U.S. patent application number 11/705044 was filed with the patent office on 2008-03-27 for conformation-activity relationship of apoptosis-inducing phosphodiester oligonucleotides.
This patent application is currently assigned to Bioniche Life Sciences Inc.. Invention is credited to Mario C. Filion, Zdenek Richard Holan, Nigel C. Phillips, Stephanie Reader.
Application Number | 20080077372 11/705044 |
Document ID | / |
Family ID | 23215136 |
Filed Date | 2008-03-27 |
United States Patent
Application |
20080077372 |
Kind Code |
A1 |
Phillips; Nigel C. ; et
al. |
March 27, 2008 |
Conformation-activity relationship of apoptosis-inducing
phosphodiester oligonucleotides
Abstract
The present invention facilitates in silico evaluation of
molecules for biological activity by providing a computer-based
method to predict whether oligonucleotides may possess biological
activity and the efficacy of the biological activity based on the
three-dimensional structure and charge characteristics of the
oligonucleotides. Biological activities include, but are not
limited to, cellular proliferation, induction of cell cycle arrest
and apoptosis.
Inventors: |
Phillips; Nigel C.;
(Pointe-Claire, CA) ; Filion; Mario C.; (Laval,
CA) ; Holan; Zdenek Richard; (Montreal, CA) ;
Reader; Stephanie; (Ste-Julie, CA) |
Correspondence
Address: |
JOHN S. PRATT, ESQ;KILPATRICK STOCKTON, LLP
1100 PEACHTREE STREET
ATLANTA
GA
30309
US
|
Assignee: |
Bioniche Life Sciences Inc.
|
Family ID: |
23215136 |
Appl. No.: |
11/705044 |
Filed: |
February 9, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10223234 |
Aug 19, 2002 |
7200531 |
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11705044 |
Feb 9, 2007 |
|
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60313290 |
Aug 17, 2001 |
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Current U.S.
Class: |
703/11 |
Current CPC
Class: |
A61P 11/06 20180101;
A61P 43/00 20180101; G01N 33/48 20130101; G16B 15/00 20190201; A61K
38/00 20130101; A61P 29/00 20180101; C12N 2310/33 20130101; A61P
19/02 20180101; A61P 35/00 20180101; A61P 9/10 20180101; C12N
2310/335 20130101; A61P 35/02 20180101; C12N 15/117 20130101; C07H
21/04 20130101 |
Class at
Publication: |
703/011 |
International
Class: |
G06G 7/48 20060101
G06G007/48 |
Claims
1. A computer-based method of predicting biological activity of an
oligonucleotide sequence comprising analysis of the oligonucleotide
sequence to determine if the oligonucleotide sequence contains at
least one centrum.
2. The method of claim 1, wherein the analysis comprises examining
the sequence for electronegativity, electropositivity,
intra-molecular hydrogen bonding and inter-atomic distances.
3. A computer-based method of predicting biological activity of an
oligonucleotide sequence comprising: drawing the sequence using
chemical drawing software; checking the drawn sequence for errors;
creating a three dimensional model of the drawn sequence;
minimizing energy of the three dimensional model; calculating
molecular dynamics using chemical analytical software; displaying a
solvent accessible surface of the three dimensional model on a
display means; identifying globular and linear domains in the three
dimensional model; identifying intramolecular hydrogen bonds;
identifying phosphate groups capable of forming an electronegative
centrum; evaluating spatial orientation of bases in the three
dimensional model for electropositive framing with respect to the
phosphate groups in the electronegative centrum; measuring
interatomic distances of amino/amido groups, and 3' and 5'
hydroxyls from phosphate groups; and, predicting whether the
sequence possesses the biological activity.
4. The method of claim 3, wherein measuring interatomic distances
of amino/amido groups and 3' and 5' hydroxyls from phosphate groups
comprises: measuring a first interatomic distance X between
phosphates associated with the centrum; measuring a second
interatomic distance alpha between phosphates and a farthest atom
in a participating base; measuring a third interatomic distance
beta as the furthest inter-atomic distance beta between amido or
amino groups; measuring a fourth interatomic distance Z between a
front to a rear of the centrum; and, measuring a fifth interatomic
distance Y as a longest distance between a phosphate backbone and
the farthest atom in a participating base.
5. The method of claim 4, wherein a centrum is present in the
sequence if Y is equal to or between about 780 to 2200 pm; Z is
equal to or between about 400 to 1300 pm; alpha is equal to or
between about 900 and 1500 pm; beta is equal to or between about
300 to 1700 pm; and, X is between about 700 to 1360 pm.
6. The method of claim 1, wherein the biological activity is
inhibition of cellular proliferation, induction of cell cycle
arrest or induction of apoptosis.
7. The method of claim 3, wherein the biological activity is
inhibition of cellular proliferation, induction of cell cycle
arrest or induction of apoptosis.
8. The method of claim 1, further comprising testing the sequence
for the biological activity.
9. The method of claim 3, further comprising testing the sequence
for the biological activity.
Description
PRIOR RELATED APPLICATIONS
[0001] The present application claims priority to U.S. Provisional
patent application Ser. No. 60/313,290 filed Aug. 17, 2001.
FIELD OF THE INVENTION
[0002] The present invention provides a computer-based method to
predict whether oligonucleotides may induce apoptosis in cancer
cells based on the three-dimensional structure and charge
characteristics of the oligonucleotides.
BACKGROUND OF THE INVENTION
[0003] Cancer is an aberrant net accumulation of atypical cells,
which can result from an excess of proliferation, an insufficiency
of cell death, or a combination of the two.
[0004] Proliferation is the culmination of a cell's progression
through the cell cycle resulting in the division of one cell into
two cells. The five major phases of the cell cycle are G.sub.0,
G.sub.1, S, G.sub.2, and M. During the G.sub.0, phase, cells are
quiescent. Most cells in the body, at one time, are in this stage.
During the G.sub.1 phase, cells, responding to signals to divide,
produce the RNA and the proteins necessary for DNA synthesis.
During the S-phase (SE, early S-phase; SM, middle S-phase; and SL,
late S-phase) the cells replicate their DNA. During the G.sub.2
phase, proteins are elaborated in preparation for cell division.
During the mitotic (M) phase, the cell divides into two daughter
cells. Alterations in cell cycle progression occur in all cancers
and may result from over-expression of genes, mutation of
regulatory genes, or abrogation of DNA damage checkpoints
(Hochhauser D., Anti-Cancer Chemotherapeutic Agents, 8:903,
1997).
