, generated with the ms program, were used as
inputs to the SPHGEN program. The resulting sphere cluster was used
as input to DOCKv2.0 and a subset of the Cambridge crystallographic
database was search using the contact scoring algorithm.
N,N'-bis(2-dimethylaminoe- thyl)-3,4,9,10-perylenetetracarboxylic
acid diimide was found to have one of the highest contact scores in
the .about.2000 compounds examined.
[0129] (B) Compounds may be selected for their ability to interact
with human DNA G-quadruplex as indicated by UV/VIS spectroscopy. To
a 10 .mu.M solution of
N,N'-bis(2-dimethylaminoethyl)-3,4,9,10-perylenetetracarboxyl- ic
acid diimide in 20 mM phosphate buffer containing 100 mM KCl, pH
7.0 in a quartz cuvette was added 10 .mu.L aliquots of a 3 mM
solution of d(TTAGGGT).sub.4. After each addition the UV/VIS
spectrum was recorded. Pronounced changes in the UV/VIS spectrum of
the compound were noted at wavelengths 488 nm (.about.40%
hypochromicity), 510 nm .about.50% hyperchromicity), and 548 nm
(.about.200% hyperchromicity).
[0130] (C) Compounds may be selected for their ability to interact
with human DNA G-quadruplex as indicated by NMR spectroscopy. The
imino proton spectrum (9-12 ppm) of a solution of d(TTAGGG)4 in
D2O/H2O (10:90) was determined at 500 MHz. Aliquot of
N,N'-bis(2-piperdinoethyl)-3,4,9,10-per- ylenetetracarboxylic acid
diimide were added and the imino proton spectrum recorded. At an
overall stoichiometry of 1:1 the G6 imino resonance becomes
significantly broader and shifts >0.2 ppm upfield.
[0131] (D) Compounds may be selected for their ability to interact
with human DNA g-quadruplex as indicated by an increase in the
melting temperature of the G-quadruplex structure. Thermal
denaturation of the parallel four-stranded G-quadruplex structure
formed by the d[T.sub.2AG.sub.3T] (7-mer) (125 mM KCl, 25 mM
KH.sub.2PO.sub.4, 1 mM EDTA, pH 6.9) monitored by NMR. The spectrum
for DNA alone and in the presence of
N,N'-bis(2-dimethylaminooethyl)-3,4,9,10-perylenetetracarboxy- lic
acid diimide. The molar ratio of
N,N'-bis(2-dimethylaminoethyl)-3,4,9,- 10-perylenetetracarboxylic
acid diimide to quadruplex was 4:1. The imino proton signals have
been assigned previously (Laughlan et al., 1994) as G6, G5, and G4
from high to low field. The presence of drug leads to line
broadening and an upfield shift of the imino proton signals
indicative of intercalation. Furthermore, the melting temperature
of the DNA G-quadruplex is increased significantly in the presence
of the compound. Spectra were acquired in 90% H.sub.2O/10% D.sub.2O
on a Bruker AMX 500 MHz spectrometer at various 15-85.degree. C.
using a 1-1 echo pulse sequence with a maximum excitation centered
at 12.0 ppm. A total of 128 scans was obtained for each spectrum
with a relaxation delay of 2 s. Before acquiring the spectrum at
each temperature, the sample was allowed to equilibrate at the new
temperature for at least 10 min. The data were processed with an
exponential window function using 2 Hz of line broadening. The data
indicate that N,N'-bis(2-dimethylaminoethyl)-3,4,9,1-
0-perylenetetracarboxylic acid diimide increases the melting
temperature of the G-quadruplex by at least 20.degree. C.
Example 3
Synthesis of
N,N'-bis(2-piperdinoethyl)-3,4,9,10-perylenetetracarboxylic acid
diimide
[0132] N,N'-bis(2-piperdinoethyl)-3,4,9,10-perylenetetracarboxylic
acid diimide was prepared by mixing three g of
3,4,9,10-perylenetetracarboxyli- c acid dianhydride with 2.5 mL of
1-(2-aminoethyl)piperidine in 10 mL of DMA and 10 mL of
1,4-dioxane. The mixture was heated under reflux for 6 hours, and
the solvents removed under reduced pressure. The residue was
dissolved in -100 mL of distilled water, and insoluble components
were removed by filtration. The pH of the resulting solution was
adjusted to -3 with the addition of HCl, and the solution was
allowed to stand overnight. Precipitated impurities was removed by
filtration, and the resulting solution was adjusted to pH 1-12 with
the addition of NaOH. The precipitated product was isolated by
filtration, washed with water and dried under vacuum.
