U.S. patent application number 11/699758 was filed with the patent office on 2009-03-05 for programmable genotoxic agents and uses therefor.
This patent application is currently assigned to Massachusetts Institute of Technology. Invention is credited to Robert G. Croy, John M. Essigmann, Marshall Morningstar, Kevin J. Yarema.
Application Number | 20090062236 11/699758 |
Document ID | / |
Family ID | 22902083 |
Filed Date | 2009-03-05 |
United States Patent
Application |
20090062236 |
Kind Code |
A1 |
Essigmann; John M. ; et
al. |
March 5, 2009 |
Programmable genotoxic agents and uses therefor
Abstract
The compositions and methods disclosed herein provide
heterobifunctional programmable genotoxic compounds that can be
designed to kill selected cells present in a heterogenous cell
population. The present compounds comprise a first agent that
inflicts damage on cellular DNA, and a second agent that attracts a
macromolecular cell component such as a protein, which in turn
shields genomic lesions from repair. Unrepaired lesions therefore
persist in the cellular genome and contribute to the death of
selected cells. In contrast, lesions formed in nonselected cells,
which lack the cell component, are unshielded and thus are
repaired. As a result, compounds described herein are less toxic to
nonselected cells. Compounds of this invention can be designed to
cause the selective killing of transformed cells, viral-infected
cells and the like.
Inventors: |
Essigmann; John M.;
(Cambridge, MA) ; Croy; Robert G.; (Belmont,
MA) ; Yarema; Kevin J.; (Albany, CA) ;
Morningstar; Marshall; (Frederick, MD) |
Correspondence
Address: |
WOLF GREENFIELD & SACKS, P.C.
600 ATLANTIC AVENUE
BOSTON
MA
02210-2206
US
|
Assignee: |
Massachusetts Institute of
Technology
Cambridge
MA
|
Family ID: |
22902083 |
Appl. No.: |
11/699758 |
Filed: |
January 29, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10299029 |
Nov 18, 2002 |
7169611 |
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11699758 |
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09103671 |
Jun 23, 1998 |
6500669 |
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10299029 |
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08434664 |
May 4, 1995 |
5879917 |
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09103671 |
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08239428 |
May 4, 1994 |
5882941 |
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08434664 |
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Current U.S.
Class: |
514/83 ; 435/325;
435/375; 514/245; 514/492; 514/590; 514/672; 514/711 |
Current CPC
Class: |
A61K 47/64 20170801;
A61P 31/00 20180101; A61K 47/554 20170801; A61K 47/55 20170801;
A61K 47/557 20170801; A61K 47/54 20170801; A61P 31/12 20180101;
A61P 43/00 20180101; A61P 35/00 20180101 |
Class at
Publication: |
514/83 ; 435/325;
435/375; 514/590; 514/672; 514/492; 514/711; 514/245 |
International
Class: |
A61K 31/675 20060101
A61K031/675; C12N 5/00 20060101 C12N005/00; C12N 5/02 20060101
C12N005/02; A61K 31/175 20060101 A61K031/175; A61K 31/131 20060101
A61K031/131; A61K 31/28 20060101 A61K031/28; A61K 31/10 20060101
A61K031/10; A61K 31/53 20060101 A61K031/53 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] Work described herein was supported by Federal Grant No.
5R35-CA52127, awarded by the National Institutes of Health. The
Government has certain rights in the invention.
Claims
1. A cell membrane permeant heterobifunctional compound effective
in destroying selected cells in a heterogenous cell population,
comprising i) a first agent that mediates binding of said compound
to cellular DNA to form a genomic lesion, wherein said first agent
is linked, via a linkage stable under intracellular conditions, to
ii) a second agent that mediates binding of a protein to said
compound, wherein said protein is preferentially present in
selected cells of the population, such that a three-membered
complex forms between the cellular DNA of selected cells, said
compound and said protein, said complex being effective to
preferentially inhibit repair of genomic lesions formed in selected
cells by the binding of said compound to cellular DNA.
2. A compound of claim 1 wherein said first agent binds covalently
to DNA.
3. A compound of claim 1 wherein said first agent intercalates into
DNA.
4. A compound of claim 1 wherein said first agent is
photoactivated.
5-6. (canceled)
7. A compound of claim 1 wherein said first agent is a
synthetically or naturally sourced antibiotic.
8-9. (canceled)
10. A compound of claim 1 wherein said first agent is a
chloroethylnitrosourea or a nitrogen mustard.
11-12. (canceled)
13. A compound of claim 1 wherein said first agent is a heavy metal
coordination compound.
14. (canceled)
15. A compound of claim 1 wherein said first agent is selected from
a group consisting of: busulfan, hepsulfan, mitoguazone,
procarbazine, hexamethylmelamine, triethylenemelamine,
triethylenephosphoramide, triethylenethiophosphoramide, and any
analog or derivative of any of the foregoing.
16-19. (canceled)
20. A compound of claim 1, wherein said linkage stable under
intracellular conditions comprises a covalent bond.
21. A compound of claim 1, wherein said linkage stable under
intracellular conditions comprises an organic linker comprising up
to about 20 carbon atoms.
22-39. (canceled)
40. A pharmaceutical composition comprising: (a) the compound of
claim 1, dispensed in (b) a pharmaceutically acceptable
carrier.
41. A method of killing cells comprising the step of administering
the cell membrane permeant heterobifunctional genotoxic compound of
claim 1 to said cells.
42. The method of claim 41 wherein said cells are tumorigenic.
43. The method of claim 41 wherein said cells are of reproductive
tract origin.
44. The method of claim 41 wherein said cells are of prostate
origin.
45. The method of claim 41 wherein said cells are of testicular
origin.
46. The method of claim 41 wherein said cells are of breast
origin.
47. The method of claim 41 wherein said cells are of ovarian
origin.
48. The method of claim 41 wherein said cell permeant
heterobifunctional genotoxin is provided in a pharmaceutically
acceptable carrier.
49. The compound of claim 1 wherein said second agent is selected
from the group consisting of: peptide isolates of an epitope
library; oligonucleotides; chemical isolates of a combinatorial
synthesis library; and, functional equivalents of the foregoing.
Description
RELATED APPLICATIONS
[0001] This application is a Continuation-in-Part of co-pending
U.S. patent application Ser. No. 08/434,664, filed May 4, 1995.
Attorney Docket No. N41T-018CP, which is now allowed, which is a
Continuation-in-Part of Ser. No. 08/239,428 filed May 4, 1994,
Attorney Docket No. MIT-018.
FIELD OF THE INVENTION
[0003] The present invention relates generally to compounds and
methods for the selective destruction of cells in a heterogenous
cell population. The compounds feature, in pertinent part, a
genotoxic agent that damages cellular DNA.
BACKGROUND OF THE INVENTION
[0004] Frequently, a need arises in biological investigations and
clinical or veterinary practice for selectively killing a
subpopulation of cells in a heterogenous cell population. For
example, to attain a strain or culture of cells having desirable
characteristics, available in vitro techniques can be applied for
selectively killing a subpopulation of cells in a heterogenous cell
population that comprises cells that possess a desired
characteristic. In this manner, cells that have undesirable
characteristics can be eliminated from the population. Hybridoma
cell lines producing desired monoclonal antibodies and stable
genetic transfectant cell lines expressing the products of
heterologous cloned genes are customarily established in this
manner. See generally, Sambrook et al., Molecular Cloning: A
Laboratory Manual (2d ed. 1989). A mixed population of cells
comprising the desired hybridoma or transfectant is maintained in
culture for a period of time in the presence of one or more
genotoxic drugs, such as aminopterin or methotrexate. The desired
cells are resistant to the genotoxic effects of the drug employed.
In contrast, other cells in the population are susceptible to the
drug and fail to survive. These techniques rest on the creation of
cells having a defined phenotype that confers resistance to a
particular, preselected genotoxic drug. Thus, although significant
advances in biology and biotechnology have been achieved through
the use of these techniques, limits remain to their
flexibility.
[0005] Another general context in which practitioners desire to
kill cells selectively involves heterogenous cell populations
comprising cells of tango or more phylogenetically different
species of organisms. Here, it may be desirable to destroy
selectively the cells of one species while preserving viability of
another. In this manner, a desired species can be enriched in the
population or an offensive species, such as an infectious agent,
can be removed. Here again, the desired objective is often
accomplished by treating the cell population with a drug, such as
an antibiotic, antiviral, antifungal or antiparasitic drug, to
which the undesired species is susceptible. Cells of the undesired
species succumb to the effects of the drug and die. Conversely, the
desired species (e.g., a human or other host animal) must have the
capacity to resist the chosen drug. Although a wide choice of drugs
useful for such purposes has historically been available, recent
reports have documented the appearance of drug resistance in
undesirable species. For example, resistant strains of the
organisms responsible for septic wound infections,
hospital-acquired infections, tuberculosis, malaria, dysentery and
a host of other contagious diseases have arisen in recent years.
Harrison's Principles of Internal Medicine, Part 5 Infectious
Diseases, Ch. 78, 79, and 83-88 (12th ed. 1991). The emergence of
such strains greatly complicates the treatment of infection, and
limits choices available to the practitioner.
[0006] The need to manage or alleviate cancer provides yet another
general setting in which practitioners require means for
selectively killing cells in a heterogenous cell population. Here,
the population comprises normal and neoplastic (malignant or
transformed) cells in an individual's tissues. Cancer arises when a
normal cell undergoes neoplastic transformation and becomes a
malignant cell. Transformed (malignant) cells escape normal
physiologic controls specifying cell phenotype and restraining cell
proliferation. Transformed cells in an individual's body thus
proliferate, forming a tumor (also referred to as a neoplasm). When
a neoplasm is found, the clinical objective is to destroy malignant
cells selectively while mitigating any harm caused to normal cells
in the individual undergoing treatment. Currently, three major
approaches are followed for the clinical management of cancer in
humans and other animals. Surgical resection of solid tumors,
malignant nodules and or entire organs may be appropriate for
certain types of neoplasia. For other types, e.g., those manifested
as soluble (ascites) tumors, hematopoeitic malignancies such as
leukemia, or where metastasis of a primary tumor to another site in
the body is suspected, radiation or chemotherapy may be
appropriate. Either of these techniques is also commonly used as an
adjunct to surgery. Harrison's Principles of Internal Medicine,
Part 11 Hematology and Oncology. Ch. 296, 297 and 300-308 (12th ed.
1989).
[0007] Chemotherapy is based on the use of drugs that are
selectively toxic to cancer cells. Id. at Ch. 301. Several general
classes of chemotherapeutic drugs have been developed, including
drugs that interfere with nucleic acid synthesis, protein
synthesis, and other vital metabolic processes. These are generally
referred to as antimetabolite drugs. Treatment regimes typically
attempt to ensure inactivation of a particular pathway in cancer
cell metabolism by coadministering two or more suitable
antimetabolite drugs. Other classes of chemotherapeutic drugs
inflict damage on cellular DNA. Drugs of these classes are
generally referred to as genotoxic. The repair of damage to
cellular DNA is an important biological process carried out by a
cell's enzymatic DNA repair machinery. Unrepaired lesions in a
cell's genome can impede DNA replication or impair the replication
fidelity of newly synthesized DNA. Thus, genotoxic drugs are
generally considered more toxic to actively dividing cells that
engage in DNA synthesis than to quiescent, nondividing cells. In
many body tissues, normal cells are quiescent and divide
infrequently. Thus, greater time between rounds of cell division is
afforded for the repair of damage to cellular DNA in normal cells.
In this manner, practitioners can achieve some selectivity for the
killing of cancer cells. Many treatment regimes reflect attempts to
improve selectivity for cancer cells by coadministering
chemotherapeutic drugs belonging to two or more of these general
classes.
[0008] In some tissues, however, normal cells divide continuously.
Thus, skin, hair follicles, buccal mucosa and other tissues of the
gut lining, sperm and blood-forming tissues of the bone marrow
remain vulnerable to the action of genotoxic drugs. These and other
classes of chemotherapeutic drugs can also cause severe adverse
side effects in drug-sensitive organs, such as the liver and
kidneys. These and other adverse side effects seriously constrain
the dosage levels and lengths of treatment regimens that can be
prescribed for individuals in need of cancer chemotherapy. Id. at
Ch. 301. See also Loehrer and Einhorn (1984), 100 Ann. Int. Med.
704-714 and Jones et al. (1985), 52 Lab. Invest. 363-374. Such
constraints can predjudice the effectiveness of clinical treatment.
For example, the drug or drug combination administered must contact
and affect cancer cells at times appropriate to impair cell
survival. Genotoxic drugs are most effective for killing cancer
cells that are actively dividing when chemotherapeutic treatment is
applied. Conversely, such drugs are relatively ineffective for the
treatment of slow growing neoplasms. Carcinoma cells of the breast,
luna and colorectal tissues, for example, typically double as
slowly as once every 100 days. Id. at Table 301-1. Such slowly
growing neoplasms present difficult chemotherapeutic targets.
[0009] Moreover, as with the emergence of resistant strains of
pathogenic organisms, transformed cells can undergo further
phenotypic changes that increase their resistance to
chemotherapeutic drugs. Cancer cells can acquire resistance to
genotoxic drugs through diminished uptake or other changes in drug
metabolism, such as those that occur upon drug-induced gene
amplification or expression of a cellular gene for multiple drug
resistance (MDR). Id. at Ch. 301. Resistance to genotoxic drugs can
also be acquired by activation or enhanced expression of enzymes in
the cancer cell's enzymatic DNA repair machinery. Therapies that
employ combinations of drugs, or drugs and radiation, attempt to
overcome these limitations. The pharmacokinetic profile of each
chemotherapeutic drug in such a combinatorial regime, however, will
differ. In particular, permeability of neoplastic tissue for each
drug will be different. Thus, it can be difficult to achieve
genotoxically effective concentrations of multiple chemotherapeutic
drugs in target tissues.
[0010] Needs remain for drugs that can selectively destroy cells in
a heterogenous cell population. Particular needs remain for drugs,
including genotoxic drugs, that can selectively destroy cells of a
pathogenic or undesired organism while preserving relatively
unimpaired the viability of cells of a host or desired organism.