[0005] Apoptosis or programmed cell death is the physiological
process for the killing and removal of unwanted cells and the
mechanism whereby chemotherapeutic agents kill cancer cells.
Apoptosis is characterized by distinctive morphological changes
within cells that include condensation of nuclear chromatin, cell
shrinkage, nuclear disintegration, plasma membrane blebbing, and
the formation of membrane-bound apoptotic bodies (Wyllie et al.,
Int. Rev. Cytol., 68: 251, 1980). The translocation of
phosphatidylserine from the inner face of the plasma membrane to
the outer face coincides with chromatin condensation and is
regarded as a cellular hallmark of apoptosis (Koopman, G. et al.,
Blood, 84:1415, 1994). The actual mechanism of apoptosis is known
to be mediated by the activation of a family of cysteine proteases,
known as caspases. However, most prior art anti-cancer therapies
are directed to induction of apoptosis, have proven to be less than
adequate for clinical applications. Many of these therapies are
inefficient or toxic, have adverse side effects, result in
development of drug resistance or immunosensitization, and are
debilitating for the recipient. Many diseases or conditions are
characterized by undesired cellular proliferation and are know to
one of ordinary skill in the medical or veterinary arts.
[0006] Induction of programmed cell death via the induction of
senescence (Dimri et al., Proc. Natl. Acad. Sci. USA 92:20, 1995)
or apoptosis (Wyllie et al., Int. Rev. Cytol. 68:251, 1980) is
important for the treatment of disorders that involve aberrant
accumulation of unwanted cells such as, but not limited to, cancer,
autoreactive, autoimmune, inflammatory and proliferative disorders.
However, most prior art anti-cancer therapies, whether directed to
induction of apoptosis or to stimulation of the immune system, have
proven to be less than adequate for clinical applications. Many of
these therapies are inefficient or toxic, have adverse side
effects, result in development of drug resistance or
immunosensitization, and are debilitating for the recipient. New
methods are needed for evaluating molecules to predict whether they
will possess a desired biological activity.
[0007] Synthetic oligonucleotides are polyanionic sequences that
are internalized in cells (Vlassov et al., Biochim. Biophys. Acta,
11197:95, 1994). Synthetic oligonucleotides are reported that bind
selectively to nucleic acids (Wagner, R., Nature, 372:333, 1994),
to specific cellular proteins (Bates et al., J. Biol. Chem.,
274:26369, 1999) and to specific nuclear proteins (Scaggiante et
al., Eur. J. Biochem, 252:207, 1998) in order to inhibit
proliferation of cancer cells.
[0008] Synthetic 27 base sequences containing guanine (G) and
variable amounts of thymine (T) (oligonucleotides GTn) wherein n is
.gtoreq.1 or .ltoreq.7 and wherein the number of bases is
.gtoreq.20 (Scaggiante et al., Eur. J. Biochem., 252:207, 1998),
are reported to inhibit growth of cancer cell lines by sequence
specific binding to a 45 kDa nuclear protein, whereas GTn, wherein
the number of bases is .ltoreq.20, are reported to be inactive
against cancer cell lines (Morassutti et al., Nucleosides and
Nucleotides, 18:1711, 1999). Two synthetic GT-rich oligonucleotides
of 15 and 29 bases with 3' aminoalkyl modifications are reported to
form G-quartets that bind to nucleolin and to inhibit proliferation
of cancer cell lines (Bates et al., J. Biol. Chem., 274:26369,
1999). The synthetic six base TTAGGG-phosphorothioate, having a
sequence identical to that of the mammalian telomere repeat
sequence, is reported to inhibit proliferation of Burkitt's
lymphoma cells in vitro and in vivo (Mata et al., Toxicol. Applied
Pharmacol., 144:189, 1997). However, the synthetic six base
TTAGGG-phosphodiester nucleotide is reported to have no
anti-telomerase activity (U.S. Pat. No. 5,643,890).
[0009] Deoxyribonucleotides with biological activity such as
antisense DNA (mRNA binding or triplex-forming DNA) or
immunostimulatory CpG motifs are characterized by sequence-specific
linear motifs, often stabilized by intramolecular base-pair
bonding. Backbone modification, such as phosphorothioate
substitution, does not adversely affect and often enhances the
activity of these molecules.
[0010] We have previously described a composition and method
comprising 2 to 20 base 3'-OH, 5'-OH synthetic oligonucleotides
selected from the group consisting of (G.sub.xT.sub.y).sub.n,
(T.sub.yG.sub.x).sub.n, a(G.sub.xT.sub.y).sub.n,
a(T.sub.yG.sub.x).sub.n, (G.sub.xT.sub.y).sub.nb,
(T.sub.yG.sub.x).sub.nb, a(G.sub.xT.sub.y).sub.nb,
a(T.sub.yG.sub.x).sub.nb, wherein x and y is an integer between 1
and 7, n is an integer between 1 and 12, a and b are one or more
As, Cs, Gs or Ts, wherein the sequence is between 2 and 20 bases
and wherein the sequence induces a response selected from the group
consisting of induction of cell cycle arrest, inhibition of
proliferation, induction of caspase activation and induction of
apoptosis in a number of cancer cells (PCT CA00/01467, WO
01/44465).
[0011] Computational procedures allow a correlation of three
dimensional molecular structures with biological activity, and
facilitate prediction of the conformation of active molecules.
Modeling entails the use of mathematical equations that are capable
of representing accurately the phenomenon under study. Molecular
mechanics analysis (Allinger, N. L., J. Comput. Chem., 12,
844,1991) can be used to analyze structural and conformational
relationships. The fundamental assumption of molecular mechanics is
that data determined experimentally for small molecules (bond
length, bond angles, etc.) can be extrapolated to larger molecules.
Molecular modeling approaches have been used to determine structure
activity relationships and to enable the prediction of active three
dimensional molecular conformations (N. Evrard-Todeschi et al., J.
Chem. Inf. Comput. Sci., 38:742, 1998; Chen H. et al., J. Med.
Chem. 36:4094, 1993; A. Guarna et al., J. Med. Chem., 40:3466,
1997; M. Read, et al. Proc. Natl. Acad. Sci. USA, 98:4844,
2001).
[0012] Therefore, there is a continuing need for the identification
of novel 3-dimensional conformations or structural motifs in
oligonucleotides that are useful in predicting their biological
activity, particularly with regard to their capability to induce
cellular responses in cells. What is needed is the ability to
predict cellular responses including responses such as apoptosis in
cancer cells.