Example 4
DNA Synthesis Arrest Assay
[0133] It has been shown that DNA sequences with quadruplex-forming
potential present obstacles to DNA synthesis by DNA polymerases in
a K.sup.+ dependent manner. This K.sup.+ dependent block to DNA
polymerase is a selective and sensitive indicator of the formation
of intramolecular quadruplexes (Weitzmann, et al., 1996). This
assay has been adapted to demonstrate the stabilization of
quadruplex by small molecules and used to screen potential
G-quadruplex-interactive compounds.
[0134] The assay is a modification of that described by Weitzmann,
et al. Briefly, primers (24 nM, sequence: 5'-TAATACGACTCACTATAG-3')
labeled with [y-.sup.32P]ATP were mixed with template DNA PQ74(12
nM,
[0135] sequence:TCCAACTATGTATACTTGGGGTTGGGGTTGGGG
[0136] TTGGGGTTGGGGTTAGCGGCACGCAATTGCTATAGTGAGTCGTATTA-3') in a
Tris-HCl buffer (10 mM Tris, pH8.0) containing 5 mM K.sup.+ and
heated at 90.degree. C. for 4 min. After cooling at room
temperature for 15 min. potential G-quadruplex-interactive
compounds were then added to various concentrations. The primer
extension reactions were initiated by adding dNTP (final
concentration 100 .mu.M), MgCl.sub.2 (final concentration 3 mM) and
Taq polymerase (2.5 U/reaction, Boehringer Mannheim). The reactions
were incubated at 55.degree. C. for 15 min. then stopped by adding
an equal volume of stop buffer (95% formamide, 1 mM EDTA, 10 mM
NaOH, 0.1% xylene cyanol. 0.1% bromophenol blue). The products were
separated on a 12% polyacrylamide sequencing gel. The gels were
then dried and visualized on a phosphorimager (Molecular Dynamics
model 445 S1).
[0137] To validate the assay, G-quadruplex interactive compounds
such as porphyrins and perylenes were tested. The results were
consistent with NMR and telomerase inhibition data. FIG. 8 shows
the DNA synthesis arrest induced by Quinobenzoxazine analogs (QQ23,
1130, QQ31) and a perylene compound (APPER).
Example 5
Photocleavage Assay to Detect Quadruplex DNA Interactions
[0138] (i) Design and Synthesis of an Intramolecular Quadruplex
DNA
[0139] The oligonucleotide G4A employed was synthesized on a
Perseptive DNA synthesizer and deprotected following the routine
phosporamidite procedures the DNA was purified by polyacrylamide
gel electrophoresis (PAGE). The sequence for this 39 oligomer
single strand DNA is:
2 5' CATGGTGGTTTGGGTTAGGGTTAGGGTTAGGGTTACCAC 3'.
[0140] This human telomere repeat-containing DNA was designed to
form an intramolecular quadruplex which can be stabilized by the
stem region in (FIG. 5). A sticky end was added so that unusual
secondary structures could be detected by ligation assay once they
are formed.
[0141] (ii) Photocleavage Assay
[0142] The G4A DNA was labeled with .sup.32P at the 5' end and
stored in 1.times.TE buffer at 3000 cpm/.mu.l. For each
photocleavage reaction, 10.mu. of DNA (.about.5 ng) was mixed with
10 .mu.l of 200 mM KCl or 200 mM NaCl and boiled for 10 min before
cooled down to room temperature. For the no porphyrin control
samples, 10 .mu.l of distilled water was added instead. The
mixtures were transferred to a 96 well plate and added with 2 .mu.l
of 1 .mu.M TMPyP.sup.4 aqueous solution. The samples were then
exposed to 24 watts fluorescent daylight under a glass filter for
different periods of time. Then the reactions were stopped with 100
.mu.l of calf thymus DNA (0.1 .mu.g/.mu.l). After phenol-chloroform
extraction, the samples were subjected to strand breakage treatment
and ethanol precipitation. The DNA samples were loaded onto a 16%
polyacrylamide gel for electrophoresis and visualized with
PhosphorImager (from Molecular Dynamics, Inc.). A typical result
for the photocleavage assay is shown in FIG. 6.