Still more poignant needs remain for chemotherapeutic drugs,
including genotoxic drugs, that can selectively destroy neoplastic
or virally infected cells yet not significantly impair the
viability of normal healthy cells in the body of an individual
afflicted with cancer or a viral disease.
SUMMARY OF THE INVENTION
[0011] It is an object of this invention to provide a
heterobifunctional compound that is genotoxic to selected cells in
a heterogenous cell population. It is an object of this invention
to provide a heterobifunctional compound that inflicts genomic
lesions on selected cells in a heterogenous cell population. It is
an object of this invention to provide a heterobifunctional
compound that inflicts genomic lesions and impairs cellular repair
of said lesions in selected cells in a heterogenous cell
population. It is an object of this invention to provide a
genotoxic agent or drug that can be "programmed" to destroy
selected cells that are phenotypically distinguishable from
nonselected cells in a heterogenous cell population. Another object
of this invention is to expand the range of chemotherapeutic drugs
available for the treatment of infectious and neoplastic diseases.
Yet another object of this invention is to expand the range of
infectious and neoplastic diseases that are susceptible to
chemotherapy. These and other objects, along with advantages and
features of the invention disclosed herein, will be apparent from
the description, drawings and claims that follow.
[0012] In one aspect, the invention features a cell membrane
permeant heterobifunctional compound suitable for destroying
selected cells in a heterogenous cell population. The selected
cells possess a cell component, such as a protein, that is absent
or is present at significantly diminished levels in other,
nonselected cells of the heterogenous cell population. Preferably,
the cell component is intracellular. Most preferably, it is located
within the cell nucleus or is naturally translocated to the nucleus
from another intracellular site. In preferred embodiments, the cell
component is a diffusible macromolecule having a molecular weight
of at least about 25 kDa, more preferably at least about 40 kDa and
still more preferably at least about 80 kDa. The present
heterobifunctional compound is actively or passively transported
across cell membranes, or diffuses through cell membranes. Thus, it
can internalize within cells. It comprises a first agent that binds
to cellular DNA to form a genomic lesion. The genomic lesion can be
formed at a random or site-specific locus in cellular DNA. In
certain embodiments, the first agent damages cellular DNA by
forming one or more covalent bonds with nucleotide bases, the
sugar-phosphate DNA backbone, or both. In other embodiments, the
genomic lesion is formed by intercalation of the first agent into
cellular DNA. Optionally, the first agent is a precursor that is
converted into a DNA-reactive intermediate spontaneously or by
exposure to physiological conditions, a cellular or secreted
enzyme, product or byproduct of cellular metabolism, ionizing or
nonionizing radiation, light energy, or the like. The genomic
lesion so formed by interaction of the first agent with cellular
DNA is potentially repairable by the cell's enzymatic DNA repair
machinery.
[0013] The first agent is linked to a second agent that binds to
the cell component that is preferentially present in selected cells
of the population. In some embodiments, the first and second agents
are linked by a covalent bond. In other embodiments, the first and
second agents are linked indirectly by covalent bonds to an organic
linker. In still other embodiments, the first and second agents are
linked by noncovalent interactions, such as electrostatic or
hydrophobic interactions. Thus, in certain embodiments, the first
and second agents become linked upon or following binding of the
first agent to cellular DNA. The second agent forms a stable
complex with the cell component. That is, the second agent
interacts specifically with the cell component. Interaction can be
noncovalent or covalent, and is energetically favored under
intracellular, e.g., nuclear, conditions. As noted, the cell
component is preferably a diffusible macromolecule, such as a
protein. Alternatively, it can be a metabolite, ligand or cofactor
that is specifically bound by a protein or another diffusible
macromolecule present in the cell. In either circumstance, the
complex comprises a macromolecular cell component found
preferentially in the selected cells. The second agent thus
localizes a sterically large cell component in the immediate
vicinity of the genomic lesion. Preferably, the cell component is
large enough to sterically obscure a segment of adjacent
nucleosides extending from the lesion site for at least about five
base pairs, more preferably at least about eight base pairs, still
more preferably at least about twelve base pairs in both the 5' and
3' directions. As a result, the complex between the cell component
and the second agent is effective for shielding or inhibiting
repair of the genomic lesion formed by the binding of the first
agent to cellular DNA. Formation of a sterically large complex at
the lesion site hinders access by the cell's enzymatic DNA repair
machinery. As a result, shielded lesions persist in the genome and
prejudice DNA replication, the expression of genes relevant to cell
survival, and the like. Thus, the heterobifunctional compounds of
the present invention are fatal to selected cells of the
heterogenous cell population.
[0014] In certain embodiments, the second agent interacts
specifically with a cell component that is relevant to the survival
or proliferation of the selected cells. For example, the second
agent can interact with a regulatory protein or enzyme involved in
the control of cell proliferation. These include, but are not
limited to, oncogene products (e.g., myc, ras, abl, and the like),
tumor suppressor gene products (e.g., the nuclear phosphoprotein
p53), and proteins that regulate initiation and progress through
the cell cycle (e.g., cyclins and cyclin-dependent kinases).
Alternatively, the second agent can interact with a transcription
factor that controls or modulates the expression of one or more
genes that are relevant to metabolic or secretory processes carried
out by the selected cell. One such transcription factor is upstream
binding factor (UBF), which controls the expression of ribosomal
RNA genes and thus is pivotal to the function of the cell's protein
synthesis machinery. Second agents that specifically interact with
transcription factors preferably mimic or resemble the natural
genomic binding site for the particular transcription factor. That
is, the transcription factor binds to the second agent with an
affinity near (e.g., within about 100-fold) or preferably exceeding
its affinity for the natural genomic binding site. Such second
agents are referred to herein as "transcription factor decoys".
Certain transcription factors, in addition to binding an endogenous
genomic binding site, also bind to soluble ligands. Binding of
these transcription factors to their cognate ligands modulates
binding of the transcription factors to their endogenous genomic
binding sites. That is, ligand binding confers or abrogates ability
of the transcription factor to bind its cognate genomic site, or
enhances or suppresses its ability to do so. Such transcription
factors are accordingly referred to herein as ligand-responsive
transcription factors. They have sometimes been referred to in the
art as intracellular or nuclear receptors for soluble ligands.
Second agents that recognize and bind to these transcription
factors can mimic an activating or repressing ligand, such as
estrogen or an estrogen analog or derivative. Heterobifunctional
compounds comprising transcription factor decoys or ligand mimics
thus are doubly fatal to the selected cell.
[0015] In another aspect, the present invention provides a method
for the destruction of selected cells in a heterogenous cell
population. The heterogenous cell population can comprise
phenotypically distinguishable cells of a single phylogenetic
species, or cells of two or more different phylogenetic species.
The phylogenetic species can be unicellular or multicellular. The
population can comprise cells in culture, cells withdrawn from a
multicellular organism (e.g., a blood sample or tissue biopsy), or
cells present in tissue or organs of a multicellular organism. It
should be understood that the term "multicellular organism"
embraces mammals, including humans. The heterogenous cell
population can comprise cells of both normal and transformed
phenotypes. Thus, the population can comprise neoplastic or
malignant cells. In the present method, selected cells of the
heterogenous population are killed. "Selected cells" are
phenotypically distinguishable from other, nonselected cells in the
heterogenous population in that they possess a cell component that
is absent or is present at significantly diminished levels in
nonselected cells. For example, the cell component is made or
accumulates in the selected cells to levels that are about 5-fold
in excess of the levels of the same or a similar cell component in
nonselected cells. Preferably, the selected cells possess about a
10-fold excess of the cell component. More preferably, the selected
cells possess about a 100-fold or higher excess of the cell
component. In certain embodiments, the cell component is the
expression product of a cellular or viral oncogene. In certain
other embodiments, the cell component is the expression product of
a mutant tumor suppressor gene. In still other embodiments, the
cell component is a regulatory or enzymatic element of a nuclear
protein complex that controls initiation of or progress through the
cell cycle.
[0016] The present method involves contacting the heterogenous cell
population with the cell membrane permeant heterobifunctional
compound described herein. The population is incubated with the
compound for a period of time sufficient for the compound to cross
cell membranes and internalize within cells, including the selected
cells. The first agent of the compound binds to cellular DNA,
inflicting a genomic lesion. As noted above, the genomic lesion is
potentially repairable. In selected cells, the second agent of the
compound binds to the cell component, forming a complex at the
genomic lesion site that sterically hinders access to the lesion by
the cell's DNA repair machinery, thereby inhibiting repair or
"shielding" the lesion. As a result, genomic lesions persist in the
selected cells and contribute to their demise. That is, the present
compound is preferentially genotoxic to the selected cells. In
contrast, lesions in nonselected cells do not form complexes at the
site of the genomic lesion, or form complexes with much lower
frequency than in selected cells. Lesions in nonselected cells are
therefore predominantly unshielded and remain accessible to the
cellular DNA repair machinery. As a result, genomic lesions in
nonselected cells are repaired. Lesion repair contributes to the
survival of the nonselected cells. That is, the present compounds
are relatively less genotoxic to nonselected cells. It is
understood herein that the present compounds also may be
internalized selectively by selected cells, regardless of whether
the intracellular complex indeed is formed at the genomic lesion
site. Selective internalization is expected to arise from the
influence of intracellular complex formation in selected cells on
chemical equilibrium dynamics between extracellular and
intracellular levels of the present genotoxic compounds. Thus, the
compounds of the present invention can be used to enhance
selectively the uptake of DNA damaging first agents by selected
cells in a heterogenous cell population. This process further
contributes to the demise of selected cells.
[0017] As a result of the present method, the heterogenous cell
population becomes depleted of selected cells. Embodiments of the
present method wherein the selected cell component that is
sequestered at the lesion site is a transcription factor are
referred to as "transcription factor hijacking". In such
embodiments, hijacking or sequestration of the transcription factor
by the second agent at sites other than the factor's natural
genomic binding site still further contributes to the death of
selected cells, by inducing disarray in one or more of the cell's
metabolic or secretory functions.
[0018] An advantage of the invention described herein is that
heterobifunctional compounds can be engineered that are selectively
fatal (genotoxic) to a great phenotypic and phylogenetic variety of
selected cells. The term, "programmable genotoxic drugs" thus aptly
sums up the flexibility and adaptability of the inventive concept
disclosed herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] The foregoing and other objects, features and advantages of
the present invention, as well as the invention itself, will be
more fully understood from the following description of preferred
embodiments, when read together with the accompanying drawings, in
which:
[0020] FIG. 1 is a schematic illustration showing basic features of
the heterobifunctional programmable genotoxic compounds of the
present invention and their anticipated mode of action in mediating
steric hinderance of the repair of genomic lesions.
[0021] FIG. 2, Panels A-D, presents autoradiograph results of
Southwestern (A, B and C) and Western Blot (D) studies of the
binding of an HMG-box transcription factor, hUBF (human UBF), to a
structural decoy comprising a cisplatin 1,2-dinucleotide
intrastrand DNA adduct. (WCE=whole cell extract; hUBF obtained from
in vitro translation; Anti-NOR-90=antiserum against hUBF.)
[0022] FIG. 3, Panels A and B, presents autoradiograph results of
DNase I footprinting studies showing that hUBF protects a region of
the decoy symmetrically spanning the cisplatin adduct site.
[0023] FIG. 4, top and bottom panels, presents autoradiograph
results of studies establishing the affinity of hUBF for its
endogenous genomic binding site.
[0024] FIG. 5 presents autoradiograph results of additional
footprinting studies that revealed similarity in the hUBF-protected
regions of this transcription factor's cognate genomic binding site
and the cisplatin decoy (compare FIG. 3).
[0025] FIG. 6 presents autoradiograph results demonstrating binding
of streptavidin to U-17 monoadducted with a heterobifunctional
TMP-biotin conjugate. Panel A presents an autoradiograph of the
results of a gel mobility shift assay. 3200 cpm (.about.0.1 nM) of
the radiolabeled TMP-biotin lesioned DNA was used in each lane. 0
nM, 0.4 nM, 1 nM, 2 nM, 5 nM, 10 nM, 50 nM and 50 nM of
streptavidin were used in lanes 1 to 8 respectively. In lane 8,
free d-biotin was also added to the final concentration of 0.4 nM.
Panel B is a binding curve created by plotting the percentages of
bound probe (Panel A) against streptavidin concentrations.
[0026] FIG. 7 presents autoradiograph results demonstrating
inhibition of uracil gylcosylase by lesion-bound streptavidin.
Bands (a) represent the full-length and intact probe used in each
reaction. Bands (c) represent the products of uracil glycosylase
treatments and the subsequent piperidine cleavages. Bands (b) and
bands (d) are the breakdown products of bands (a) and bands (c)
respectively due to the alkali liability of the adducts.
[0027] FIG. 8 presents autoradiograph results of a DNase I
protection assay. 5000 cpm (.about.1.5 fmoles, .about.0.15 nM) of
the .sup.32P end-labeled, TMP-biotin lesioned probe was used in
each lane. 0 nM, 0.4 nM, 2 nM, 10 nM, 50 nM and 50 nM of
stretavidin were used in lanes 2 to 7 respectively. In lane 7, 0.4
nM of free d-biotin was also included in the incubation. Where
indicated, 5 pg of DNase I (final concentration, 0.4 mg/ml) was
used in each digestion. The boxed thymidine base shown at right
marks the position of the TMP-biotin monoadduct.
[0028] FIG. 9 presents a flow chart summarizing a chemical
synthesis scheme for preparing a preferred heterobifunctional
compound of the invention, demonstrated herein to be toxic
selectively to cells that express estrogen receptor.
[0029] FIG. 10 presents a quantitative plot of results of
competition studies establishing the relative affinities of
heterobifunctional compounds comprising a 2-phenylindole second
agent for the estrogen receptor (ER).
[0030] FIG. 11 presents a quantitative plot of results of
competition studies establishing that ER affinity of two of the
compounds assessed in FIG. 10 is retained when the compounds are
adducted covalently to DNA.