SUMMARY OF THE INVENTION
[0013] The present invention fulfills this need by providing a
computer-based method useful for predicting whether oligonucleotide
sequences possess apoptotic activity. This in silico method also
predicts the relative efficacy of the oligonucleotide sequence to
induce apoptosis. This invention provides a rational basis for in
silico evaluation or screening of oligonucleotide compositions for
their ability to induce apoptosis, thereby providing a means to
select specific oligonucleotide compositions for further testing in
vivo or in vitro. This invention provides significant savings in
the cost of drug design and development by: a) identifying
oligonucleotide compositions with specific predicted biological
activity; b) predicting the efficacy of the oligonucleotide
compositions with the specific predicted biological activity; and,
c) reducing the number of candidate oligonucleotide compositions to
be tested in vitro and in vivo for apoptotic activity.
[0014] Prediction of the capability of a sequence to induce
apoptosis is desired in several diseases and conditions including
but not limited to the following: cancer; hyperproliferative
disorders; autoimmune disease; arthritis; rheumatoid arthritis;
inflammation; lymphoproliferative disorders; restenosis of vessels
after angioplasty; and, asthma. Prediction of the capability of a
sequence to induce apoptosis is particularly desirable in cancers
including but not limited to, squamous cell carcinoma,
fibrosarcoma, sarcoid carcinoma, melanoma, mammary cancer, lung
cancer, colorectal cancer, renal cancer, osteosarcoma, cutaneous
melanoma, basal cell carcinoma, pancreatic cancer, bladder cancer,
brain cancer, ovarian cancer, prostate cancer, leukemia, lymphoma
and metastases derived therefrom.
[0015] The unexpected ability to predict apoptosis-inducing
activity in silico with a high degree of precision (>95%)
reduces the need for costly high-throughput chemical synthesis and
apoptosis-inducing screening, thus enabling the identification of
biologically active molecules in a much more efficient and cost
effective manner.
[0016] An advantage of the present invention is that it accelerates
the discovery of new therapeutic compositions. Another advantage of
the present invention is that it decreases the cost of discovering
new therapeutic compositions by providing candidate oligonucleotide
sequences for biological testing in vivo and in vitro. These
savings directly affect the cost of therapeutic drugs for patients
and throughout the health care industry for humans and animals.
Still another advantage of the present invention is that it
decreases the cost of discovering new therapeutic compositions by
predicting the efficacy of oligonucleotide sequences, thereby
providing a prioritization for biological testing in vivo and in
vitro.
[0017] Accordingly, it is an object of the present invention is to
provide a computer-based method useful for evaluation of
oligonucleotide sequences.
[0018] It is another object of the present invention to provide a
computer-based method useful for evaluation of oligonucleotide
sequences to predict whether they possess the ability to induce a
response in a cell such as inhibition of cellular proliferation,
induction of cell cycle arrest or induction of apoptosis.
[0019] It is a specific object of the present invention to provide
a computer-based method useful for evaluation of oligonucleotide
sequences to predict whether they possess the ability to induce
apoptosis in cells.
[0020] Yet another object of the present invention is to provide a
computer-based method useful for evaluation of oligonucleotide
sequences to predict whether they possess the ability to induce
apoptosis in cancer cells.
[0021] Yet another object to the present invention is to provide a
method useful for identifying oligonucleotide sequences that will
be useful in the treatment of disease.
[0022] Another object to the present invention is to provide a
method useful for identifying oligonucleotide sequences that will
be useful in the treatment of diseases and conditions characterized
by undesired cellular proliferation.
[0023] Still another object to the present invention is to provide
a method useful for identifying oligonucleotide sequences that will
be useful in the treatment of diseases and conditions characterized
by undesired cellular proliferation such as autoimmune disease,
inflammation, arthritis, asthma, restenosis of vessels after
angioplasty, hyperproliferative disorders, lymphoproliferative
disease, and cancer.
[0024] Yet another object to the present invention is to provide a
method useful for identifying oligonucleotide sequences that will
be useful in the treatment of cancer.
[0025] Another object to the present invention is to provide a
method that allows the identification of molecules with
apoptosis-inducing activity in silico without resort to high
throughput chemical synthesis and biological activity
screening.
[0026] The unexpected and surprising ability of the present
invention to predict the capability and efficacy of an
oligonucleotide sequences to induce a cellular response, and
particularly to inhibit cell proliferation, to arrest the cell
cycle progression and/or to induce apoptosis in cells addresses a
long unfulfilled need in the medical arts and provides an important
benefit for animals and humans.
[0027] These and other objects, features and advantages of the
present invention will become apparent after a review of the
following detailed description of the disclosed embodiment and the
appended claims.
BRIEF DESCRIPTION OF THE FIGURES
[0028] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawings will be provided by the Office upon
request and payment of the necessary fee.
[0029] FIG. 1. Spatial definition of the general orientation of a
centrum in three dimensions (front, rear, ventral, dorsal, lateral
left and lateral right.
[0030] FIG. 2a. Schematic representation of framed centrum of
electronegativity in three dimensions.
[0031] FIG. 2b. Schematic representation of framed centrum of
electronegativity in three dimensions showing x, y, z, alpha
(.alpha.) and beta (.beta.) variables as defined in the
specification and the 5' and 3' orientation. Also shown are areas
of electronegativity from phosphate groups, areas of
electropositivity from amines and amido groups, phosphates,
deoxyribose, and bases.
[0032] FIGS. 3-13. The following FIGS. 3-13 each consist of views
as follows:
[0033] a) Position 1 as starting position
[0034] b) Position 2 after 90.degree. rotation along the x-axis
from the starting position
[0035] c) Position 3 after 180.degree. rotation along the x-axis
from the starting position
[0036] d) Position 4 after 270.degree. rotation along the x-axis
from the starting position
[0037] e) Position 1 as starting position in black and white
showing the solvent accessible surface.
[0038] The first position is presented twice as a solid, black and
white picture and as a color picture with dots as the solvent
accessible surface. The hydrogen atoms are suppressed for better
clarity. The following colors are used to represent the atoms in
the 3-dimensional sequences shown in the figures: Blue=nitrogen;
Grey=carbon; Pink=phosphorus; Red=oxygen; Yellow=sulfur.
The four rotational views (a, b, c, and d) are provided as examples
to demonstrate the globular nature of the centrum in different
orientations.