Example 6
Selection of G-Quadruplex Selective Ligand
[0143] The
N,N'-bis(3-morpholinopropyl)3,4,9,10-perylenetetracarboxilic acid
diimide (KeTEL01) was synthesized from
3,4,9,10-perylenetetracarboxy- lic acid dianhydride and
3-morpholinopropylamine using a procedure analogous to that
described above in example 5.3 for the synthesis of
N,N'-bis(2-piperdinoethyl)-3,4,9,10-perylenetetracarboxylic acid
diimide. A solution of KeTEL01 was prepared by dissolving 1 mg of
KeTEL01 in 300 .mu.L of 1N HCl. To this solution was added 11 mL of
a pH 7.0 buffer containing 20 mM sodium phosphate, 100 mM KCl, 1 mM
EDTA, and 0.02% hydroxypropyl-.beta.-cyclodextrin. Aliquots of this
stock solution of KeTEL01 were transferred to 8 different quartz
cuvettes and diluted into pH 7.0 mM sodium phosphate, 100 mM KCl, 1
mM EDTA buffer to afford solutions in which the concentration of
KeTEL01 was 20 .mu.M. To each of the cuvettes was added a solution
of [d(TTAGGGT)]4 so that the final concentration of [d(TTAGGGT)]4
in each of the cuvettes was 0, 4, 8, 12, 16, 20, 50, and 80 .mu.M.
These solutions were allowed to stand overnight in the dark, and
the UV/VIS spectrum of each was determined. Pronounced,
G-quadruplex concentration-dependent changes in the UV/VIS spectrum
were noted at wavelengths 488 nm (.about.40% hypochromicity), 510
nm (.about.40% hyperchromicity) and 548 nm (.about.100%
hyperchromicity). In a parallel study, changes in the UV/VIS
spectrum of a 20 .mu.M solution o f KeTEL01 in a pH 7.0 20 mM
phosphate buffer containing 100 mM KCl and 1 mM EDTA were
determined upon the addition of 10 .mu.M aliquots of a 3 mM (base
pair) solution of calf thymus DNA. No changes in the UV/VIS
spectrum of this solution were noted, indicating that KeTEL01 does
not interact with double-stranded DNA.
Example 7
Kinetics of Interaction of KeTEL01 with G-Quadruplex DNA
[0144] A solution of 5 .mu.M KeTEL01 in 20 mM phosphate buffer, 100
mM KCl, pH 7.0 was placed in a quartz cuvette and the UV/VIS
spectrum determined. An aliquot of a solution of [d(TTAGGGT)]4 was
added to the cuvette to afford a final concentration of 50 .mu.M.
The cuvette was quickly inverted several times and placed in the
spectrophotometer. The absorption of the sample at 488 nm was
continuously monitored for 3 hours, during which time, the
absorption decreased in a multiexponential function. The time
required for the absorption at 488 nm to reach one-half of its
equilibrium value was 60 min.
Example 8
Telomerase Inhibition by UT-SK-02 (Diethylthiocarbocyanine
Iodide)
[0145] Using the DOCK screening methods above, the carbocyanine
group of compounds were identified as potential G-quadruplex
interactive agents. A number of these compounds were assayed using
the DNA Synthesis Arrest Assay described in example 5.4. Each
compound was assayed at a concentration of 20 .mu.M. The results of
this study are summarized in Table 1 ahead:
3 TABLE 1 Stop Stop Compound (F + P).sub.rel* (P/T).sub.rel**
IBT-129A 68% 91% UT-SK-001 86% 83% UT-SK-002 88% 139% UT-SK-003 29%
32% UT-SK-004 71% 88% UT-SK-006 33% 12% *Relative amount of both
full-length and paused products. **Relative ratio of the amount of
paused products as compared to the total amount of products.
[0146] Of the compounds tested, only one, UT-SK-002
(diethylthiocarbocyanine iodide) demonstrated a specific
interaction with G-quadruplex DNA, as indicated by a relative ratio
of paused to total DNA product greater than 100% and a relative
amount of DNA products, both paused and full-length, that is close
to 100%. In confirmatory tests, only this compound inhibited
telomerase, with an inhibition of 10-35% at a concentration of 50
.mu.M.