[0031] FIG. 12 presents a quantitative plot of results of cellular
toxicity studies establishing that a preferred heterobifunictional
ER decoy compound (I) of the present invention having the structure
shown at the right of FIG. 12 (inset), is toxic selectively in
human breast carcinoma cells expressing ER.
[0032] FIG. 13 presents a quantitative bar graph of results of 4
hour cellular toxicity studies establishing that a preferred ER
ligand decoy compound (I) is toxic selectively to HeLa cell
transformants expressing functional ER (ER+), and is relatively
nontoxic to HeLa cells transformed with a control, antisense ER
expression vector (ER-).
[0033] FIG. 14 presents a quantitative bar graph of results of 4
day cellular growth inhibition studies establishing that a
preferred ER ligand decoy compound (I) is growth inhibitory to ER+
transformed HeLa cells, and is relatively nontoxic to ER
transformed HeLa cells.
[0034] FIG. 15 presents a quantitative bar graph of results of
control toxicity studies establishing that the selective toxicity
results set forth in FIG. 13 depend upon the genotoxicity of the
first agent (e.g., chlorambucil), rather than on a possible
antiestrogenic activity of the second agent (2-phenylindole).
[0035] FIG. 16 presents a flow chart summarizing a chemical
synthesis scheme for preparing another preferred heterobifunctional
compound 10 of the invention, demonstrated herein to be toxic
selectively to cells that express estrogen receptor.
[0036] FIG. 17 presents a scatterplot of results of competition
studies establishing the relative affinity of preferred
heterobifunctional compound 10 for estrogen receptor.
[0037] FIG. 18 is a scatterplot of results of clonogenic survival
assays in which estrogen receptor positive (MCF-7) and estrogen
receptor negative (MDA-MB231) breast cancer cell lines were exposed
to the indicated concentrations of
7.alpha.-estradiol-C.sub.6NC.sub.2-mustard compound 10 for 2
hours.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0038] Broadly, the selectively genotoxic compounds disclosed
herein comprise a first agent that inflicts genomic lesions on
cellular DNA, linked to a second agent that attracts or sequesters,
preferably at the genomic lesion site, a sterically large cell
component preferentially present in selected cells of a
lieterogenous cell population The cell component is sterically
large enough to effectively hinder access to the lesion site by
elements of the cell's enzymatic DNA repair machinery, thereby
shielding the lesion from repair. In preferred embodiments, the
cell component is a protein, such as a cell circle control factor,
a transcription factor, an oncogene product or a mutant tumor
suppressor gene product, that is normally engaged in the control of
one or more genes relevant to the cell's growth or survival, or to
secretory processes carried out by the selected cell. FIG. 1
illustrates the basic principle of repair shielding by the
heterobifunctional "programmable genotoxic" compounds of the
present invention. A heterobifunctional compound 3 of the present
invention is shown bound to cellular DNA of a nonselected cell 1 or
of a selected cell 2. In each cell, binding of the compound to
cellular DNA results in a potentially repairable genomic lesion.
The compound 3 comprises a first agent 5 that binds to cellular
DNA, linked, optionally by linker 7, to a second agent 9 that binds
to a cell component 12 preferentially present in selected cells of
a heterogenous cell population comprising selected and nonselected
cells. If unrepaired, the genomic lesion contributes to the
destruction of cells. Lesion repair is carried out by the cell's
enzymatic DNA repair machinery, which includes one or more
sterically large repair enzymes 10. In the absence of the cell
component 12, repair enzymes 10 access and repair the lesion.
However, in selected cells, the cell component 12 binds to the
second agent 9, effectively shielding the lesion from repair by
presenting a steric obstacle to repair enzyme 10 access.
Genotoxic Agents Useful as First Agents
[0039] The present compounds employ as first agent 5, genotoxic
drugs that preferably are known in the art and can readily be
prepared according to published techniques, or are commercially
available. Many of these genotoxic drugs currently are used to
treat infections and neoplastic diseases in mammals, e.g., humans.
Analogs or derivatives of these drugs readily can be prepared that
are suitable for linkage to cell component binding second agent 9
to obtain heterobifunctional genotoxic compound 3 of the present
invention. It is anticipated that novel genotoxic drugs also can be
developed that will be suitable for use as first agents herein.
Compounds comprising such novel first agents are considered to be
encompassed by the present invention.
[0040] Two general classes of compounds that are suitable for use
as first agent 5 are DNA alkylating agents and DNA intercalating
agents. Optionally, the first agent can be a precursor that becomes
reactive with cellular DNA spontaneously or following exposure to
an activating stimulus, such as a cellular or secreted enzyme, cell
metabolite or metabolic byproduct, ionizing or nonionizing
radiation, light energy, etc. For example, the first agent can be
photoactivated. One class of photoactivatable first agents is
represented by the drug psoralen, a tricyclic furocoumarin that
produces pyrimidine base adducts and crosslinks in cellular DNA.
Tricyclic furocoumarin analogs and derivatives of psoralen can also
be used as first agents. Thus, for example, trimethylpsoralen (TMP)
can be used herein. Psoralens are known to be useful in the
photochemotherapeutic treatment of cutaneous diseases such as
psoriasis, vitiligo, fungal infections and cutaneous T cell
lymphoma. Harrison's Principles of Internal Medicine, Part 2
Cardinal Manifestations of Disease, Ch. 60 (12th ed. 1991). Another
class of photoactivatable first agents is represented by
dacarbazine and includes analogs and derivatives thereof.
[0041] Another general class of first agents, members of which can
alkylate or intercalate into DNA, includes synthetically and
naturally sourced antibiotics. Of particular interest herein are
antineoplastic antibiotics, which include but are not limited to
the following classes of compounds represented by: amsacrine;
actinomycin A, C, D (alternatively known as dactinomycin) or F
(alternatively KS4); azaserine; bleomycin; caminomycin (carubicin),
daunomycin (daunorubicin), or 14-hydroxydaunomycin (adriamycin or
doxorubicin); mitomycin A, B or C; mitoxantrone; plicamycin
(mithramycin); and the like. Each class of antineoplastic
antibiotics includes analogs and derivatives of the foregoing
representative compounds. Antineoplastic antibiotics are known to
be useful in the treatment of a variety of neoplasms and viral
diseases. Neoplasias currently manageable by the foregoing include
leukemias, lymphomas, myelomas, neuroblastomas, neoplasias of
bladder, testicular, endometrial, gastric, or lung origin, and
others listed in Tables 301-6 and 301-7 of Harrson's Principles of
Internal Medicine, Part 11 Hematology and Oncology (12th ed. 1991).
A given neoplasm is "manageable" by a given drug if treatment with
the drug alone or in combination with another drug confers some
clinically recognized benefit on the afflicted individual.
Optimally, a partial or total remission is achieved. Drugs that
contribute to a stabilization of the individual's clinical status
or slow the progress of disease, however, are also considered
beneficial and are used in the management of neoplasias.
[0042] Still another general class of first agents, members of
which alkylate DNA, includes the haloethylnitrosoureas, especially
the chloroethylnitrosoureas Representative members of this broad
class include carmustine, chlorozotocin, fotemustine, lomustine,
nimustine, ranimustinie and streptozotocin. Haloethylnitrosourea
first agents can be analogs or derivatives of any of the foregoing
representative compounds. Neoplasias currently manageable by the
foregoing include Hodgkin's, non-Hodgkin's and Burkitt's lymphomas,
myelomas, glioblastomas and medulloblastomas, pancreatic islet cell
carcinomas, small cell lung carcinomas and the like. Id.
[0043] Yet another general class of first agents, members of which
alkylate DNA, includes the sulfur and nitrogen mustards. These
compounds damage DNA primarily by forming covalent adducts at the
N7 atom of guanine. Representative members of this broad class
include chlorambucil, cyclophosphamide, ifosfamide, melphalan,
mechloroethamine, novembicin, trofosfamide and the like. Nitrogen
mustard or sulfur mustard first agents can be analogs or
derivatives of any of the foregoing representative compounds.
Nitrogen mustards are generally understood to comprise the moiety
N(CH.sub.2H.sub.2X).sub.2, wherein X is a halogen, preferably
chlorine. In mechloroethamine, X is chlorine, and the moiety is
covalently bonded to a methyl (CH.sub.3) group. Typically, then,
nitrogen mustards such as chorambucil and mephalen have two
reactive groups that can form covalent bonds with the N7 atoms of
guanine residues. Thus, these drugs can form intrastrand or
interstrand DNA crosslinks, and can crosslink DNA to nucleophilic
atoms in proteins. Each type of genomic lesion is thought to
contribute to the lethal effects of nitrogen mustards. Neoplasias
currently manageable by the foregoing include Hodgkin's,
non-Hodgkin's, Burkitt's and other lymphomas, leukemias, myelomas,
medullomas, neuroblastomas, small cell lung carcinoma, osteogenic
sarcoma, neoplasias of breast, endometrial and testicular tissue,
and the like. Id. U.S. Pat. No. 3,299,104 (issued Jan. 17, 1967)
and Niculescu-Duvaz et al. (1967), J. Med. Chem., 172-174, disclose
estrogen, progesterone, androgen and steroid conjugates of
mechloroethamine. Muntzing and Nilsson (1972), 77 J. Krebsforch.
166-170, report histologic studies conducted on cells of patients
receiving one such conjugated methochloroethamine compound.
[0044] Yet a further general class of first agents, members of
which form covalent DNA adducts, includes heavy metal coordination
compounds, including platinum compounds. Generally, these heavy
metal compounds bind covalently to DNA to form, in pertinent part,
cis-1,2-intrastrand dinucleotide adducts. Generally, this class is
represented by cis-diamminedichloro-platintum(II) (cisplatin), and
includes cis-diammine-(1,1-cyclobutane-dicarboxylato)platinum(II)
(carboplatin), cis-diammino-(1,2-cyclohexyl)-dichloroplatinum(II),
and cis-(1,2-ethylene-diammine)dichloroplatinum(II). Platinum first
agents include analogs or derivatives of any of the foregoing
representative compounds. Neoplasias currently manageable by
platinum coordination compounds include testicular, endometrial,
cervical, gastric, squamous cell, adrenocortical and small cell
lung carcinomas along with medulloblastomas and neuroblastomas.
trans-Diamminedichloroplatinum (II) (trans-DDP) is clinically
useless owing, it is thought, to the rapid repair of its DNA
adducts. The use of trans-DDP as a first agent herein likely would
provide a compound with low toxicity in nonselected cells, and high
relative toxicity in selected cells.
[0045] Other classes of first agents, members of which alkylate
DNA, include the ethylenimines and methylmelamines. These classes
include altretamine (hexamethylmelamine), triethylenephosphoramide
(TEPA), triethylenethiophosphoramide (ThioTEPA) and
triethylenemelamine. Additional classes of DNA alkylating first
agents include the alkyl sulfonates, represented by busulfan; the
azinidines, represented by benzodepa; and others, represented by,
e.g., mitoguazone, mitoxantrone and procarbazine. Each of these
classes includes analogs and derivatives of the respective
representative compounds.
[0046] Oligonucleotides or analogs thereof (e.g., phosphorothioate
oligonucleotides, oligonucleotides incorporating
O.sup.6-methylguanine and/or O.sup.4-methylguanine and the like)
that interact covalently or noncovalently with specific sequences
in the genome of selected cells can also be used as first agents,
if it is desired to select one or more predefined genomic targets
as the locus of a genomic lesion. Suitable oligonucleotides that
intercalate nonclassically into cellular DNA to form triple helices
or other complex structures are disclosed in Riordan and Martin
(1991), 350 Nature 442-443, the teachings of which are incorporated
by reference. These compounds are expected to be useful for the
management of neoplasias whose growth characteristics are traceable
to the aberrant activation of particular genes, such as cyclin
genes, oncogenes and mutant tumor suppressor genes.
[0047] Each of the foregoing classes of suitable first agents
comprises analogs and derivatives of the representative compounds
mentioned herein. An analog of a representative compound can be a
structurally related compound, optionally a precursor of a
representative compound or a derivative of a precursor. For
example, trimethylpsoralen (TMP) is an analog of the representative
compound psoralen. An analog can also be a known or novel compound
bearing substituents that are structurally and/or functionally
analogous to those of a representative compound. For example, if a
representative compound has a chlorine substituent, an analog can
have another halogen substituent (e.g., bromine or fluorine). A
known or novel derivative of a representative compound is
chemically, physicochemically or metabolically synthesized from a
representative compound, and can comprise a greater or lesser
number and complexity of substitutents than the representative
compound. Appropriate substituents to the basic structure of the
representative compounds in each class will be known or can be
determined through no more than routine experimentation or
comparative inspection of the structures of two or more
representative members of a particular class. Substitutents
suitable for use in the various classes of first agents listed
above thus include linear, branched or cyclic alkyl, aryl or mixed
alkyl and aryl groups; organic or inorganic acids, bases or neutral
moieties. Substituents present in analogs and derivatives of the
representative compound can modulate DNA binding activity (e.g.,
enhance or impair activity), but should not abrogate such
activity.
Preferred Classes of Cell Components Bound by Second Agent 9
[0048] Turning to second agent 9 of heterobifunctional compound 3,
it should be noted that, in all preferred embodiments, the second
agent serves to mediate attachment of a cellular macromolecule
preferably to the site of genomic damage caused by binding of the
first agent 5 to cellular DNA. The bound cellular macromolecule
thus preferably sterically shields the lesion from repair. The
second agent either binds directly to the cellular macromolecule,
or to a ligand, cofactor or metabolite to which the cellular
macromolecule in turn binds with high affinity. In either
circumstance, the second agent mediates formation of a stable
complex between the cellular macromolecule and the
heterobifunctional compound. As the complex is stable under
intracellular, e.g., nuclear, conditions, it preferably acts as a
persistent steric shield, preventing repair of the genomic lesion
for a sufficiently long period of time for the lesion to contribute
to the demise of selected cells.