[0039] FIG. 3. TGT
[0040] FIG. 4. GGGGGG
[0041] FIG. 5. GGGTGG phosphorothioate backbone
[0042] FIG. 6. GGG
[0043] FIG. 7. TTGTGG
[0044] FIG. 8. GGGTGGGG
[0045] FIG. 9. GGGTGG.sub.--3P (3'-phosphate)
[0046] FIG. 10. 5P_GGGAGG (5'-phosphate)
[0047] FIG. 11. 5P_GGGTGG (5'-phosphate)
[0048] FIG. 12. GGGTGG
[0049] FIG. 13. GGGGTGG
DETAILED DESCRIPTION OF THE INVENTION
[0050] The present invention provides a computer-based method
useful for predicting whether oligonucleotide sequences possess the
ability to induce a cellular response, and particularly to inhibit
cell proliferation, to arrest the cell cycle progression and/or to
induce apoptosis in cells. In a preferred embodiment, this in
silico method predicts whether oligonucleotide sequences possess
the ability to induce apoptosis. This in silico method also
predicts the relative efficacy of the oligonucleotide sequence to
induce a cellular response, and particularly to inhibit cell
proliferation, to arrest the cell cycle progression and/or to
induce apoptosis in cells. In a preferred embodiment, this in
silico method predicts the relative efficacy of the oligonucleotide
sequence to induce apoptosis in cells. This invention provides a
rational basis for in silico evaluation or screening of
oligonucleotide compositions to predict their ability to inhibit
cell proliferation, to arrest the cell cycle progression, to
activate caspases, to cleave PARP, and/or to induce apoptosis in
cells, thereby providing a means to select specific oligonucleotide
compositions for further testing in vivo or in vitro.
[0051] The method of the present invention is used to determine
whether an oligonucleotide sequence possesses one or more centrums
which are useful in predicting whether the sequence has apoptotic
biological activity.
[0052] As used herein, sequence refers to an association of bases,
deoxyribose and phosphodiester groups in an oligonucleotide
sequence forming an identifiable globular 3-dimensional structure
(that is based on the centrum of negatively charged phosphate
groups framed by positive charges of amino/amido groups of bases at
the opposite side) that is used to predict the capability of the
sequence to induce apoptosis in cells.
[0053] As used herein, "centrum" refers to the absence or presence
of intramolecular substructure comprising two or more phosphate
groups and two or more adjacent bases (Type A centrum) or
non-adjacent bases (Type B centrum), with or without stabilizing
intermolecular hydrogen bonding. The centrum is defined as adjacent
(type A) if bases are adjacent with a perpendicular orientation in
the same or opposite plane to the phosphate necklace. The centrum
is defined as non-adjacent (type B) if the order of the bases is
not consecutive. A preferred orientation is type A or B, a more
preferred orientation is type A and B, a most preferred orientation
is type A with the bases in the same plane perpendicular to the
phosphate necklace. If a sequence has one centrum at the 5' end and
a second centrum at the 3' end then the subscript index, e.g.
A.sub.1 refers to the type A centrum at the 5' end. The subscript
A.sub.2 refers to the type A centrum at the 3' end. The same
indexing is applied to the type B centrum. The centrum is
considered as framed if there is presence of amino or amido or both
groups at the opposite sites of the phosphate necklace.
[0054] The following notation is used to describe the sequence of
bases in the oligonucleotide sequences: A=Adenine; C=Cytosine;
G=Guanine; T=Thymine.
[0055] The following parameters are used in describing the
3-dimensional oligonucleotide sequences: A) All distances are in
pm; B) Intramolecular hydrogen bonds are assumed to form if the
mutual distance of participating atoms is less than 300 pm; and, C)
The molecular dynamics values are in kcal/mole.
[0056] As used herein, a cellular response refers generally to
inhibition of proliferation, to arrest in cell cycle progression
and/or to induction of apoptosis in cells. A preferred response is
induction of apoptosis. Cells include any cell, particularly cells
exhibiting undesired proliferation. Such cells may be found in
hyperproliferative disorders; autoimmune disease; arthritis;
rheumatoid arthritis; inflammation; lymphoproliferative disorders;
cancer; and, asthma. Cancer cells include, but are not limited to,
cells from squamous cell carcinoma, fibrosarcoma, sarcoid
carcinoma, melanoma, mammary cancer, lung cancer, colorectal
cancer, renal cancer, osteosarcoma, cutaneous melanoma, basal cell
carcinoma, pancreatic cancer, bladder cancer, brain cancer, ovarian
cancer, prostate cancer, leukemia, or lymphoma and metastases
derived therefrom.
[0057] As used herein, the phrases "therapeutic treatment" and
"amount effective to" refer to an amount of a 3-dimensional
oligonucleotide sequence effective to inhibit cell proliferation,
arrest the cell cycle progression or induce apoptosis in cells,
including cancer cells.
[0058] Administration of a composition comprising an effective
amount of a oligonucleotide sequence of the present invention to an
animal, including a human, is a therapeutic treatment that
prevents, treats or eliminates a disease, including, but not
limited to, cancer, arthritis, rheumatoid arthritis,
hyperproliferative disorders, restenosis of vessels after
angioplasty, lymphoproliferative disorders and asthma.
[0059] Induction of apoptosis is particularly desirable in cancers
including but not limited to, squamous cell carcinoma,
fibrosarcoma, sarcoid carcinoma, melanoma, mammary cancer, lung
cancer, colorectal cancer, renal cancer, osteosarcoma, cutaneous
melanoma, basal cell carcinoma, pancreatic cancer, bladder cancer,
brain cancer, ovarian cancer, prostate cancer, leukemia, lymphoma
and metastases derived therefrom.
[0060] Although not wanting to be bound by the following statement,
it is proposed that a molecular combination in an oligonucleotide
sequence of negative charges or electronegativity (from for example
phosphate groups), positive charges or electropositivity (from for
example amine and amido groups), in conjunction with appropriate
intra-molecular hydrogen bonding and inter-atomic dimensions as
defined herein, will possess the ability to induce apoptosis in
cancer cells.
Computer Hardware and Software
[0061] It is to be understood that the present invention may be
practiced using any computer with sufficient memory and computing
speed to operate chemical drawing software used by those of
ordinary skill in the art and to measure the parameters of the
centrum. Measurements of the various parameters of the centrum are
accomplished by means of the software translates drawn structures
into 3-d structures.
[0062] Using the software, interatomic distances are measured in pm
(picometers) in the 3-dimensional structure where the first atom is
selected and at the tip of the cursor (pointer) the information
about the distance between those atoms is shown.