Example 9
Reduced Cellular Proliferation by Selected Compounds
[0147] The ability of these compounds to inhibit the proliferative
capacity of human cancer cells was determined by a standard MTT
assay. Briefly, cells were incubated for 72 hours in the presence
of various concentrations of compound, and the cell viability was
determined by monitoring the formation of a colored formazan salt
of the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide (MTT) by viable cells. KeTEL01 showed no
cytotoxic effect up to the highest concentrations tested (100
.mu.M), whereas KeTEL03
(N,N'-bis(2-dimethylaminoethyl)-3,4,90,10-perylenetetracarboxylica
acid diimide, see Example 1) showed good cytotoxicity in a variety
of human cancer cell lines. Thus, KeTEL01, which is a selective
G-quadruplex interactive agent, has no acute (72 hr) cytotoxic
effect, but the structurally analogous KeTEL03, which does interact
with double-stranded DNA as well as G-quadruplex, is cytotoxic
under these assay conditions.
4 Cytotoxicity - IC.sub.50 MTT Cell Lines KeTEL01 KeTEL03 MCF-7
>100 .mu.M 18.4 .mu.M BT-20 >100 .mu.M 3.8 .mu.M PC-3 >100
.mu.M 3.8 .mu.M Raji >100 .mu.M 0.4 .mu.M
Example 10
DNA Oligonucleotides
[0148] The DNA primer extension sequence P18
(5'-TAATACGACTCACTATAG-3') and the template sequences shown in
Table 1 were synthesized using a PerSeptive Biosystems Expedite
8909 synthesizer and purified with denaturing polyacrylamide gels.
The template DNA was diluted to 5 ng/.mu.L and dispensed into small
aliquots.
Example 11
DMS Methylation Protection Assay
[0149] The .sup.32P-labeled PQ74 and HT4 templates were denatured
by heating at 90.degree. C. for 5 min and then cooled down to room
temperature in 50 mM Tris-HCl buffer with or without 100 mM of
K.sup.+. One microliter of 1:4 ethanol-diluted DMS was added to 1
.mu.g (300 .mu.L) of annealed DNA. Aliquots were taken at time
points as indicated in the figures, and modification reactions were
stopped by adding 1/4 volume of stop buffer containing 1M of
.beta.-mercaptoethanol and 1.5 M of sodium acetate. The
modification products were ethanol precipitated twice and treated
with piperidine. After ethanol precipitation, the cleaved products
were resolved on a 16% polyacrylamide gel.
Example 12
DNA Synthesis Arrest Assay
[0150] This assay is a modification of that described by Weitzmann
and co-workers (Weitzmann et al., 1996). Briefly, primers (P18, 24
nM) labeled with [.gamma.-.sup.32P] were mixed with template DNA
(12 nM)) in a Tris-HCl buffer (10 mM Tris, pH 8.0) containing
K.sup.+ (5 mM for the PQ74 template and 50 mM for the HT4 template)
and denatured by heating at 90.degree. C. for 5 min. After cooling
down to room temperature, BSU-1051 was added at various
concentrations and incubated at room temperature for 15 min. The
primer extension reactions were initiated by adding dNTP (final
concentration 100 .mu.M), MgCl.sub.2 (final concentration 3 .mu.M),
and Taq DNA polymerase (2.5 U/reaction, Boehringer Mannheim). For
sequencing reactions, the TaqTrack Sequencing System (Promega,
Wis., Madison) was used. The sequencing reaction buffer was changed
to 50 mM Tris-HCl, pH 9.0, 10 mM MgCl, and 50 mM K.sup.+. The
reactions were stopped by adding an equal volume of stop buffer
(95% formamide, 10 mM EDTA, 10 mM NaOH, 0.1% xylene cyanol, 0.1%
bromphenol blue). For the temperature-dependent studies, the ligand
concentration was fixed and the primer extension reactions were
carried out at the temperatures indicated in methods. The products
were separated on a 12% polyacrylamide sequencing gel. The gels
were then dried and visualized on a PhosphorImager (Molecular
Dynamics model 445 S1).