[0049] In preferred embodiments, the second agent binds directly to
a cell component that is a macromolecule, such as a protein,
preferentially associated with selected cells in a heterogenous
cell population. This protein provides a phenotypic distinction
between selected and nonselected cells of the population. It can be
a protein of endogenous cellular origin (expressed from the
cellular genome), or of viral origin (expressed from the genome of
a virus infecting the selected cells). Selected cells are
phenotypically distinguished from on selected cells by the
qualitative or quantitative association of the second
agent-recognized protein. Thus, nonselected cells lack the protein,
or are associated with diminished amounts thereof. The recognized
protein can be a phylogenetic species or tissue-type variant of a
corresponding protein associated with nonselected cells. It can be
a protein, the expression of which is developmentally regulated or
dysregulated in selected cells in a manner different from its
regulation in nonselected cells. It can also be a mutant of a
protein normally associated with nonselected cells. Examples of
recognized proteins thus include bacterial, fungal, parasitic and
viral intracellular proteins. Other examples include developmental
stage specific proteins, including proteins expressed upon
dedifferentiation or malignant transformation of nonselected cells
into selected cells. Still other examples include proteins
preferentially expressed by dividing cells, or proteins that are
relevant to the process of cell division (cell cycling). Other
examples include proteins that can be induced in selected cells by
irradiation or other stimuli to which selected cells respond.
[0050] Selected cells with which the second-agent recognized
protein is associated, therefore, can be dividing cells, e.g.,
transformed cells. Preferably, selected cells have at least about a
5-fold excess of the recognized protein, over the amount of the
same or a corresponding protein in nonselected cells. More
preferably, the excess is at least about 10-fold. Still more
preferably, the excess exceeds about 100-fold. Even more
preferably, the recognized protein is undetectable in nonselected
cells. In many preferred embodiments, the recognized protein is
intracellular. For example, the recognized protein is a nuclear
protein or is a protein that is normally translocated to the
nucleus, e.g., when bound to a transport protein or an activating
or suppressing ligand. Preferred classes of second agent-recognized
intracellular proteins therefore include but are not limited to
cyclins, cyclin dependent kinases, oncogene products, mutant tumor
suppressor gene products, and transcription factors.
[0051] Oncogenes are genes encoding proteins that are relevant to
the process of malignant transformation of a normal cell into a
malignant (cancerous) cell. Thus, oncogenes encode proteins
relevant to the proliferation and differentiation states of a cell.
Molecular Cell Biology. Ch. 24 Cancer, 967 and 984-994 (2d ed.
1990). Oncogenes can be found in the cellular genome, or in the
genome of a virus infecting the cell. Infection with certain
tumorigenic viruses causes the infected cell to undergo malignant
transformation. Examples of such viruses include the adenoviruses
and papovaviruses (e.g., SV40 and polyoma), retroviruses (e.g.,
Rous sarcoma virus, mouse mammary tumor virus, human T-cell
leukemia virus-1, Epstein-Barr virus, and the papilloma viruses).
Id., Ch. 24 Cancer, 967-980. Oncogenes encoding intracellular
proteins (e.g., src, yes, fps, abl, met, mos and crk), particularly
those encoding nuclear proteins (e.g., erbB, abl, jun, fos, myc,
N-myc, myb, ski and rel) are of particular interest herein. Id. at
Table 24-1. That is, certain preferred second agents 9 bind to
nuclear oncogene products. Oncogenes of both viral and cellular
origin have been implicated in the etiology of numerous neoplasias.
Harrison's Principles of Internal Medicine, Part 1 Biological Basis
of Disease, Ch. 10 (12th ed. 1991). These include, but are not
limited to Burkitt's lymphoma (Epstein-Barr virus; activation of
endogenous myc), chronic myelogenous leukemia (activation of abl),
anogenital cancers (papilloma viruses), and pancreatic carcinomas
(ras). Id. at Table 10-3.
[0052] Tumor suppressor genes are also known as "anti-oncogenes" or
"repressive oncogenes" because they encode proteins (gene products)
that affirmatively maintain cells in an appropriately
differentiated state, and/or restrain cells from embarking on
unbridled rounds of proliferation. These desirable properties can
be lost upon mutation of a tumor suppressor gene, freeing the cell
from normal growth controls. Tumor suppressor gene products are
thought to bind either to cellular DNA (and thus may themselves be
transcription factors), or to other proteins, e.g., oncogene
products. Two examples of tumor suppressor genes are Rb, the
retinoblastoma gene (Molecular Cell Biology, Ch. 24 Cancer, 996 (2d
ed. 1990); Harrison's Principles of Internal Medicine, Part 1
Biological Basis of Disease, Ch. 10, 68-69) and the nuclear
phosphoprotein p53 (Hollstein et al. (1991), 253 Science 49-53).
p53 is of particular interest herein, as somatic mutations of p53
have been reported in sporadic and inherited neoplasms of breast,
colon, lung, esophagus, liver, brain, blood-forming (myeloid and
lymphoid), reticuloendothelial and other tissues (Id.). Indeed,
somatic mutations of p53 are thought to play a role in up to
one-half of all new malignancies documented yearly in Britain and
the United States, making this protein the most frequent target for
mutation in human cancers (Vogelstein (1990), 348 Nature 681-682:
Marx (1990), 250 Science 1209). Studies investigating gene-line
mutations of p53 in familial Li-Fraumeni syndrome, an inherited
susceptibility to cancers associated with p53 mutation, have shown
that small deletions, transpositions and point mutations affecting
conserved regions of the protein convert p53 from a suppressive
growth regulatory protein into a transdominant oncogene, which can
bind to and inactivate wildtype p53 (Gannon et al. (1990), 9 EMBO
J. 1595-1602; Malkin et al. (1990), 250 Science 1233-1238; and
Srivastava et al. (1990), 348 Nature 747-749). Second agents 9
which bind to mutant, but not wildtype, p53 are accordingly
preferred in certain embodiments of the present invention. The
precise nature and locations of transforming mutations in p53 has
been the subject of intense investigation, and is reviewed in
Hollstein et al. (1991), 253 Science 49-53, the teachings of which
are herein incorporated by reference. At least one monoclonal
antibody, PAb240, that recognizes mutant but not wildtype p53 has
been isolated, and the recognized epitope characterized (Gannon et
al. (1990), 9 EMBO J. 1595-1603; Stephen and Lane (1992), J. Mol.
Biol. 577-580; the teachings of both of which are incorporated
herein by reference). Second agents 9 that bind the epitope
recognized by PAb240 are particularly preferred in certain
embodiments of the invention.
[0053] Cyclins, cyclin dependent kinases, and cyclin associated
proteins together form nuclear complexes that control initiation
and progress through the cell cycle. Keyomarsi and Pardee (1993),
90 Proc. Natl. Acad. Sci. USA 1112-1116, and Xiong et al. (1993), 7
Genes and Dev. 1572-1583, the teachings of each of which are
incorporated herein by reference. Cyclins and cyclin dependent
kinases are classified, according to the presence therein of
conserved amino acid sequence motifs, as members of evolutionarily
conserved multigene families that determine and regulate cell
proliferation. The particular cyclins and cyclin dependent kinases
that are associated in nuclear cell cycle control complexes shift
subtly at different stages of the cell cycle (e.g., upon transition
from G.sub.1 to S or upon transition from G.sub.2 to M). Xiong et
al. report that expression and association patterns of cyclins and
associated proteins are deranged in transformed cells. Thus, as for
tumor suppressor gene products, malignant transformation may be
associated with the inappropriate display of a cryptic epitope in
one or more cyclins, cyclin dependent kinases or cyclin associated
proteins. Such a cryptic epitope might prevent normal association
between cyclins and cyclin dependent kinases, or might promote
inappropriate associations. In certain embodiments, then, second
agents 9 of the present heterobifunctional compounds bind
selectively to such cryptic cyclin-related epitopes. Keyomarsi and
Pardee report that one cycling Cyclin E, is significantly
overexpressed in breast carcinoma cells, relative to its expression
level in normal breast tissue. Accordingly, the accumulation of
cyclin E offers a phenotypic distinction between selected
(transformed) and nonselected (normal) cells in breast tissue. In
certain embodiments, second agents 9 of the present invention that
bind to Cyclin E thus offer the ability to selectively destroy
breast carcinoma cells.
[0054] Transcription factors are proteins that bind to specific
sites in cellular DNA (e.g., specific sequences, structures or a
combination thereof) and thereby regulate the expression of one or
more genes. Such sites in the cellular DNA are referred to herein
as endogenous genomic binding sites. Transcription factors can, by
binding to their cognate endogenous genomic binding sites, promote,
enhance or repress gene expression. Molecular Cell Biology, Ch. 11
Gene Control and Development in Eukaryotes, 400-412 (2d ed. 1990).
Transcription factors can be grouped into the following classes,
based upon similarities in protein structure in the regions thought
to interact with DNA: helix-turn-helix or homeobox proteins;
zinc-finger proteins; and amphipathic helical proteins, such as
leucine-zipper proteins. Second agents can thus be designed
according to the principles set forth herein to bind to one or more
structurally similar transcription factors. In some embodiments,
second agents mimic soluble modulating ligands that affect the
binding of ligand-responsive transcription factors, such as the
estrogen receptor, to the factor's endogenous genomic binding site.
In other embodiments, second agents mimic the endogenous genomic
binding sites. Such binding site mimics are referred to herein as
transcription factor decoys or ligand decoys. Binding affinity of a
given transcription factor for a decoy is preferably near the
affinity of the factor for its endogenous genomic binding site or
modulating ligand. That is, binding affinity of the factor for the
decoy is within about 100-fold of its affinity for the cognate site
or ligand (e.g., if K.sub.d(app) for the cognate site is 1 nM, the
K.sub.d(app) for the decoy is at most about 100 nM). Preferably,
affinity of the decoy is within about 10 fold that of the cognate
site or ligand. More preferably, affinity of the decoy exceeds that
of the cognate site or ligand. Particular transcription factor
decoys that mimic the sequences of endogenous genomic binding sites
for particular transcription factors are disclosed in Bielinska et
al. (1990), 250 Science 997-1000, and in Chu and Orgel (1992), 20
Nucl. Acids Res. 5857-5858, the teachings of each of which are
incorporated herein by reference. The present invention extends
these teachings to encompass transcription factor decoys that mimic
the structures of endogenous genomic binding sites for
transcription factors, including structures that are
sequence-independent.
[0055] In appropriate embodiments, second agents 9 are employed
that are decoys for transcription factors that control the
expression of genes relevant to the growth or survival of selected
cells, or to metabolic or secretory processes carried out by
selected cells. Exposure of selected cells to such compounds
results in transcription factor hijacking. That is, a transcription
factor bound to the decoy is titrated away from its natural genomic
binding site and becomes sequestered at the site of a genomic
lesion. Preliminary studies were carried out to confirm that a
vital transcription factor could be "hijacked" in this manner and
caused to bind to a genomic lesion. These preliminary studies
demonstrated that an HMG box transcription factor, upstream binding
factor (UBF), binds to cisplatin 1,2-d(GpG) intrastrand crosslinks
(G G) and to its natural genomic binding site with comparable
affinities. For these studies, human upstream binding factor (hUBF)
was used. hUBF binds to the upstream control element or UCE of the
human ribosomal RNA (rRNA) promoter and is an important positive
regulator of rRNA transcription (Jantzen et al. (1990), 344 Nature
830-836). rRNAs are required elements of the cellular protein
synthesis machinery. Thus, hUBF/promoter binding is relevant to the
proper functioning of the cell's protein synthesis machinery.
[0056] It should be noted that the particular nucleic acid used as
an hUBF decoy in the preliminary studies lacked sequence similarity
to the sequence of the endogenous genomic binding site for hUBF;
thus, transcription factor hijacking was accomplished by
structural, rather than sequence-specific, recognition.
Southwestern and Western blot analysis yielded results, presented
in FIG. 2, showing that in vitro translated hUBF bound to DNA
globally modified by cisplatin but failed to recognize unmodified
DNA or DNA containing adducts of the genotoxically inactive isomer,
trans-DDP. Proteins in crude HeLa cell extracts having molecular
weights that correlate to the known sizes of hUBF species displayed
a similar binding preference. DNase I footprinting analysis (FIG.
3) of the cisplatin-modified nucleic acid decoy showed that hUBF
protected the 14 bp DNA region that symetrically flanks the site of
a defined cisplatin G G adduct (Panel A, Lane 1). These results
directly demonstrate that hUBF binding prevented access to the
cisplatin lesion site by a sterically large DNA processing enzyme.
Competition studies (results presented in FIG. 5) established that
the cisplatin decoy efficiently inhibited the formation of
[hUBF-promoter] complexes. That is, the structural decoy was shown
to be an effective competitive inhibitor of the proper binding of
hUBF to its cognate, sequence specific, genomic binding site (the
UCE). The affinity of hUBF for G G was substantial (K.sub.d(app)=60
.mu.M; see FIG. 3, Panel B). For comparison, the K.sub.d(app) of
another HMG box protein, HMG1, for the cisplatin G G adduct has
been shown to be 370 nM (Pil and Lippard (1992), 256 Science
234-237). FIG. 5 also reveals a significant nonspecific binding
component of hUBF for its promoter. This is also observed with
other HMG box proteins, including lymphoid enhancer factor-1
(LEF-1), which binds with nominal specificity (20-40 fold) to its
putative genomic recognition sequence (Giese et al. (1991), 5 Genes
& Dev. 2567-2578). Footprinting studies (results presented in
FIG. 5) also established similarity between the hUBF protected
regions of the cognate genomic binding sequence (the UCE) and the
cisplatin decoy. From these results, it can be predicted that the
levels of cisplatin that accumulate in cellular DNA in vivo upon
treatment of a cancer patient with a cisplatin chemotherapeutic
regime are sufficient to titrate hUBF away from the ribosomal RNA
promoter.