[0063] Further, any chemical software that facilitates
determination of the components of the centrum described below may
be used. Such software is commonly known to those of ordinary skill
in the art. In one embodiment of the present invention, ChemDraw
software is employed. The Chem3D software is supplied by
Cambridgesoft.Com, Cambridge, Mass., USA. Other software packages
known to one of ordinary skill in the art of molecular modeling may
be employed, such as Sybill (from Tripos), Charm and Inside II
(from Accelrys).
[0064] The method of the present invention may be practiced using
personal computers such as commonly available to consumers, for
example, desktop units and laptops manufactured by Dell, Apple,
Compaq, Hewlett-Packard, Gateway, IBM or more sophisticated
computers such as Silicon Graphics or Cray computers.
[0065] The computer is operationally connected to a means for entry
of information, such as a keyboard, touchscreen or other entry
device known to one of skill in the art. The computer is
operationally connected to means such as CD read write devices,
disk drive or other means known to one of skill in the art for
accessing and inputting information. Further, computer may be
operationally connected to the internet, to remote databases, or to
other servers that provide access to databases of chemical
structures so that information concerning specific molecular
structures may be obtained rapidly.
[0066] The computer is operationally connected to a means for
display or output of information, such as monitors, printers and
other display means known to one of skill in the art. Such means
permit visualization of three dimensional structure of
oligonucleotide sequences and various parameters associated with
their structure, such as globular shape, shape of the phosphate
backbone orientation and location of phosphate groups,
2-deoxyribose, purines, pyrimidines, amino groups, amido groups,
hydroxyl groups at 3' and 5' ends, and centrums.
General Method of the Invention for in Silico Identification of
Centra in Oligonucleotides
[0067] The following steps describe the general method of the
present invention for identifying centra in an oligonucleotide
sequence.
1) Draw the molecule. The oligonucleotide molecule was drawn using
ChemDraw v.5 software
2) Check for errors Structures were examined to ensure that the
atoms, bonds and valences were correct.
[0068] 3) Translation and analysis of drawn model by Chem3D
software. The structure was drawn and then opened using the Chem3D
software structure to create a 3-dimensional model. If an error was
found (e.g., double bonds instead of single bonds) a warning
message was generated indicating that something was not correct. In
such case the ChemDraw program was used to make changes in the
drawn structure, and the procedure was repeated by opening the
drawn (corrected) structure in Chem3D software.
[0069] 4) Minimization of energy. Minimization of energy was
required for locating stable conformations. From the MM2 menu in
CHem3D software, the "Minimize energy" choice was made. The default
value of 0.1000 was a reasonable compromise between accuracy and
speed. The result contained the values of the following parameters:
bond stretching energy; angle bending energy; torsional energy;
non-bonded energy; van der Waals energy; electrostatic energy;
dipole/dipole contribution; dipole/charge contribution;
out-of-plane bending; and, stretch-bend parameters. The
stretch-bend parameters are force constants for the stretch-bend
interaction terms in the prior list of elements. Parameters are
already installed as a part of the software. X and y represent any
non-hydrogen atom. when an angle is compressed, the MM2 force field
uses the stretch-bend force constants to lengthen the bonds from
the central atom in the angle to the other two atoms in the
angle.
[0070] The Total Steric Energy for the given conformation is
expressed a summary of the values mentioned above (bond stretching
energy, angle bending energy, torsion energy, van der Waals energy,
electrostatic energy and stretch-bend energy) in units of kcal/mol.
Stretch bend cross terms are used when a coupling occurs between
bond stretching and angle bending. The sum of these energies gives
the resulting total steric energy.
5) Calculation of molecular dynamics of molecule at 310.degree. K
Molecular Dynamics calculations used Newtonian mechanics to
simulate motion of atoms, adding or subtracting kinetic energy as
the model moves from lower to higher temperature or vice versa.
[0071] The Molecular Dynamics was computed from the Menu MM2 by
choosing Molecular Dynamics. The present computation used the
default parameters as follows: step interval: 2 fs; frame interval:
10 femtosecond (fs); terminate after: 10,000 steps; heating/cooling
rate: 1 kcal/atom/1 picosecond (Ps); target temperature set at
300.degree. Kelvin (K).
[0072] The target temperature was set at 300.degree. K. after a set
of experiments to determine variance between 300.degree. K. and
310.degree. K. with respect to the shape of molecules and the
temperature range of each molecule. It was found that the default
value of 300.degree. K. covered the range of the temperature up to
310.degree. K. (300.degree. K. corresponded to the temperature of
37.degree. C. at which the experiments were performed). It was
observed that if the temperature was set to 310.degree. K. the
range of calculated values usually exceeded the 310.degree. K.
range.
[0073] 6) Display of solvent accessible surface to identify
molecular conformation, base fingers and necks between them, and to
identify globular or linear domains of given molecule. Solvent
accessible surface displays provide information about entire
molecules instead of specific atom and bond information. The
solvent accessible surface represents the portion of the molecule
that solvent molecules can access. Common solvents have different
values of radius. The default value of water (140 pm) was used.
Surfaces display information about the molecule's physical and
chemical properties. Surfaces display aspects of the external
surface interface (or electron distribution) of a molecule.
Molecular surface types are solid, wire mesh, dots, or translucent.
To display the molecular surfaces the View menu was employed with
four choices: A) solid the surface is displayed as an opaque form.
This was useful to examine details of the surface itself and not
particularly in the underlying atoms and bonds. B) Wire mesh was
displayed as a connected net of lines. The wire mesh displays
surface features and permits visibility of the atoms and bonds. C)
Dots were displayed as a series of unconnected dots. This was we
used to view the underlying structure. D) Translucent surface is
displayed in solid form but is partially transparent so that the
atoms and bonds are visible.
7) Identification of intramolecular hydrogen bonds. Hydrogen bonds
are capable of being formed if the distance is equal to or less
than 300 pm.
[0074] 8) Identification of the presence of phosphate groups
forming a phosphate necklace or bead-like appearance as the basis
for the formation of a strong electronegative centrum. If a
phosphate necklace was present, the size of the centrum was
calculated.
9) Determination of the spatial orientation of bases for
electropositive framing with respect to phosphate groups in an
electronegative centrum.
[0075] 10) Measurement of interatomic distances of amino/amido
groups, and 3' and 5' hydroxyl groups from phosphate groups. Using
the software, interatomic distances were measured in pm
(picometers) in the 3-dimensional structure where the first atom is
selected and at the tip of the cursor (pointer) the information
about the distance between those atoms is shown. Interatomic
distances: The relative position of each atom in our models was
determined by a set of measurements called internal coordinates of
Z-matrix. The internal coordinates for any particular atom consists
of measurements, in the present case bond length between it and
other atoms (more detailed analyses optionally include bond angles
and dihedral angles).