Example 13
Results
[0151] (i) The G-rich Regions of the PQ74 and HT-4 Templates Form
Intermolecular G-quadruplex Structures In K.sup.+ Buffer
[0152] To determine the nature of the G-quadruplex structures
formed by the template sequences used in this study (see Table 1),
dimethylsulfate (DMS) was used to probe the accessibility of N7 of
guanine in the DNA templates (Maxam and Gilbert, 1980). When the
PQ74 template was methylated in 1.times.TE buffer, there was no
apparent protection of any guanine N7. However, with the exception
of the first guanine in each of the four TTGGGG repeats, all the
guanines in the G-rich region of the PQ74 template are protected
from reacting with DMS in 100 mM K.sup.+ buffer, whereas guanines
located outside the four repeats react strongly with DMS. This DMS
protection pattern for the G-rich region of the PQ74 template in
K.sup.+ buffer suggests that only three guanines in each of the
four TTGGGG repeats are involved in G-tetrad formation. This DMS
reaction pattern is different from that observed previously by
Henderson and co-workers (Henderson et al., 1990) with the
d(TTGGGG).sub.4 G-quadruplex in which only the first guanine of the
third repeat (corresponding to G9 in the PQ74 template) is
hypersensitive to DMS methylation. On the basis of the results from
the inventors' study, they propose a model for the G-quadruplex
structure formed by the G-rich region of the PQ74 sequence
consisting of d(TTGGGG).sub.4. In this model, the first guanine of
the first repeat is located in the 5' overhang region and is
therefore open to DMS methylation. However, the first guanines of
the second, third, and fourth repeats (G5, G9, and G13,
respectively) are located in the loop regions of the G-quadruplex.
Although the N7 groups of these three loop guanines are not
involved in hydrogen bonds, steric inaccessibility may protect them
from DMS methylation. The DMS footprinting pattern shows that while
they are partially protected from DMS methylation, this protection
is less than that for the other guanines in the repeat.
[0153] The TTAGGG repeats in the G-rich region of the HT-4 template
also showed high DMS methylation protection in K.sup.+ buffer. In
this particular case, all three guanines in each repeat were almost
evenly protected from methylation, indicating that all of them are
involved in G-tetrad formation. This DMS methylation pattern is
consistent with the intramolecular G-quadruplex structure proposed
by Patel and co-workers for the d[AG.sub.3(T.sub.2AG.sub.3).sub.3]
sequence based on NMR studies (Wang and Patel, 1993).
[0154] (ii) BSU-1051 Binds to G-quadruplex DNA and Blocks DNA
Synthesis In a Concentration Dependent Manner
[0155] Although it has been shown that G-quadruplex structures
block primer extension by DNA polymerase in a K.sup.+ dependent
manner (Weitzmann et al., 1996), the inventors are unaware of any
reports showing enhanced blockage by G-quadruplex--interactive
agents. To determine if BSU-1051 binding to G-quadruplex enhances
the block to DNA synthesis, primer extension reactions were carried
out in the absence and presence of BSU-1051. Taq DNA polymerase
primer extension on DNA templates containing four repeats of either
TTGGGG (PQ74) or TTAGGG (HT4) in the presence of different
concentrations of BSU-1051 at 55.degree. C. were performed. In
these studies, K.sup.+ was added at low concentrations (5 mM of
K.sup.+ for the PQ74 template and 20 mM of K.sup.+ for the HT4
template) in order to prevent overwhelming polymerase pausing due
to formation of highly stable G-quadruplex structures. In the
absence of BSU-1051, there is only a slight pausing of the Taq DNA
polymerase when it reaches the 3'-end of the G-rich site on the
template DNA at 55.degree. C. However, upon increasing the
concentration of BSU-1051, enhanced pausing is observed at the same
site as that seen with low K.sup.+ concentrations. This suggests
that BSU-1051 enhances the polymerase pausing by stabilizing the
G-quadruplex structure formed in the K.sup.+ buffer. At high
BSU-1051 concentrations, the inventors not only observed enhanced
pausing at the 3'-end of the G-quadruplex site but also increased
premature termination resulting from nonspecific interactions
between BSU-1051 and the single-stranded template DNA. At a
BSU-1051 concentration of 100 .mu.M, the primer extension is
completely inhibited due presumably to nonspecific interactions
between BSU-1051 and the single- and/or double-stranded DNA or
between BSU-1051 and the polymerase itself. In addition to the
primary pausing site at the beginning of the G-quadruplex site, two
other secondary pausing sites at the second and third G-rich
repeats are observed at high BSU-1051 concentrations. These
pausings are probably induced by other structures formed by this
G-rich sequence. Given the fact that secondary pausing beyond the
first G-tetrad is not seen in the sequencing lanes that contain 50
mM K.sup.+, it is likely that these secondary pausings are caused
by hairpin structures that are stabilized by BSU-1051 but not
K.sup.+. This suggests that BSU-1051 has a relatively higher
affinity for G-quadruplex DNA over other DNA secondary structures
or single- and double-stranded DNA.