[0057] The above-summarized preliminary studies lend insight into
the structural features of hUBF-cisplatin decoy complexes. The
cisplatin adduct was approximately centered within the 14 bp
protected region (FIG. 3), suggesting that the DNA binding
domain(s) is symmetrically placed relative to the adduct. As
discussed hereinabove, cisplatin adducts cause structural
distortions in duplex DNA. 1,2-Dinucleotide adducts are bent and
partially unwound in the area immediately associated with the
platinum coordination complex. The resulting angular structure thus
has an "elbow" at the lesion site. This elbow appears to remain
solvent exposed, even when the lesion is shielded by bound hUBF:
the phosphodiester bond immediately 5' to the lesion remained
sensitive to DNase I. This result is consistent with binding of
hUBF to the minor groove of duplex DNA, on the convex side of the
DNA bend. Others have reported similar findings concerning the
binding of other HMG box transcription factors, particularly LEF-1
and SRY (the testis determining factor) to their cognate genomic
binding sites through minor groove interactions (Giese et al.
(1991), 5 Genes & Dev. 2567-2578; van de Wetering and Clevers
(1992), 11 EMBO J. 3039-3044; King and Weiss (1993), 90 Proc. Natl.
Acad. Sci. U.S.A. 11990-11994).
[0058] hUBF, like SRY, exhibits both sequence-specific and
structure-specific modes of DNA recognition. The footprinting data
suggest that the structure-specific [hUBF-G G decoy] and
sequence-specific [hUBF-UCE] complexes share structural features.
In each case, a protected region symmetrically flanks a nuclease
sensitive site. DNA bending is the likely common feature of these
complexes. Indeed, a hallmark of the HMG domain is its propensity
to interact with bent DNA and also to induce bending in linear
sequences. SRY, to give one example, efficiently recognizes four
way DNA junctions with sharp angles (Ferrari et al. (1992), 11 EMBO
J. 4497-4506). Furthermore, SRY induces a sharp bend (85.degree.)
in a specific DNA sequence upon binding (Giese et al. (1992), 69
Cell 185-195). The specific interactions of the HMG domain with
bent DNAs may be attributed to its "L" shaped cleft, as reported
recently (Weir et al. (1993), 12 EMBO J. 1311-1319). hUBF probably
also bends DNA, although detailed structural studies have yet to be
performed. The DNase I hypersensitive site induced in the UCE upon
hUBF binding may indicate DNA bending because DNase I activity is
sensitive to structural features of DNA, including the width of the
minor groove (Drew and Travers (1984), 37 Cell 491-502). The
putative bend site is centered within a UCE region that is
protected from DNase I; interestingly, the GIG-induced DNA bend is
also centered within a DNase I-resistant region. Thus, it appears
that the bent and unwound DNA structure induced by G G mimics a
favorable DNA conformation that occurs during the formation of a
stable [hUBF-rDNA promoter] complex. A similar model was proposed
recently to explain structure-specific recognition by SRY (King and
Weiss (1993), 90 Proc. Natl. Acad. Sci. U.S.A. 11990-11994).
[0059] Results of in vitro competition assays (shown in FIG. 5)
further established that the cisplatin decoy interacts with hUBF by
substituting for the transcription factor's endogenous genomic
binding site (the UCE) in the rDNA promoter. By logical
implication, introduction of cisplatin decoys into the cellular
milieu is expected to hijack hUBF and induce disarray of cellular
processes normally dependent on proper [hUBF-promoter] complexing.
In particular, the formation of high affinity [hUBF-decoy]
complexes should reduce the amount of hUBF available for promoter
binding. The steep relationship between promoter occupancy and
nuclear hUBF concentration (FIG. 4) indicates that even a small
degree of sequestration of hUBF by cisplatin lesions can
significantly impair expression of nucleolar genes encoding rRNAs.
Thus, rRNA transcription, and therefore cellular protein synthesis,
will be compromised. Furthermore, binding of a sterically large
protein, such as hUBF, to cisplatin lesions impedes or inhibits DNA
repair. Indeed, studies have shown that, although G G adducts are
excised from cellular DNA in human cells (Fichtinger-Schepman et
al. (1987), 47 Cancer Res. 3000-3004), this repair process is
inefficient (Szymkowski et al. (1992), 89 Proc. Natl. Acad. Sci.
U.S.A. 10772-10776). Results presented herein showed that the 14 bp
region symetrically flanking the G G lesion in the cisplatin decoy
was strongly protected from nuclease cleavage. From this, it is
reasonable to predict that this region would also be shielded from
components of the enzymatic DNA repair machinery. In further
support of this prediction, the XPAC protein, which recognizes
damaged DNA and is essential for human nucleotide excision repair,
has a relatively low affinity for G G cisplatin lesions
(K.sub.d(app)>600 nM)(Jones and Wood (1993), 32 Biochemistry
12096-12104). XPAC, therefore, should not displace hUBF, which
binds much more avidly to cisplatin lesions. hUBF therefore acts as
an effective shield protecting cisplatin genomic lesions from
repair.
[0060] Both DNA repair and protein synthesis are likely to be more
critical for proliferating cells, such as those of tumors, than for
quiescent cells, such as those of normal differentiated tissue
(Mauck and Green (1973), 70 Proc. Natl. Acad. Sci. U.S.A.
2819-2822; Fraval and Roberts (1979), 39 Cancer Res. 1793-1797).
The numbers of intracellular hUBF molecules, and of cisplatin
genomic lesions formed in a typical round of chemotherapy, have
been calculated. Both are in the range of about
5.times.10.sup.4/cell (Bell et al. (1988), 241 Science 1192-1197;
Reed et al. (1993), 53 Cancer Res. 3694-3699). Biologically
significant and synergistic assaults on the viability of selected
cells should therefore follow from the cisplatin-hUBF interactions
predicted by both the hijacking and shielding models for cisplatin
genotoxicity.
[0061] Still other classes of proteins for which second agents 9 of
the present invention can be designed comprise nucleic acid
processing proteins, e.g., ribonucleic acid (RNA) processing
proteins, including proteins involved in splicing RNA gene
transcripts to produce messenger RNA (mRNA). In addition, second
agents can be designed that bind to other cellular macromolecules,
e.g., RNA transcripts or portions thereof of genes that are
actively expressed in selected cells but not in nonselected
cells.
Cell-Component Binding Compounds Useful as Second Agent 9
[0062] Turning now to the structural features of second agent 9, it
has been disclosed above that the second agent can be a ligand or
an analog or derivative thereof, that is bound by the
above-discussed protein preferentially associated with selected
cells. Such ligands include estrogens, progesterones, androgens,
glucocorticoids and other soluble hormones, toxins, clinical useful
analogs and metabolites thereof, both of intracellular and
extracellular origin, whether presently known or novel.
Heterobifuntional ligand-genotoxic agent compounds have been
prepared and subjected to preliminary studies that support
extension of the cisplatin-based lesion shielding concept according
to the first model described above. In one such compound, the
ligand biotin was linked, through the use of standard techniques,
to a genotoxic first agent. The particular first agent selected was
the photoactivatable drug trimethylpsoralen (TMP), but it should be
understood that any of the first agents disclosed herein could have
been used. Psoralen compounds intercalate into double-stranded DNA
at d(TpA) dinucleotides and form mono- and diadducts therewith upon
exposure to near-UV irradiation (Cimino et al. (1985), 54 Ann. Rev.
Biochem. 1151-1193). Biotin binds with extraordinarily high
affinity to the proteins avidin and strepatavidin (K.sub.d(app)
10.sup.-15M, Green (1975), 25 Adv. Protein Chem. 85-133) and thus
is widely used in research and clinical assays, such as
enzyme-linked immunosorbent assays (ELISA), that capitalize on
specific protein interactions to detect or quantitate a protein of
interest. In this study, the biotin-TMP compound was mixed with
duplex DNA comprising an additional, defined genomic lesion
(deoxyuridine, the deamination product of cytosine) located in the
immediate vicinity (within three base pairs) of the TMP lesion
site. As described more fully in the Examples, the biotin-TMP
compound effectively bound concurrently to duplex DNA and
streptavidin, although both binding affinities were lower in the
heterobifunctional compound than in the unconjugated precursors of
the first and second agents. Quantitative gel shift assays revealed
a K.sub.d of .about.1.5 nM between streptavidin and the immobilized
biotin. This K.sub.d value differs significantly from that of free
biotin for streptavidin or avidin (the K.sub.d for free biotin with
streptavidin or avidin is about 10.sup.-6 nM). Assuming that the
biotin domain in the TMP-biotin conjugate behaved similarly to free
biotin, the significant increase of the K.sub.d value (in other
words, the decrease in binding affinity between biotin and
streptavidin) was probably caused by the covalent binding of
TMP-biotin conjugates to DNA. This effect likely can be minimized
by further optimizing the linkage technique and nature of the
linker employed. Techniques such as those described herein can be
applied or adapted through no more than routine experimentation to
accomplish this goal. As described, an optimal distance between a
second agent ligand and the DNA helix should allow tight binding
between the ligand and the cell component and yet adequately shield
the DNA region vicinal to the adduct site (i.e., the genomic
lesion). Streptavidin, when bound to damaged DNA at genomic lesion
sites, shielded adjacent deoxyuridine lesions from repair by the
appropriate DNA repair enzyme, uracil gylcosylase. These results
are presented below in the Examples. The size of the shielded
region, at least 20 adjacent nucleosides, was comparable to that of
the DNA patch typically released by DNA excision repair
enzymes.
[0063] Other heterobifunctional ligand-genotoxic agent compounds,
in this case estradiol-chorambucil conjugates, have been prepared.
These programmed genotoxic compounds are shown herein to mediate
adherence of intracellular estrogen receptors (ER) to genomic
lesions inflicted by chorambucil and to produce selective
cytotoxicity. Again, it should be understood that, through
appropriate standard techniques, the estrogen ligand could have
been linked to any of the genotoxic first agents disclosed herein.
Guidance is presented herein for confirming that estrogen receptors
also can be used to shield genomic lesions effectively from repair
by the cellular enzymatic DNA repair machinery, or to enhance
uptake of the genotoxic compound by estrogen receptor positive
cells, thereby contributing to the demise of selected cells that
express estrogen receptors. According to the principles of the
present invention, heterobifunctional compounds programmed to
recruit the estrogen receptor to become a shield for genomic
lesions comprise genotoxic first agent, an optional linker, and a
second agent that affixes the receptor protein to the site of a
lesion in cellular DNA caused by the genotoxic agent. Preferably,
the genotoxic first agent is itself bifunctional and thus offers
the capability of forming intra- or interstrand crosslinks in
cellular DNA. Linking groups of varying length and molecular
composition allow the practitioner to optimize the present
compounds for concurrent binding of estrogen receptors and DNA. For
example, the molecular composition of the linker can be adjusted so
as to enhance solubility of the compounds under physiological
conditions, or to enhance cell membrane permeability thereof. The
length of the linker similarly can be adjusted to accommodate
accessibility of the ligand second agent to the hydrophobic ligand
binding pocket of the estrogen receptor while the genotoxic first
agent is bound to cellular DNA.
[0064] There is precedent indicating that estrogen ligands that are
affixed to large carrier molecules or to a solid support such as
agarose, can still attract and bind the estrogen receptor from
solution. The estrogen precursor estradiol has been linked at
either the 7.alpha. or 17.alpha. position to agarose, creating a
means to isolate the estrogen receptor from cell extracts by
affinity chromatography (Sica et al. (1973), 248 J. Biol. Chem.
6543-6558; Bucort et al. (1978). 253 J. Biol. Chem. 8221-8228;
Redeuilh et al. (1980), 106 Eur. J. Biochem. 481-493). DNA and
agarose both have polysaccharide character. Unlike agarose,
however, DNA monomer units of deoxyribose are linked by charged
phosphodiester groups and are also bonded to heterocyclic purines
or pyrimidines containing both nitrogen and oxygen atoms. Hydrogen
atoms bonded to nitrogens and oxygens form hydrogen bonds within
the helical DNA molecule, and can also form such bonds with
proteins and other diffusible molecules. Such associations could
assist in the formation of a lesion-shielding complex between the
cell component, the heterobifunctional compound and cellular DNA,
by analogy to interactions between cellular DNA and nuclear
proteins that determine DNA structure and regulate gene expression.
It is also possible, however, that hydrogen bonding could adversely
affect the ability of the estrogen ligand to bind its receptor.
Optimization of a linker disposed between the estrogen ligand and
the genotoxic agent should, however, project the ligand
sufficiently away from the DNA molecule, facilitating a high
affinity interaction with the bound shielding protein.
[0065] Because appropriate precursors are readily available, the
preparation of 17a linked derivatives of estradiol are the simplest
from a synthetic chemical viewpoint. For example, starting with
estrone (3-hydroxy-1,3,5[10]-estriene-17-one), substitution of a
short amino alcohol at the 17.alpha. position of estradiol can be
achieved using the Grignard reaction. Alternatively,
17.alpha.-ethynylestradiol can be used as a starting point for
attachment of a short alkyl amine. The reported synthetic routes to
7.alpha. estradiol derivatives are more complex, but should still
be within the abilities of those skilled in the art. Charpentier et
al. (1988), 52 Steroids 609-621, synthesized
7.alpha.-carboxymethyl-9(11)-ene derivatives of estrone and
estradiol starting with adrenosterone, in which a carboxymethyl
group was first introduced at the 7.alpha. position and then the A
ring was aromatized. In another published synthesis of 7.alpha.
derivatives, Bucort et al. (1978), 253 J. Biol. Chem. 8221-8228
described the conjugate addition of a Grignard reagent to a
canrenoate methylester, to ultimately produce a 7.alpha. carboxylic
acid derivative of estradiol.