[0076] Internal coordinate values were obtained by choosing values
from the tools menu, pointing to show model tables, and then
choosing internal coordinates). The first three atoms in a Z-matrix
were defined as follows: 1) the origin atom was the first atom in
Z-matrix, and all other atoms in the model were positioned in terms
of this atom; 2) the first positioned atom was positioned only in
terms of the origin atom. The first positioned atom position was
specified by the distance from the origin atom; (in the present
case it was the measurement of interatomic distances between
phosphate groups, between phosphate groups and amino/amido groups,
and between amino/amido groups of involved bases); 3)
[0077] The second positioned atom is positioned in terms of the
origin atom and the first positioned atom. the entire set of
internal coordinates was obtained from the tools menu by pointing
to show model table and choosing internal coordinates.
11) Comparison of model prediction with actual degree of
apoptosis-induction.
[0078] A comparison was made of the globular or linear shape and
interatomic distances of the electronegative centrum framed by
amino/amido groups, and measured apoptotic activity.
12) If two centra were found, then there is a high probability of a
higher degree of apoptosis-induction.
Description of Electronegative Centrum Framed by Amino/Amido Groups
of Bases as well as by Hydroxyl Groups at 5' and 3'Ends.
[0079] The following description of a centrum includes its key
features. FIGS. 2a and 2b displays these features. FIG. 1 provides
general orientation of a centrum (front, rear, ventral, dorsal,
lateral left and lateral right).
[0080] Orientation of bases in the centrum. The centrum is defined
as adjacent (type A) if bases are adjacent with a perpendicular
orientation in the same or opposite plane to the phosphate
necklace. The centrum is defined as non-adjacent (type B) if the
order of the bases is not consecutive. A preferred orientation is
type A or B, a more preferred orientation is type A and B, a most
preferred orientation is type A with the bases in the same plane
perpendicular to the phosphate necklace. one sequence can possess
both type A and type B. If two centra were found in the given
sequence then A.sub.1 represents the first centrum at the 5' end
and A.sub.2 represents the second centrum at the 3' end.
[0081] Centrum phosphorus atoms. the x-axis represents the
inter-atomic distance between phosphorus atoms associated with the
centrum. If two phosphate atoms are involved (e.g.
5'-N.sub.1PN.sub.2PN.sub.3-3' where N.sub.x represent purine or
pyrimidine deoxyribonucleosides and P represents the phosphate
group) then x is between approximately 700 to 1360 pm. If four
phosphate atoms are involved then x is between approximately 750 to
1300 pm (e.g. 5'-N.sub.1PN.sub.2PN.sub.3PN.sub.4PN.sub.5-3' where
N.sub.x represent the number of purine or pyrimidine
deoxyribonucleosides as an integer in the range 2 to 4, and P
represents the number of phosphate groups as an integer).
[0082] Phosphate backbone-base distance. The Y-axis represents the
longest distance between the phosphate backbone and the farthest
atom of a participating base. This will depend on the rotation of
the given base and which atom (group) is considered (methyl,
carbonyl, or amino group). Y is equal to or between approximately
780 to 2200 pm. There is no preferred distance for Y.
Centrum depth. The Z-axis represents the distance from the front to
the rear of the centrum when viewed from above. Z is equal to or
between about 400 to 1300 pm. There is no preferred distance.
Amine/amido framing. The next defining parameter .alpha. is the
furthest framing distance of the amino/amido group from the
phosphate groups. The .alpha. distance is equal to or between about
900 and 1500 pm.
Amine/amido distance. The next defining parameter .beta. is the
furthest inter-atomic distance of amino/amido groups. The value is
equal to or between about 300 to 1700 pm.
[0083] Intra-molecular hydrogen bonding. This bonding stabilizes
the size and shape of molecules as well as the interatomic
distances. Hydrogen bond distances should be equal to or less than
about 300 pm. The most frequently observed bonding is via carbonyl
groups and amino or amido groups. The next most frequent type of
hydrogen bonding is between carbonyl groups and hydroxyl groups at
the 3' or 5' end of the molecule. The last type of hydrogen bonding
is between carbonyl groups and the hydroxyl groups of phosphates. A
preferred type of hydrogen bonding comprises all three types, a
more preferred hydrogen bonding is via carbonyl groups and
amino/amido groups, and via carbonyl groups and phosphate groups,
and a most preferred hydrogen bonding is via carbonyl and
amino/amido groups.
Distance of the 5' or 3' End Hydroxyls to their Corresponding
Phosphate Groups.
[0084] The last parameter is the distance of the 5' or 3' end
hydroxyls to their corresponding phosphate groups. A preferred
distance is equal to or between about 320 and 650 pm, a more
preferred distance is equal to or between about 390 to 420 pm, and
a most preferred distance is about 380 pm.
[0085] Randomly oriented molecules have bases that form fingers
with necks separating individual fingers. These structures do not
possess a centrum or centra as defined above and are either
inactive or possess weak activity (<20% apoptosis-inducing
activity, see Table 4).
[0086] The present invention demonstrates that a molecular
combination of negative charges or electronegativity (for example
from phosphate groups), positive charges or electropositivity (for
example from amine and amido groups), in conjunction with
appropriate intra-molecular hydrogen bonding and with X, Y, Z, a
and P dimensions as defined above, possesses the ability to induce
apoptosis in cancer cells. Accordingly the present invention
provides a method of in silico analysis of molecular structures,
particularly oligonucleotide structures, for prediction of
apoptotic activity.
[0087] Three-dimensional computation is used to identify one or
more 3-dimensional centrum or centra in oligonucleotide sequences
or other molecules that comprise a molecular combination of
negative charges or electronegativity (from phosphate groups),
positive charges or electropositivity (from amine and amido
groups), in conjunction with appropriate intra-molecular hydrogen
bonding and with X, Y, Z, .alpha. and .beta. dimensions as defined
herein, and that on biological testing demonstrate the ability to
induce apoptosis in cancer cells.
[0088] It is proposed that such a molecular combination of negative
charges or electronegativity (from for example phosphate groups),
positive charges or electropositivity (from for example amine and
amido groups), in conjunction with appropriate intra-molecular
hydrogen bonding and with X, Y, Z, .alpha. and .beta. dimensions as
defined above, will possess the ability to induce apoptosis in
cancer cells. While such a molecular combination is described for
oligonucleotide sequences, it is clear that this approach can be
used to conduct molecular modeling and identification of apoptosis
inducing centra in other molecular species.