[0156] (iii) DNA Synthesis Arrest by the BSU-1051-Quadruplex
Complex Depends On the Stability of the G-quadruplex Structure
[0157] To further evaluate the ability of BSU-1051 to stabilize
G-quadruplex DNA, Taq DNA polymerase primer extension reactions
were carried out at five different temperatures in the presence and
absence of BSU-1051. In the absence of BSU-1051 polymerase pausing
on the PQ74 template containing four repeats of TTGGGG is almost
lost at around 65.degree. C., which is presumably the melting point
of the G-quadruplex structure formed by this G-rich region in the
template DNA. On the other hand, in the presence of 20 .mu.M
BSU-1051, the G-quadruplex structure is further stabilized, and
significant pausing is observed up to 74.degree. C. In the HT4
template containing four repeats of TTAGGG, in which the
G-quadruplex structure formed is presumably less stable, pausing
fades out at 55.degree. C. in the absence of the ligand. However,
in the presence of BSU-1051, pausing is observed up to 65.degree.
C. Thus, for both DNA sequences, .DELTA.TM upon the addition of 20
.mu.M BSU-1051 is about 20.degree. C.
[0158] In order to confirm that the pausings seen result from the
formation of a G-quadruplex structure on the template DNA, certain
guanines in the templates were substituted with 7-deaza-dG. Since
N7 of guanine is involved in hydrogen bonding in the formation of a
G-quadruplex structure, substitution of guanine with 7-deaza-dG
should preclude the formation of any G-quadruplex structure and
allow for uninterrupted primer extension on the template by Taq DNA
polymerase in the presence of either K.sup.+ or BSU-1051. As shown
in Table 1, two guanines in the TTAGGG repeat region of the HT4
template and four guanines in the TTGGGG repeat region of the PQ74
template were replaced with 7-deaza-dG. This change would allow the
formation of no more than two intramolecular G-tetrads and should
lead to destabilization of the intramolecular G-quadruplex
structure. The primer extension results with these 7-deaza-dG
substituted templates indicate that no significant pausing occurs
in either template in the presence of up to 20 mM of K.sup.+ or at
BSU-1051 concentrations of up to 50 .mu.M. This result provides
strong support for the conclusion that BSU-1051 binds to and
stabilizes intramolecular G-quadruplex DNA, leading to pronounced
DNA synthesis arrest at the G-quadruplex site in the original
G-rich templates.
Example 14
Discussion
[0159] G-rich sequences such as telomeric DNA and triplet DNA have
been reported to form parallel or antiparallel G-quadruplex
structures in the presence of monovalent cations such as Na.sup.+
and K.sup.+. Williamson and co-workers observed very strong
intramolecular UV cross-linking for the sequence d(TTGGGG).sub.4 in
a 50 mM K.sup.+ buffer (Williamson et al., 1989). Their results
indicate that this sequence forms an intramolecular structure.
Using DMS methylation, the inventors conclude that four repeats of
TTGGGG or TTAGGG within a non-G-rich sequence are capable of
forming an intramolecular G-quadruplex structure in K.sup.+ buffer.
Furthermore, the DMS methylation results indicate that of the
possible types of G-quadruplex structures that could be formed by
d(TTGGGG).sub.4, a structure consisting of three G-tetrads is the
predominant species in 100 mM of K.sup.+ buffer. The proposed
G-quadruplex structures formed by d(TTGGGG).sub.4 and
d(TTAGGG).sub.4 repeats have diagonal loops, but alternative
intramolecular G-quadruplex structures formed by foldover hairpins
consisting of three G-tetrads are also possible (Williamson, 1994;
Wang and Patel, 1995; Wang and Patel, 1994). However, the inventors
could not differentiate between these two different types of
intramolecular G-quadruplex structures by the DMS methylation
pattern alone.