[0066] From the examples presented below, one of skill can readily
adapt the methods used to demonstrate function of the
streptavidin-attracting heterobifunctional compound suitably for
demonstrating the biochemical and in vitro functionality of
heterobifunctional compounds that comprise a ligand decoy for the
estrogen receptor. Additional guidance is provided in the
prospective examples further set forth below, particularly for
assessing the ability of the ligand decoy to form interstrand
crosslinks in DNA; assessing whether an appropriate lesion
shielding complex is formed between damaged DNA and the estrogen
receptor; and assessing the present ligand-decoy compound for
selective killing in vitro of cells that express the estrogen
receptor. Similar techniques can be applied or adapted with no more
than routine experimentation to demonstrate functional properties
of heterobifunctional compounds programmed to attract other
ligand-responsive transcription factors. For example, the foregoing
techniques can be adapted routinely to demonstrate function of
heterobifunctional compounds that bind other steroid receptor
proteins, such as androgen receptors, glucocorticoid receptors,
progesterone receptors, and the like. Indeed, such studies are
appropriate for evaluating the functionality of other
heterobifunctional compounds, such as compounds designed to attract
oncogene products, tumor suppressor gene products, cyclins, and the
like and affix these cell components to the sites of genomic
lesions. Electrophoretic mobility shift and DNase I protection
analysis are suitable techniques generally for demonstrating
whether a particular heterobifunctional compound forms genotoxic
lesions, whether a chosen cell component is bound by a suitably
programmed heterobifunctional compound, and whether the resulting
complex is effective for shielding genomic lesions from the action
of enzymes that act on cellular DNA.
[0067] Still other heterobifunctional ligand-genotoxic agent
compounds that specifically recognize estrogen receptor (ER) now
have been prepared. A flowchart illustrating the preparation of a
heterobifunctional 2-phenylindole-chlorambucil compound,
1-[6-(N-2-ethoxy-(O-(3-(4-(N,N-bis(2-chloroethyl)-aminophenyl)propylamine-
)carbamoyl)amine)hexyl]-5-O-hydroxy-2-(4-hydroxyphenyl)indole (1)
is shown in FIG. 9 and discussed in Example 12. Heterobifunctional
ER-ligand decoy compound 1 is presently a preferred embodiment of
the invention, and has been demonstrated to bind to mammalian ER
both when in solution and when affixed to double-stranded DNA
(Example 13). Further, preferred compound 1 has been demonstrated
to be cytotoxic selectively to mammalian cells that express ER
(Example 14). This selective toxicity depends upon the chlorambucil
first agent portion of compound 1 and is not due to antiestrogenic
activity (Example 15). Thus, ER-ligand decoy compounds of the
present invention do not kill ER-expressing cells selectively in
the same manner as the therapeutic antiestrogen, tamoxifen. These
results illustrate practice of the invention with a
heterobifunctional compound programmed to affix a ligand-responsive
transcription factor (ER) to genomic lesion sites.
[0068] As is apparent from the preliminary studies carried out with
hUBF, second agent 9 can, in other embodiments, be a nucleic acid
that mimics an endogenous genomic binding site of a transcription
factor or other protein to be sequestered at genomic lesion sites.
Nucleic acid second agents can be single stranded, double stranded,
linear, branched, circular or a combination of these
configurations. Either RNA or DNA can be used. Through intrastrand
base pairing, linear or circular nucleic acid second agents can
adopt stable hairpin or dumbell configurations (Chu and Orgel
(1992), 20 Nucl. Acids Res. 5857-5858). Certain second agents
(transcription factor decoys) can resemble either the sequence or
the structure of the recognized transcription factor's endogenous
binding site. That is, the nucleotide sequence of the decoy can
comprise the sequence of the endogenous site, or a sequence
sufficiently homologous thereto to confer protein-binding activity
on the decoy. For example, the decoy sequence can be a conservative
variant of the endogenous sequence, such as a sequence that binds,
under stringent hybridization conditions, to the endogenous
sequence. Preferably, the decoy sequence is more than 50% identical
to the endogenous sequence. More preferably, it is more than 70%
identical, and even more preferably, it is more than 90% identical.
It is well known that the binding avidity of many nucleic acid
binding proteins that recognize specific nucleotide sequences can
be enhanced by nucleic acid regions adjacent to the actual binding
site. These flanking regions can be disposed 3' or 5' to the
specific, recognized sequence. As the nucleic acid binding protein
need not interact directly or strongly with nucleotides in the
flanking region, greater sequence variability can be tolerated at
such locations than in the binding site itself. Thus, decoys can be
constructed that comprise a core, conserved binding sequence
flanked by adjacent regions that modulate, e.g., enhance, binding
preference of the protein to the decoy relative to the endogenous
site. Similar principles can be applied to the construction of
nucleic acid decoys that mimic nucleic acid structures rather than
sequences. Structural features recognized by the protein can be
produced by folding, looping, kinking, adoption of higher ordered
structures (e.g., cruciforms) or of nonclassical helix
configurations (e.g., ZDNA) by the nucleic acid decoy. Optionally,
these structural features can be stabilized by non-nucleic acid
components of the decoy, such as crosslinking agents. Still further
variation can be introduced, and favorable properties (e.g.,
stability under in vivo conditions) emphasized, by the use of
nucleotide analogs or derivatives such as phosphorothioate analogs,
or O.sup.6- and/or O.sup.4-methylguanine derivatives, in the decoy
sequence.
[0069] An important class of nucleic acid second agents 9 includes
those known in the art as "aptamers". Aptamers are the products of
directed, also known as in vitro, molecular evolution. The term
"aptamer" was originally coined by Ellington and Szostak to
describe the RNA products of directed molecular evolution, a
process in which a nucleic acid molecule that binds with high
affinity to a desired ligand is isolated from large library of
random DNA sequences (Ellington and Szostak (1990), 346 Nature
818-822). The process involves performing several tandem iterations
of affinity separation, e.g., using a solid support to which the
desired ligand is bound, followed by polymerase chain reaction
(PCR) to amplify ligand-eluted nucleic acids. Each round of
affinity separation thus enriches the nucleic acid population for
molecules that successfully bind the desired ligand. In this
manner, Ellington and Szostak "educated" an initially random pool
of RNAs to yield aptamers that specifically bound organic dye
molecules such as Cibacron Blue (Id. at FIG. 2). Certain of the
aptamers obtained could discriminate between Cibacron Blue and
other dyes of similar structure, demonstrating specificity of the
technique. Aptamers can even be engineered to distinguish between
stereoisomers that differ only by optical rotation at a single
chiral center (Famulok and Swstak (1992), 114 J. Am. Chem. Soc.
3990-3991). Originally, it was thought that RNA aptamers would be
more suitable for ligand recognition, in view of established
knowledge of naturally occurring RNAs with higher ordered
three-dimensional structures (e.g., rRNA or transfer RNA, tRNA).
However, single-stranded DNA molecules produced by asymmetric PCR
amplification were also shown to be effective (Ellington and
Szostak (1992), 355 Nature 850-852). It should be noted that
aptamers can be prepared from nucleotide analogs, such as
phosphorothioate nucleotides, which can offer increased aptamer
stability under physiological conditions. Standard techniques are
available for linking nucleic acids, such as transcription factor
decoys and aptamers, to other chemical moieties, such as genotoxic
drugs, without substantial loss of protein-recognition capability
and genotoxicity.
[0070] The principles of directed molecular evolution encompass the
production of aptamers that bind with high affinity to proteins,
such as DNA binding proteins, including transcription factors
(Tuerk and Gold (1990), 249 Science 505-510; Famulok and Szostak
(1992). 31 Angew. Chem. Intl. Ed. Engl. 979-988, the teachings of
which are herein incorporated by reference). Recently, an aptamer
has been reported that binds with high affinity to the
extracellular protein thrombin (Bock et al. (1992), 355 Nature
564-566), and can impair thrombin catalyzed blood clot formation.
High affinity aptamers can be generated even against proteins for
which there is little or no structural or ligand-recognition
information available (Famulok and Szostak (1992), 31 Angew. Chem.
Intl. Ed. Engl. 979-988; see discussion concerning the HIV Rev
protein). Thus, aptamer second agents can be generated, through
available techniques, that bind to virtually any desired
selected-cell associated protein, whether or not the protein has a
known natural ligand or endogenous genomic binding site. This
flexibility offers great promise in the design of programmable
genotoxic drugs useful in selectively destroying neoplastic or
virally infected cells, such as cells infected with the human
immunodeficiency virus (HIV) or tumorigenic adenoma and papilloma
viruses. The aptamer-recognized protein can be a member of any of
the general classes discussed herein: transcription factors,
ligand-responsive transcription factors, oncogene products, tumor
suppressor gene products, cell cycle regulatory proteins, nucleic
acid processing proteins, nuclear structural proteins, and the
like. A preferred aptamer binds to the nuclear phosphoprotein p53.
A particularly preferred aptamer binds to a region of
tumor-associated mutant p53 that is cryptic in wildtype p53, such
as the PAb240 epitope (Gannon et al. (1990) 9 EMBO J. 1595-1602;
Steven and Lane (1992), 255 J. Mol. Biol. 577-583). As described
more fully in the examples, a population of aptamers that bind
selectively to the PAb240 epitope has been prepared.
Heterobifunctional compounds comprising an aptamer amplified from
this pool and thus programmed to bind mutant p53 can be assessed
for biomolecular and in vitro function through appropriate routine
adaptation of the techniques and guidelines set forth below in the
actual and prospective examples.
[0071] Yet another general class of second agents 9 includes
peptide ligands selected from so-called epitope libraries.
Libraries of random peptides of defined average length are
available, as are techniques for preparing additional such
libraries. Such libraries have been used for determining the
precise epitope recognized by an antibody of interest (Geysen et
al. (11984). 81 Proc. Natl. Acad. Sci. USA 3998-4002. Fodor et al.
(1991). 251 Science 767-773, the teachings of each of which are
incorporated herein by reference). At least one "living library"
has been constructed, from filamentous bacteriophage expressing
random peptide epitopes cloned into a viral coat protein (Scott and
Smith (1990), 249 Science 386-390, the teachings of which are
incorporated herein by reference). This technology offers the
advantages that phage displaying a peptide with favorable binding
characteristics can be affinity purified against a desired protein
component of selected cells (e.g., a transcription factor, cyclin,
intracellular receptor, or tumor suppressor gene product),
propagated in vivo using a bacterial host, and subjected to
techniques such as site-directed mutagenesis to improve further the
binding affinity for the desired protein. Through appropriate
genetic engineering techniques, a peptide optimized for binding in
this manner can be introduced into a high-expression host cell
(e.g., a bacterial host such as E. coli), optionally produced as a
cleavable fusion protein, and isolated in high yield. In this
manner, large amounts of a peptide second agent can be prepared and
linked, through standard techniques, to a genotoxic first agent to
produce the heterobifunctional compound disclosed herein.
Appropriate techniques for linking peptide second agents to
genotoxic first agents without incurring substantial loss of
protein-recognition capability or genotoxicity are known and
available.
[0072] Still another general class of second agents 9 include
organic and inorganic compounds isolated from libraries of
synthetic organic and inorganic compounds prepared by combinatorial
synthesis (Needels et al. (1993), 90 P.N.A.S. USA 10700-10704;
Ohlmeyer et al. (1993), 90 P.N.A.S. USA 10922-10926). Combinatorial
chemistry involves the simultaneous production of large sets of
related molecules which are then screened for a desired trait.
Early in its development, combinatorial chemistry was used to
generate libraries of variants of benzodiazepines which can target
a wide variety of receptors and enzymes, as described in Ellman et
al., U.S. Pat. No. 5,288,514, the disclosure of which is
incorporated by reference herein. More recent developments include
the generation and screening of a library of about 500 mercaptoacyl
prolines to identify a new treatment for hypertension and heart
disease. Gallop, et al. (1995), 114 J. Am. Chem. Soc. 7029 (1995).
For the purposes of this invention, a combinatorial library would
be created and then assayed for its binding affinity to a protein
preferentially associated with selected cells. The techniques of
combinatorial chemistry are well known in the art, and kits for
performing combinatorial chemistry are commercially available. For
example, Novabiochem, publishes a catalog of components for
generating combinatorial chemistry libraries. (Novabiochem, The
Combinatorial Chemistry Catalog including Solid Phase Organic
Chemistry Handbook, March 1998). Another vendor of reagents for
combinatorial chemistry technology is Irori of La Jolla, Calif.
Linkage Between First and Second Agents
[0073] In the heterobifunctional compounds disclosed herein, the
above-described first and second agents are linked together,
preferably covalently. In many embodiments, the first and second
agents are linked through covalent linker 7. In other embodiments,
linkage of the first and second agents is accomplished by
noncovalent association. In such embodiments, the first and second
agents optionally become linked to form compound 3 intracellularly.
Linkage thus can occur after either the first agent has bound to
cellular DNA, or the second agent has become complexed with the
cell component. One example of a noncovalent linker comprises
complimentary oligonucleotide strands (e.g., oligo(dG)/oligo(dC))
covalently attached, respectively, to the first and second
agents.
[0074] In most embodiments, however, the first and second agents
are linked directly by a covalent bond or indirectly through
covalent bonds to an organic linker. This organic linker comprises
a linear, branched or cyclic, aliphatic, aromatic or mixed
aliphatic and aromatic organic compound comprising preferably up to
about 20 carbon atoms, optionally in association with other atoms,
such as oxygen, nitrogen or sulfur, that occur naturally in
biological molecules. The organic linker can be, for example, a
peptide, oligosaccharide, oligonucleotide, carbamate or urea
derivative, such as an oligocarbamate peptide analog. Additional
examples of linkers include polymers assembled from linkable
monomers independently selected from ethyleneglycols, alkyldiamines
and the like such as polyethylene glycol, ureas; or
spermine/spermidine. The linker serves to space apart the binding
moieties of the first and second agents such that the
heterobifunctional compound disclosed herein can sterically
accommodate concurrent binding to cellular DNA and the cell
component. Yet, the linker does not separate the first and second
agents so far as to obviate shielding of the genomic lesion by the
cell component that is bound to the second agent. In certain
embodiments, the organic linker comprises up to about 12 carbon
atoms. In other embodiments, it comprises up to about 8 carbon
atoms. Yet in still other embodiments, such as where the second
agent must access a deep cleft or pocket in a recognized cell
component protein, the linker can comprise up to about 30 carbon
atoms. Whether covalent linkage of the first and second agents is
direct or indirect (through the optional linker), the linkage is
stable under physiological conditions, particularly intracellular
conditions. That is, the linkage is resistant to cleavage by
hydrolysis or other biochemical processes, including enzymatic
processes. For this reason, linkers comprising amide or ester bonds
are not presently preferred. Conversely, linkers comprising
carbamate or urea moieties are preferred herein due to their
stability and hydrophilicity characteristics. For example,
oligocarbamate peptide analogs comprised of aminocarbamate monomers
linked through a carbamate backbone have been reported to be stable
for at least 150 minutes in the presence of trypsin or pepsin (Cho
et al. (1993), 261 Science 1303-1305).