[0089] In addition to the assay presented in Example 3 involving
Annexin staining, other in vitro assays may optionally be employed
to evaluate centrum-predicted biological activity of sequences. In
vivo assays may also be employed. Various assays useful for this
purpose are described in PCT CA00/01467, WO 01/44465, the entirety
of which is incorporated herein by reference. Additional assays for
evaluation of the efficacy of the sequences are described by
Oncogene Research Products, P.O. Box 12087, La Jolla, Calif., 92039
(Apoptosis Catalog and Technical Guide 2002-2003, especially pages
5-295) the entirety of which is incorporated herein by reference.
Such assays include assays designed to analyze DNA fragmentation,
apoptosis, mitochondrial markers, endoplasmic reticulum markers,
free nucleosomes, nuclear matrix proteins, detection and activity
of numerous caspases and related proteins, including but not
limited to caspases 1 through 14, glutathione, superoxide
dismutase, members of the bcl-2 family, analysis of the Fas/TNR-R
super family, PARP related products, analysis of apoptotic signal
transducers, analysis of various signaling receptors including
death receptors, Apo2, decoy receptors, analysis of apoptotic
membrane proteins, nervous system apoptotic markers, numerous
markers for cell cycle and cellular proliferation, mitotic kinases,
bromodeoxyuridine assays, and p53 assays. The evaluation of the
efficacy of the sequences identified with the analytical method of
the present invention may also be determined in the presence of
other agents, and therapeutic agents, such as inducers of apoptosis
and cell synchronization reagents as described by Oncogene Research
Products, P.O. Box 12087, La Jolla, Calif., 92039 (Apoptosis
Catalog and Technical Guide 2002-2003, especially pages 99-104 and
pages 214-255, the entirety of which is incorporated herein by
reference). Such agents include but are not limited to actinomycin
D, amphidocolin, A23187, caffeine, camptothecin, cycloheximide,
dexamethasone, doxorubicin, 5-fluorouracil, hydroxyurea,
paclitaxel, staurosporine, thymidine, vinblastine, retinoic acid,
etoposide, okadaic acid, vincristine and methotrexate.
[0090] The following examples will serve to further illustrate the
present invention without, at the same time, however, constituting
any limitation thereof. On the contrary, it is to be clearly
understood that resort may be had to various embodiments,
modifications and equivalents thereof which, after reading the
description herein, may suggest themselves to those skilled in the
art without departing from the spirit of the invention.
EXAMPLE 1
Preparation of Deoxyribonucleic Acid Sequences
[0091] Phosphodiester and phosphorothioate sequences were prepared
by Sigma-Genosys (Woodlands, Tex.) using Abacus Segmented Synthesis
Technology. Unless stated otherwise, the sequences were dispersed
in autoclaved deionized water or in a pharmaceutically acceptable
buffer such as, but to limited to, saline immediately prior to
use.
EXAMPLE 2
Cells and Treatment
[0092] Human Jurkat T cell leukemia cells were obtained from the
American Type Culture Collection (Rockville, Md.). The Jurkat T
cells were maintained RPMI 1640 medium, supplemented with 10%
heat-inactivated (56.degree. C., 30 min) fetal bovine serum (all
from Sigma Aldrich, Canada) in an atmosphere of 5% CO.sub.2 at
37.degree. C. Cells were seeded at 2.times.10.sup.5 cells/ml medium
in 6-well float-bottomed tissue culture plates and incubated with
oligonucleotides at a final concentration of 53 .mu.M. Testing of
other concentrations of the oligonucleotides demonstrated that they
induced apoptosis in a concentration dependent manner.
EXAMPLE 3
Analysis of Apoptosis
[0093] Apoptosis was measured by staining cells with Annexin V FITC
and propidium iodide (PI) (BD Pharmingen, San Diego, Calif. USA)
according to the manufacturer's instructions. Flow Cytometry (FCM)
determined cellular fluorescence. The FCM and data analysis were
carried out using a FACSCalibur instrument (excitation 488 nm,
emission 530 nm for Annexin-V and 580 nm for PI) using the program
CELLQuest.
EXAMPLE 4
3 Dimensional Molecular Modeling
[0094] Chem3D version 5.0 and 6.0 software (CabridgeSoft
Corporation, Cambridge, Mass.) was used to create 3-dimensional
images of specific sequences. Molecular mechanics computation of
minimal energy conformations (MM2; Allinger N. L., J. Comput.
Chem., 1993, 14:755-68) was carried out at a default value 3000K
using Newtonian mechanics to simulate motion of atoms, adding
kinetic energy as the model's temperature increased. The values of
molecular dynamics as well as the temperature range in which the
molecular dynamics is valid are mentioned. 3-dimensional modeling
was carried out in order to identify the absence or presence of
intramolecular grouping, defined as a centrum. The spatial
arrangement of electronegative charges (phosphate and base carbonyl
groups, and electropositive charges (amino, amido, or hydroxyl
groups at 3' and 5' ends) were also analyzed. When intramolecular
grouping was observed in the 3-dimensional models, spatial
characteristics were defined according to localization of phosphate
groups, localization of amine/amido groups, and position of
hydroxyl groups, or intramolecular grouping(s) in the
oligonucleotides. The resulting structures are presented with the
5'-end at the left and 3'-end at the right.
[0095] The spatial orientation is characterized as shown in FIG. 1.
Although not wanting to be bound by the following hypothesis, it is
thought that the simplified ideal 3-dimensional sequence shown in
FIG. 2a consists of phosphate groups (circles) and 2-deoxyribose
units (cylinders) at the ventral position and horizontally oriented
bases (prisms) at the dorsal side.
EXAMPLE 5
ODN with Relatively Weak (<20%) Apoptotic Activity
[0096] The results of the computational analysis and correlation
with apoptosis-inducing activity are summarized in Table 1.
Oligonucleotides containing between 3 and 8 bases and apoptosis
values of less than 20% do not possess an identifiable
3-dimensional framed centrum. Typical illustrative 3-dimensional
structures of sequences with weak apoptosis activity are shown in
FIGS. 3, 4, and 5. TABLE-US-00001 TABLE 1 Date of sequence with
weak (<20%) apoptotic activity Number Framed of bases Sequence
Apoptosis (%) centrum H bonds 3 TGT 13 0 No 6 CCGTCC 5 0 Yes CTGTCT
14 0 Yes GGGCGG 17 0 Yes GGGGGG 4 0 No TCGTTC 9.5 0 Yes GGGTGG 0 0
Yes phosphorothioate 7 GGGGGTG 11 0 No 8 GGGGGTGG 19 0 Yes
EXAMPLE 6
ODN with Intermediate (>20%<40%) Apoptotic Activity
[0097] The results of the computational analysis and correlation
with apoptosis-inducing activity are summarized in Table 2.