[0160] G-rich sequences that are capable of forming G-quadruplexes
in vitro can be found in telomeric sequences (Blackburn, 1991;
Sundquist and Klug, 1989; Kang et al., 1992), immunoglobulin switch
regions (Sen and Gilbert, 1988), the insulin gene (Hommond-Kosack
et al., 1993), the control region of the retinoblastoma
susceptibility gene (Murchie and Lilley, 1992), the promoter region
of c-myc gene (Simonsson et al., 1998), fragile X syndrome triplet
repeats (Nadel et al., 1995; Fry and Loeb, 1994), and HIV-1 RNA
(Awang and Sen, 1993). It has been suggested by Sen and Gilbert
that telomeric DNA sequences may associate to initiate the
alignment of four sister chromatids by forming parallel guanine
quadruplexes (Sen and Gilbert, 1990). Furthermore, the discovery of
G-quadruplex-forming sequences in the promoter region of certain
genes suggests that G-quadruplex structures may play a role in the
transcription regulation of these genes. Another possible role of
G-quadruplex DNA is the regulation of telomere length, since a
telomeric overhang that forms a G-quadruplex structure would not be
a good substrate for telomerase (Henderson and Blackburn, 1989;
Zahler et al., 1991). The inventors recently have demonstrated that
BSU-1051 inhibits primer extension by telomerase only when the
substrate (telomeric DNA) reaches four or more repeats in length
(Sun et al., 1997). In this report, the inventors show that
BSU-1051 is able to bind to and stabilize the intramolecular
G-quadruplex structure formed by four telomeric repeats. Thus, it
is reasonable to postulate that BSU-1051 inhibits telomerase by
interacting with its substrate (G-quadruplex-forming telomeric
repeats) rather than telomerase itself. If G-quadruplex structures
play important roles in other biological processes, then
G-quadruplex-interactive compounds such as those described here,
which stabilize these structures, may have a variety of biological
effects. A series of 2,6-diamidoanthraquinones, including BSU-1051,
has been reported to moderate conventional cytotoxicity in a range
of tumor cells (Collier and Neidle, 1988; Agbandje et al., 1992)
and to inhibit human telomerase (Perry et al., 1998). The
G-quadruplex binding property of those compounds provides a
possible mechanism for their action, although other mechanisms
involving targeting of duplex DNA are also likely.
[0161] The inventors have recently proposed a model for a
perylene--quadruplex complex based on NMR evidence (Fedoroff et
al., 1998). By analogy with this structure and that proposed for a
TMPyP.sub.4--G-quadruplex structure (Wheelhouse et al., 1998), it
seems most likely that the binding site of the BSU-1051 is external
to the lower G-tetrad and within the diagonal loop (see FIG.
5).
[0162] The block of DNA synthesis by G-quadruplex structures is not
polymerase specific. Woodford and co-workers showed that the
K.sup.+ dependent DNA synthesis arrest by G-quadruplex structures
is similar for various polymerases (Woodword et al., 1994). The
inventors have found that the BSU-1051--induced DNA synthesis
arrest pattern is virtually identical when Taq DNA polymerase, E.
coli DNA polymerase I (Klenow fragment), or AMV reverse
transcriptase is used. Given the fact that many G-rich DNA
sequences are capable of forming G-quadruplexes in-vitro
(particularly some cancer related genes and sequences such as c-myc
and telomeres), G-quadruplexs are targets for anticancer
chemotherapy. The DNA synthesis stop assay described in this report
provides a simple and rapid method for the identification of
G-quadruplex--interactive agents as lead compounds. This polymerase
stop assay also allows an internal comparison for the relative
binding of potential G-quadruplex--interactiv- e compounds with
single and double-stranded DNA targets. This is an important
comparison that may provide clues as to the relative cytotoxicity
of these compounds.