[0075] Linkage of the first agent to the second agent, either
directly or through the optional organic linker, can be
accomplished by applying routine chemical or biochemical
techniques, or modifications thereof that will be readily apparent
to those of skill in the art. The particular linkage reactions
carried out will be determined by the types of first and second
agents to be joined to produce a desired heterobifunctional
compound. It will be recognized by those of skill in the art that
the optional linker provides an opportunity to improve the
hydrophilicity or cell membrane permeability qualities of the
present compounds, by including moieties that confer these
properties within the organic linker.
Uses for Heterobifunctional Programmable Genotoxic Compounds
[0076] The heterobifunctional programmable genotoxic compounds
disclosed herein are useful in a method for destroying selected
cells in a heterogenous cell population. Broadly, the method
comprises the steps of contacting the heterogenous cell population
with a heterobifunctional compound as disclosed above, and
incubating (maintaining contact of) the cell population with the
compound for a period of time sufficient for the compound to
internalize within cells, bind to cellular DNA and bind to a cell
component preferentially associated with selected cells so as to
produce a steric shield that protects genomic lesions from repair.
As mentioned previously, the heterogenous cell population can
comprise cells of a unicellular or multicellular organism, and can
comprise cells maintained in culture, cells withdrawn from a
multicellular organism, or cells present in the tissues or organs
of a multicellular organism. That is, the method can be practiced
in vitro, ex vivo (using a sample, such as a biopsy, withdrawn from
a multicellular organism such as a mammal, e.g., a human), or in
vivo, by local or systemic administration to a multicellular
organism. The recognized cell component can be one naturally
associated with the cell, or one intentionally introduced into the
cell, e.g., by genetic engineering techniques. The present method
therefore offers the prospect of broadening the range of biological
selection methods available, e.g., for the production of
recombinant proteins or for the isolation of cells with improved or
desirable characteristics.
[0077] Extensive discussion has been devoted herein to programmable
genotoxic compounds that are appropriate for co-opting cell
components that phenotypically distinguish, for example, dividing
cells such as transformed (malignant or neoplastic) cells from
normal cells, virally infected cells from uninfected cells, and
cells of a pathogenic organism from cells of a host organism. It
should be understood that the method disclosed herein can be
practiced to achieve the selective killing of cells that are
phenotypically distinguishable from other cells of a heterogenous
cell population on any of these grounds. In particular, it should
be understood that the method can be used to achieve selective
killing of neoplastic (transformed) cells of colorectal,
reproductive tract, hepatic, lymphoid, mammary, myeloid, neurologic
or respiratory tract origin. Cells that are of reproductive tract
origin can be more specifically, of ovarian, uterine, endometrial,
cervical, vaginal, prostate, or testicular origin. Cells that are
of mammary origin can be more specifically, of breast origin. As is
apparent from the disclosure herein, selective killing of such
cells can be accomplished through the use of second agents that
recognize intracellular proteins associated with malignant
transformation. Thus, for example, heterobifunctional compounds can
be programmed or designed to selectively destroy malignant cells
that express an oncogene product (e.g., erbB, abl or myc) a mutant
tumor suppressor gene product (e.g., mutant p53) or an aberrant
cyclin or cyclin-dependent kinase. Appropriate heterobifunctional
compounds could comprise a genotoxic agent linked to a ligand, an
aptamer, a binding polypeptide or a small organic molecule, e.g.,
produced by combinatorial synthesis, that binds the target
macromolecule. Alternatively, compounds can be programmed to
selectively destroy cells whose survival or proliferation are
dependent on the expression of certain genes by incorporating a
second agent that is a transcription factor decoy. Malignant cells
whose proliferation is driven by an aberrantly expressed
ligand-responsive transcription factor, such as an estrogen
receptor, androgen receptor or progesterone receptor, can be
selectively destroyed by compounds incorporating ligand mimics as
second agents. Such ligand mimics include androgens, estrogens,
progesterones, glucocorticoids and receptor binding analogs and
derivatives thereof (e.g., the clinically relevant estrogen analog,
tamoxifen). For example, estrogen or estrogen analog-containing
heterobifunctional compounds can be used to achieve selective
killing of breast or ovarian cancer cells, progesterone compounds
can be used similarly to kill uterine or endometrial cancer cells,
and androgen compounds can be used to kill prostate cancer
cells.
[0078] Presently preferred estrogen analog-containing compounds
include 2-phenylindole compounds and analogs thereof, including
1-[6-(N-2-ethoxy-(O-(3-(4-(N,N-bis(2-chloroethyl)aminophenyl)propylamine)-
carbamoyl)amine)hexyl]-5-O-hydroxy-2-(4-hydroxyphenyl)indole (1).
Such compounds are anticipated herein to destroy selectively
malignant estrogen receptor positive cells of breast or
reproductive tract origin, especially cells of ovarian origin.
Similarly, it is anticipated that androgen and androgen
analog-containing compounds prepared by application of the
principles of the present invention will destroy selectively
malignant, androgen receptor positive cells of reproductive tract
origin, especially cells of prostate origin.
[0079] Heterobifunctional compounds of the present invention also
can be designed to destroy selectively cells of an infectious
organism, either in vitro or in vivo, that are present in a
heterogenous cell population comprising cells of a host or infected
organism, and cells of an infectious organism such as a bacterium,
a fungus, a virus or a parasite. Thus, compounds disclosed herein
are expected to be particularly useful in maintaining the health
and integrity of cultured cells (e.g., mammalian cells) in vitro,
as well as in the treatment of infectious diseases in vivo caused
by pathogenic organisms including those with acquired resistance to
currently available antibiotic, antifungal or antiparasitic drugs.
Infectious diseases for which the availability of programmable
genotoxic compounds are urgently needed accordingly include, but
are not limited to, septic astound infections, hospital-acquired
infections tuberculosis, malaria and amoebic dysentery. Other
examples, particularly of parasitic diseases, for which
programmable genotoxic compounds offer the potential to expand the
range of available genotoxic agents, include schistosomiasis,
filiariasis, Chagas disease, leishmaniasis, sleeping sickness,
toxoplasmosis, pneumocystosis, giardiasis, trichomoniasis,
croptosporidiosis, and the like. Harrison's Principles of Internal
Medicine, Part 5 Infectious Diseases, Ch. 156-172. Certain of these
diseases are relatively common among cosmopolitan communities,
while others present severe threats to the populace of developing
nations.
[0080] Alternatively, the present compounds can be used in vitro to
enrich a heterogenous cell population for cells having a desirable
characteristic, or cells lacking an undesired characteristic. This,
the present compounds offer new alternatives to current methods
for, e.g., isolating a hybridoma cell producing a desired antibody
from a heterogenous cell population comprising primary antibody
producing cells and an immortalized fusion partner cell line.
Alternatively, the present compounds expand the range of genetic
selection agents useful for separating a desired cell transfected
with heterologous nucleic acids from a cell population comprising
unsuccessful transfectants.
[0081] Those of skill in the art will readily understand and
appreciate that the incubation period needed to achieve selective
cell killing will vary widely, depending on the circumstances under
which the invention is practiced. In many instances, the time
period needed to achieve selective killing of cultured cells or
suspensions of unicellular organisms or of cells withdrawn from a
multicellular organism will be less than the time needed to achieve
selective killing of cells in vivo in a multicellular organism. For
in vivo use to destroy selected cells in the tissues of a
multicellular organism (e.g., a mammal) the protocols in which the
drugs are used will vary depending on the location of cells to be
destroyed, replicative rate of the cells, level of repair
proficiency of the cells, dose of heterobifunctional compound
administered, route of administration (generally either systemic or
local, and either enteral or parenteral), and pharmacokinetic
profiles of clearance and tissue uptake of the compound. Variables
affecting the dose thus include, but are not limited to, the nature
(e.g., species or tissue type), quantity and accessibility (i.e.,
body compartment location) of selected cells to be destroyed, and
the nature, genotoxicity, and affinity of the compound for the
recognized cell component. The present compound can be combined
with a pharmaceutically acceptable carrier or excipient for
formulation as a liquid, suspension, solid, salve, ointment or the
like, suitable for oral, nasal, intravenous, intraperitoneal,
topical, subdermal, intramuscular, or other routes of
administration. The present compound can be administered in a
single dose (e.g., a bolus injection), a series of doses of
equivalent, escalating, decreasing or intermittently varied
quantity, or infused over a period of time (e.g., by intravenous
drip or infusion), or by release from a slow-release delivery
vehicle. The appropriate dose of the present compound will of
course be dictated by the precise circumstances under which the
invention is practiced, but will generally be in the range of 0.01
ng to 10 g per kg body weight, preferably in the range of 1 ng to
0.1 g per kg, and more preferably in the range of 100 ng to 10 mg
per kg.
[0082] If desired, the degree of selective cell killing achieved
can be ascertained through standard, widely available techniques,
such as visual or microscopic inspection, biochemical, chromogenic
or immunologic methods for detecting products of selected cell
lysis, and the like. Such techniques can be used to establish both
the dose and time period effective to accomplish objectives of the
present invention under particular circumstances. Once effective
doses and time periods are established, it may be no longer
necessary to monitor the progress of selective cell killing.
[0083] Practice of the invention will be still more fully
understood from the following examples, which are presented herein
for illustration only and should not be construed as limiting the
invention in any way.
Example 1
Western and Southwestern Blotting Studies with hUBF
[0084] Probe Preparation. The DNA probe used for southwestern
blotting was a 422 base pair (bp) AvaI restriction fragment excised
from M13mp19 replicative form DNA. Platinated probes were prepared
by treating the AvaI-digested DNA with cisplatin or trans-DDP, and
the formal bound drug/nucleotide ratios (rb) were determined by
using atomic absorption: spectroscopy as described in Donahue et
al. (1990), 29 Biochemistry 5872-5880.
[0085] Western and Southwestern Blotting Technique. HeLa whole cell
extracts (WCE) were prepared by the sonication procedure of Samson
et al. (1986), 83 Proc. Natl. Acad. Sci. U.S.A. 5607-5610. The 97
kDa hUBF species was synthesized by in vitro transcription and
translation from the plasmid pTbGUBF1 as reported in Jantzen et al.
(1992), 6 Genes & Dev. 1950-1963. In vitro translated hUBF was
quantitated by the incorporation of .sup.35S-methionine. Protein
samples (75 mg WCE or 8 ng hUBF) were resolved on 5-15% gradient
SDS polyacrylamide gels and transferred to nitrocellulose
membranes. Parallel blots of HeLa whole cell extracts (WCE) and in
vitro translated hUBF (hUBF) were probed with various .sup.32P
labeled DNA fragments (southwestern analysis, panels A-C of FIG. 2)
or antiserum against hUBF (Anti-NOR-90) (panel D of FIG. 2). For
southwestern analysis, the air dried membranes were processed as
reported in Toney et al. (1989), 86 Proc. Natl. Acad. Sci. U.S.A.
8328-8332. In the probing step, the labeled DNA was present at
about 5.times.10.sup.4 cpm/ml and the nonspecific competitor
poly(dI-dC)-poly(dI-dC) at 5 mg/ml. The blot shown in Panel A was
probed with the cisplatin (cis-Pt-422) modified probe, that shown
in Panel C with the trans-diamminedichloroplatinum(II)
(trans-Pt-422) modified probe, and that shown in Panel B with
unmodified (Un-422) probe. HeLa proteins recognizing cis-Pt-422 are
listed by molecular weight to the left of Panel A. The rb values
for probes modified by cisplatin and trans-DDP were 0.043 and
0.052, respectively. During autoradiography, a 0.254 mm thick
copper sheet was used to block .sup.35S emissions selectively from
the in vitro translated hUBF. For western analysis (Panel D), the
filter was probed with a 1/250 dilution of antiserum to human
NOR-90 (hUBF) obtained as a gift from E. K. L. Chan (Chan et al.
(1991), 174 J. Exp. Med. 1239-1244). Antibody binding was
visualized through standard techniques, using a chemiluminescent
detection system commercially available from BioRad. The positions
of both HeLa and in vitro translated hUBF are shown. A 120 kDa
species of unknown identity was also visualized in the WCE with
Anti-NOR-90.
[0086] Results. Protein blots of human HeLa cell extracts (FIG. 2A)
probed with cisplatin modified DNA (southwestern analysis) revealed
species of M.sub.r(app) 97, 94 and 28 kDa. Unmodified DNA or DNA
modified with the clinically ineffective trans-DDP compound was not
bound by these proteins (Panels B and C), although a 105 kDa
nonspecific DNA binding protein was detected with each of the three
DNA probes. The 28 kDa species has recently been identified as the
abundant chromatin protein HMG1 (Pil and Lippard (1992), 256
Science 234-237; Hughes et al. (1992), J. Biol. Chem. 13520-13527).
The precise functions of HMG1 remain unclear although it has been
proposed to play roles in the maintenance of chromosome structure
and the alteration of DNA topology, and may therefore be important
for transcription and DNA replication (Bustin et al. (1990), 1049
Biochem. Biophys. Acta 231-243). Since the HMG box is a unifying
feature of many cisplatin lesion recognition proteins, it was
postulated that the 97 and 94 kDa proteins possess this DNA binding
domain. The RNA polymerase I transcription factor hUBF contains
several regions of homology to HMG1 (Jantzen et al. (1990), 344
Nature 830-836) and exists as both 97 and 94 KDa species owing to
an alternative splicing event (Chan et al. (1991), 174 J. Exp. Med.