Oligonucleotides containing between 3 and 8 bases and apoptosis
activity of >20%<40% possessed an identifiable centrum of
either type A or type B. Typical illustrative 3-dimensional
structures are shown in FIGS. 6-8. TABLE-US-00002 TABLE 2 Data of
sequences with intermediate apoptotic activity P number/ # of
Framed Centrum number of Alpha Beta bases Sequence % apoptosis
centrum type bases X(pm) Y(pm) Z(pm) (pm) (pm) H bond 3 GTG 27 1 A
3/2 721 1252 759 1150 994 Yes 4 GTGG 23 1 A 4/3 1262 1219 863 1125
936 Yes 5 GGTGG 21 1 A 3/4 1202 1403 882 1207 757 Yes 6 AAGTAA 23 1
A 2/2 728 1064 672 916 751 No ATGTAT 37 1 A 2/2 748 998 599 934 428
Yes GGCCGG 21 1 B 2/4 776 1093 882 1414 731 Yes TTGTGG 38 1 A 2/2
737 783 553 970 726 Yes GGGTGGG 29 1 A 3/3 702 1149 893 982 853 Yes
8 GGGTGGGG 23 1 A 2/2 777 1342 848 948 1157 Yes
EXAMPLE 7
ODN with High (.gtoreq.41% .ltoreq.80% Apoptosis) Activity
[0098] The results of the computational analysis and correlation
with apoptosis-inducing activity are summarized in Table 3.
Oligonucleotides containing between 3 and 7 bases and apoptosis
activity of .gtoreq.41% .ltoreq.80% possessed an identifiable
centrum of either type A or type B. Typical illustrative
3-dimensional structures are shown in FIGS. 9-13.
EXAMPLE 8
Influence of Phosphodiester Versus Phosphorothioate Backbone on
3-Dimensional Conformation and Apoptotic Activity
[0099] Comparison of the activity and 3-dimensional conformation of
sequence GGGTGG with a phosphodiester backbone (Table 3 and FIG.
12) and GGGTGG with a phosphorothioate backbone (Table 1 and FIG.
5) shows that the change in apoptosis-inducing activity (50% and 0%
respectively) correlates with a loss of a framed centrum of strong
electronegativity due to the prevailing planar orientation of the
first three Gs, as well as the amino group of G.sub.4 at the
frontal bottom and the one of G.sub.5 at the dorsal top excludes
the last two Gs from the framed centrum. TABLE-US-00003 TABLE 3
Data of sequences with high apoptotic activity P number/ # of
Framed Centrum number of Alpha beta bases Sequence % apoptosis
centrum type bases X(pm) Y(pm) Z(pm) (pm) (pm) H bond 4 GGTG 42 1 B
3/3 1123 1393 898 1118 1257 Yes 5 GGGTG 46 1 A 2/2 724 1159 652
1049 818 Yes GTGGG 46 1 A 2/3 658 1152 411 1180 826 Yes 6 GGGTGG_3P
46 1 A 3/3 1423 3132 1566 1174 967 Yes 5P_GGGTGG 62 2 A.sub.1 3/3
1250 1125 845 1719 1793 Yes 5P_GGGTGG A.sub.2 2/2 747 1085 514 821
609 Yes GGAAGG 59 1 B 3/4 901 1262 759 1451 1396 Yes GGTTGG 66 1 A
2/2 748 947 698 881 306 Yes GGGTGG 50 1 A 2/2 753 1149 674 1101 613
Yes GTGGTG 43 1 A 3/4 1440 2197 1288 1191 1333 Yes GTGTGT 67 1 A
3/3 1038 1686 1232 1222 1471 Yes TGGTTG 69 2 A.sub.1 2/2 788 1054
949 1007 631 Yes TGGTTG A.sub.2 2/2 725 881 725 937 514 Yes TGTGTG
66 1 A 3/3 887 1527 1231 1522 1086 Yes 7 GGGGTGG 44 1 A 2/2 702
1149 893 982 853 Yes
EXAMPLE 9
3' or 5' Modification does not Result in the Loss of
Apoptosis-Inducing Centra
[0100] 3' modified sequences GGGTGG-phosphate (Table 3, FIG. 9),
5'-modified sequence phosphate-GGGTGG (Table 3, FIG. 11) all
contained a framed centrum. 3',5' or 3',5'-modification of
oligonucleotides containing a centrum of activity does not result
in conformational changes that lead to the loss of such centra.
EXAMPLE 10
Prediction of the Apoptotic Efficacy of a Sequence
[0101] The sequences in Table 4 were analyzed with the method of
the present invention to determine if they possessed a centrum. A
prediction was made as to whether the sequences would possess the
ability to induce apoptosis, arbitrarily set at 20%. In other
words, a prediction of apoptotic activity implied activity greater
than 20%. Subsequently, the sequences were tested in vitro for
apoptotic activity. The results are shown in Table 4 and indicate a
very high success rate in predicting apoptotic activity. The method
was successful for each sequence analyzed. These data demonstrate
that the in silico method of the present invention identifies
sequences with different degrees of apoptotic activity, thereby
providing a basis for prioritizing selection of sequences for
biological testing. TABLE-US-00004 TABLE 4 Predictive value of the
method using new sequences with unknown apoptosis-inducing activity
PREDICTED PRESENCE OF APOPTOTIC ACTUAL % OF A CENTRUM ACTIVITY
CELLS IN SEQUENCE YES NO No Yes APOPTOSIS GCG-(3 bases) X X 25
GGAG-(4 bases) X X 35 AGTA-(4 bases) X X 12 GAGG-(4 bases) X X 17
GGAG-(4 bases) X X 35 GAGGG-(5 bases) X X 66 GGGAG-(5 bases) X X 73
GGGGAG-(6 bases) X X 33 GAGGGG-(6 bases) X X 13 GAGGGGG-(7 bases) X
X 5 GGGAGGGG-(8 bases) X X 76 GGGGGAGG-(8 bases) X X 11 AAAGTAAA-(8
bases) X X 6
[0102] It is to be understood that the foregoing relates only to a
preferred embodiment of the present invention and that numerous
modifications or alterations may be made therein without departing
from the spirit and scope of the invention as set forth in the
appended claims.
* * * * *