[0163] The inventors have successfully used the present assay in
the identification and characterization of other
G-quadruplex--interactive compounds that are also telomerase
inhibitors (Fedoroff et al, 1998; Wheelhouse et al., 1998). This
assay can be used to identify other G-quadruplex--interactive
compounds with potential clinical utility.
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[0164] The following references, to the extent that they provide
exemplary procedural or other details supplementary to those set
forth herein, are specifically incorporated herein by
reference.
[0165] Agbandje, Jenkins, McKerma, Reszka, Neidle,
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[0167] Blackburn, "Structure and function of telomeres," Nature,
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[0169] Broccoli, Young, de Lange, "Telomerase activity in normal
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RNA component of human telomerase," Science, 269:1236-1241,
1995.
[0175] Fox, Polucci, Jenkins, Neidle, "A molecular anchor for
stabilizing triple-helical DNA," Proc. Nail. Acad. Sci. USA.,
92:7887-7891, 1995.
[0176] Haq, Ladbury, Chowdry, Jenkins, "Molecular anchoring of
duplex and triplex DNA by disubstituted anthracene-9/10-diones:
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[0177] Harley, Futcher, Greider, "Telomeres shorten during aging of
human fibroblasts," Nature, 345:458460, 1990.
[0178] Harley, Kim, Prowse, Weinrich, Hirsch, West, Bacchetti,
Hirte, Counter, Greider, Wright, Shay, "Telomerase, Cell
Immortality, and Cancer," Cold Spring Harbor Syrup. Quant. Biol.,
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[0179] Hiyama, Hiyama, Ishioka, Yamakido, Inai, Gazdar, Piatyszek,
Shay, "Telomerase activity in small-cell and non-small-cell lung
cancers," Natl. Cancer Inst., 87:895-902, 1995a.
[0180] Hiyama, Hiyama, Yokoyama, Matsuura, Piatyszek, Shay,
"Correlating telomerase activity levels with human neuroblastoma
outcomes," Nature Medicine, 1:249-255, 1995a.
[0181] Kang, Zhang, Ratlift, Moyzis, Rich, "Crystal structure of
four-stranded Oxytricha telomeric DNA," Nature, 356:126-131,
1992.
[0182] Kim, Piatyszek, Prowse, Harley, West, Ho, Coviello, Wright,
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with immortal cells and cancer," Science, 266:2011-2015, 1994.
[0183] Laughlan, Murchie, Norman, Moore, Moody, Lilley, Luisi, "The
high-resolution crystal structure of a parallel-stranded guanine
tetraplex," Science, 265:520-524, 1994.
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[0189] Wang and Patel, "Guanine residues in d(T.sub.2AG.sub.3) and
d(T.sub.2G.sub.4) form parallel-stranded potassium cation
stabilized G-quadruplexes with anti glycosialic torsion angles in
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Sequence CWU 1
1
12 1 6 DNA Artificial Sequence Description of Artificial Sequence
Synthetic Primer 1 ttaggg 6 2 22 DNA Artificial Sequence
Description of Artificial Sequence Synthetic Primer 2 agggttaggg
ttagggttag gg 22 3 7 DNA Artificial Sequence Description of
Artificial Sequence Synthetic Primer 3 taagggt 7 4 8 DNA Artificial
Sequence Description of Artificial Sequence Synthetic Primer 4
ttagggtt 8 5 7 DNA Artificial Sequence Description of Artificial
Sequence Synthetic Primer 5 aatgggt 7 6 7 DNA Artificial Sequence
Description of Artificial Sequence Synthetic Primer 6 ttagggt 7 7 6
DNA Artificial Sequence Description of Artificial Sequence
Synthetic Primer 7 ttgggg 6 8 22 DNA Artificial Sequence
Description of Artificial Sequence Synthetic Primer 8 agggttaggg
ttagggttag gg 22 9 18 DNA Artificial Sequence Description of
Artificial Sequence Synthetic Primer 9 taatacgact cactatag 18 10 80
DNA Artificial Sequence Description of Artificial Sequence
Synthetic Primer 10 tccaactatg tatacttggg gttggggttg gggttggggt
tggggttagc ggcacgcaat 60 tgctatagtg agtcgtatta 80 11 39 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
Primer 11 catggtggtt tgggttaggg ttagggttag ggttaccac 39 12 18 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
Primer 12 taatacgact cactatag 18
* * * * *