1239-1244). Western blot analysis with hUBF antiserum demonstrated
that the hUBF doublet resembles the bands detected by southwestern
analysis (compare FIG. 2, Panels A and D). From these observations,
it was postulated that hUBF binds to cisplatin DNA lesions. This
postulate was confirmed by southwestern blot analysis of in vitro
translated hUBF (FIG. 2A, lane 2).
Example 2
DNase I Footprinting Studies of the [hUBF-Cisplatin] Complex
[0087] Probe Preparation. A 100 bp DNA fragment containing a
single, centrally located 1,2 intrastrand
cis-[Pt(NH.sub.3)2].sup.2+d(GpG) crosslink (G G-100) and the
analogous unmodified fragment (Un-100) were used as both competitor
DNAs and probes in hUBF footprinting experiments. These DNA
fragments were kindly provided by P. Pil and S. J. Lippard (Pil and
Lippard (1992), 256 Science 234-237). The adduct-containing strand
of G G-100 and the analogous unmodified strand of Un-100 were 5'
end-labeled with .gamma..sup.32P-ATP (>6,000 Ci/mmole), using
polynucleotide kinase according to standard procedures. The 5' end
of the unadducted strand was removed with AvaI to generate the 90
bp footprinting probes. These were purified by passage through
Sephadex G-25 Quickspin.TM. columns (Boehringer Mannheim).
[0088] DNase I Footprinting Technique. Homogeneous HeLa hUBF was
used to generate DNase I footprints on both rRNA promoter
(described below) and platinated DNA probes. Footprinting was
performed essentially as described in Bell et al. (1988), 241
Science 1192-1197. hUBF was added to footprinting reactions
containing the appropriate labeled DNA probe 10.sup.3-10.sup.4 cpm,
0.7-50 pM, depending on the experiment) and binding buffer (25 in M
Tris-HCl pH (7.9), 14 mM MgCl.sub.2, 0.5 mM dithiothreitol, 10%
glycerol, 50 mM KCl, 0.05% Nonidet-P40, 2.5 mM CaCl.sub.2) in a
total volume of 50 ml. The binding reactions were incubated for 10
min. at 30.degree. C. and then digested with DNase I (Worthington
DPFF grade) for 1 min. at 25.degree. C. The DNase I reactions were
terminated by adding a solution of 20 mM EDTA, 1% SDS, 0.2 M NaCl,
and 50 mg/ml yeast total RNA. Samples were phenol/chloroform
extracted, ethanol precipitated, and electrophoresed according to
standard procedures on denaturing wedged (0.4-1.5 mm) sequencing
gels (6% or 12% for promoter footprints or G G-100 footprints,
respectively) at 70 W. Gels were fixed, dried and exposed with an
intensifying screen to preflashed X-ray film at 80.degree. C., and
analyzed by using a Molecular Dynamics PhosphorImager.TM. imaging
machine.
[0089] Results. As shown in FIG. 3, Panel A, 400 pm hUBF was
sufficient to protect the area of the probe immediately adjacent to
the defined G G adduct from DNase I cleavage (compare lanes 1 &
2). A distinct protection pattern was observed in the 14 bp region
encompassing the adduct, providing direct evidence that hUBF
recognizes the structural distortion induced by G G. The relevant
sequence is shown to the left, and the protected residues are
displayed within the box. The broken line indicates a residue
immediately 5' to G G that remained DNase I-sensitive. The
established structural features of the G G adduct include helix
bending (34.degree.) toward the major groove (Bellon and Lippard
(1990), 35 Biophys. Chem. 179-188) and unwinding (-13.degree.)
(Bellon et al. (1991), 30 Biochemistry 8026-8035). No such
protection is afforded the analogous unmodified 100-mer (lane 3),
which gave the same cleavage pattern both in the presence and in
the absence of hUBF (lanes 3 & 4). Cleavage patterns of G G-100
and Un-100 near the cisplatin adduct should be directly comparable
(lanes 2 & 4). Panel B shows the PhorphorImager
semiquantitative profile of hUBF binding to G G-100. Y is the
fractional saturation of G G-100 and was estimated by monitoring
the intensity of three bands in the protected region at each hUBF
concentration. The data fit the equation K.sub.d=[hUBF][G
G-100]/[hUBF-G G-100] when K.sub.d=60 pM. The protein concentration
giving half-maximal binding (K.sub.d(app) is indicated by the
broken line. The labeled probe was present at 20 pM (10.sup.4 cpm).
These results indicate that [hUBF-cisplatin] complex formation is
exceptionally favorable, in energetic terms. From the shape of the
semiquantitative binding profile, it is also apparent that binding
is non-cooperative.
Example 3
DNase I Footprint Studies of the [hUBF-DNA Promoter] Complex
[0090] Probe Preparation. For footprinting studies, the
EcoRI-BstEII restriction fragment of pSBr208 containing the -208 to
+78 region of the human rRNA gene was either 5' or 3' end-labeled
on the noncoding strand. pSBr208 was digested with EcoRI and the 5'
phosphate was removed with calf intestinal phosphatase.
EcoRI-digested pSBr208 was 5' end-labeled with .gamma..sup.32P-ATP
(>6.000 Ci/rmole) and subsequently digested with BstEII. The 286
bp footprinting probes where purified on 5% polyacrylamide gels and
electroeluted. In cases where higher specific activity footprinting
probes were required, the noncoding strand was 3' end-labeled by
using the Klenow enzyme in the presence of [.alpha.-.sup.32P]-dATP,
[.alpha.-.sup.32P]-dCTP, and [.alpha.-.sup.32P]-dGTP (>6,000
Ci/mmole).
[0091] The DNase I footprinting technique described in the
preceding example was followed in the present promoter-binding
studies.
[0092] Results. The biological significance of cisplatin adduct
recognition by hUBF ultimately depends on the affinity of the
interaction. The interaction of hUBF with rDNA accordingly provides
a useful benchmark value for a biologically relevant affinity. The
upper panel of FIG. 4 shows the rDNA binding profile at hUBF
concentrations ranging from 7-78 pM. The formation of
[hUBF-promoter] complexes resulted in DNase I hypersensitivity at
positions -20 and -95 in the CORE and UCE elements, respectively.
In addition, the 40 bp region that symmetrically flanks -95 became
refractory to cleavage (Bell et al. (1988), 241 Science 1192-1197).
The degree of promoter occupancy was most easily visualized by the
increased DNase I sensitivity of the -95 position in the upstream
control element (UCE). The 3' labeled probe used to generate the
results shown in FIG. 4 was present at 0.7 .mu.M (10.sup.3 cpm).
Bands thus appear as doublets due to incomplete labeling.
[0093] hUBF binding was next quantitated by measuring the intensity
of the enhanced cleavage at -95. In the bottom panel, intensity is
reported to the left in arbitrary PhosphorImager units (PIU), and,
to the right, is expressed as the apparent fractional saturation
(Y). The protein concentration giving half-maximal binding
(K.sub.d(app), 18 .mu.M) is indicated by the broken line. Thus,
hUBF binds tightly to its endogenous site(s) in the rDNA promoter.
It should be noted that the affinities of hUBF for promoter
sequences and for the cisplatin decoy are comparable, differing by
only three-fold. This suggests that cisplatin adducts can be
effective decoys for hUBF in the cellular milieu. It should further
be noted that the UCE footprint qualitatively resembles that
observed for G G-100. In both complexes, a protected region
symmetrically flanks a nuclease sensitive site. The shape of the
hUBF-promoter binding profile reveals that the fraction of bound
promoter (Y) increases sharply over a narrow range of hUBF
concentrations, indicating that binding is cooperative. A Hill plot
of these data yielded a best fit line (r=0.997) with a Hill
constant (n.sub.H) of 2.7, indicating positive cooperativity.
Cooperativity has also been reported for Xenopus UBF binding to
enhancer repeats (Putnam and Pikaard (1992). 12 Mol. Cell. Biol.
4970-4980). An important consequence of cooperativity in the
context of the transcription factor hijacking model is that a small
decrease in the pool of free nuclear hUBF can strongly decrease
promoter occupancy.
Example 4
Competitive Inhibition of [hUBF-rDNA Promoter] Complexing by
Cisplatin Decoys
[0094] From the comparable values of the hUBF affinity constants
for cisplatin adducts and the rDNA promoter, it seemed likely that
cisplatin adducts should be effective competitive inhibitors of
[hUBF-promoter] complex formation. Accordingly, a competition study
was carried out, using the probe preparation and DNase I
footprinting techniques discussed in the preceding examples.
[0095] Competitive Technique. Purified HeLa UBF was added to all
samples, except the negative control (shown in lane 1 of FIG. 5),
to a final concentration of 160 pM. This level of hUBF is safely
above that producing an apparent fractional saturation (Y) of 1 in
the positive control (lane 2). The 5' labeled probe was present at
46 pM (10.sup.4 cpm). Un-100 (lanes 3-6) and G G-100 (lanes 7-12)
were added as unlabeled competitors to the final concentrations
(nM) listed. The competitive effect was estimated by measuring Y of
the promoter probe. Y values are shown at the bottom. Lanes 1 and 2
were used as standards to calculate Y in lanes 3-12.
[0096] Results. FIG. 5 shows that G G-100 efficiently antagonized
hUBF-promoter interactions. The reduced intensity of bands at
positions -21 and -95 in the CORE and UCE elements, and the
reappearance of bands between positions -75 and -115 illustrate
this effect (lanes 7-12). At a saturating concentration of hUBF,
the formation of promoter complexes was completely inhibited by a
platinum adduct concentration of 5.times.10.sup.9 M (lane 11),
which is well below the adduct levels in cancer patient DNA
(10.sup.4-10.sup.5/cell, or 10.sup.-7-10.sup.-6 M)(Reed et al.
(1993), 53 Cancer Res. 3694-3699). The corresponding unmodified
competitor DNA (Un-100) was a 10-30 fold weaker competitor of hUBF
than G G-100 (lanes 3-6). Since Un-100 contains up to 100
overlapping nonspecific binding sites compared to the one specific
binding site in G G-100, the preference of hUBF for a platinated
versus an unplatinated site may be as high as 1-3.times.10.sup.3
fold. These results directly support the view that cisplatin decoys
effectively hijack hUBF, sequestering this transcription factor
away from its endogenous genomic binding site and leaving the rDNA
promoter unoccupied. From these results, disarray of the cellular
protein synthesis machinery can be predicted.
Example 5
Demonstration that a Heterobifunctional Compound can Mediate
Binding of a Chosen Protein to a Genomic Lesion Site
[0097] TMP-biotin lesion conjugate. A 17-mer oligonucleotide,
referred to as U-17, was synthesized by standard phosphoramidite
chemistry. U-17 comprised a single, centrally-located 5'-TA-3'
site, along with a uracil deoxynucleotide located three bases away
from the TA site on the 3' side. The oligomer was purified on a 20%
denaturing (7 M urea) polyacrylamide gel (acrylamide/bis, 19:1) and
electroeluted by using an Amicon centrilutor. Urea was removed from
the oligomer by several distilled water washes in Amicon Centricon
3.TM. microconcentrators. Purified U-17 was 5'-end labeled with
[.gamma.-.sup.32P] ATP (6000 Ci/mmole, New England Nuclear) by
using T4 polynucleotide kinase (New England Biolabs) according to
standard techniques. Unincorporated label was removed by
centrifugation through a pre-packed G-25 column
(Boehringer-Mannheim). Labeled U-17 was then annealed to its
unlabeled complementary strand. TMP-biotin conjugate (dissolved in
50% (v/v) acetonitrile) was then added to the duplex oligmer
solution with the molar ratio of TMP-biotin to base pair at about
1000:1. After being incubated at room temperature for 10 min, the
mixture was placed on a chilled surface and subjected to near UV
irradiation with a 15-W General Electric lamp (maximum output at
365 nm). The final irradiation dose was about 85 kJ/m.sup.2. The
resulting irradiated mixture, now comprising TMP-biotin lesioned
U-17, was separated on a 20% denaturing polyacrylamide gel. A gel
slice containing monoadducted TMP-biotin U-17 strand was cut out,
and the lesioned DNA was purified by electroelution as described
above. Finally, the lesioned U-17 strand was annealed to its
cognate unlabeled complementary strand to form the double-stranded
lesioned probe.
[0098] Gel Mobility Shift Assay. The binding of streptavidin to
U-17 monoadducted with the TMP-biotin conjugate was measured by
incubating the probe with streptavidin (Pierce) in 10 .mu.l of
binding buffer [25 mM Tris-HCl (pH 7.4), 100 mM NaCl and 1.5
MgCl.sub.2] at room temperature for 10 min and electrophoresing the
mixture on a 5% nondenaturing polyacrylamide gel (acrylamide/bis,
29:1) at 4.degree. C. A constant amount of the lesioned U-17 (3200
cpm, .about.0.1 nM) was used in each incubation, with the
concentration of streptavidin varied from 0 to 50 nM. Free d-biotin
(0.4 mM) was added into the incubation(s) where indicated. After
electrophoresis, the gel was dried and exposed to x-ray film with
an intensifying screen. The dried gel was also exposed to a
PhorphorImager screen and the data were analyzed with IMAGEQUANT
software (Molecular Dynamics, Sunnyvale, Calif.).
[0099] Results. Streptavidin retarded the electrophoretic mobility
of U-17 fragments monoadducted with the TMP-biotin conjugate (FIG.
6, Panel A). The retardation was caused by the binding of
streptavidin to the biotin inserted into the DNA because free
biotin reversed the retardation, presumably by competing with the
immobilized biotin for streptavidin (lane 8 in Panel A).
Quantitation of the data by IMAGEQUANT software of Molecular
Dynamics gave rise to a binding curve (Panel B). The streptavidin
concentration for the half-maximum binding (C.sub.1/2) was about
1.5 nM, suggesting that the K.sub.d between streptavidin and the
immobilized biotin was also about 1.5 nM. Streptavidin showed
little binding activity to either U-17 oligomer or U-17
monoadducted with just a psoralen derivative (data not shown). It
should be pointed out that the K.sub.d value differs quite
significantly from that of free biotin with streptavidin or avidin.
As discussed previously, the observed increase of this K.sub.d
value was possibly attributable to steric effects exerted on the
streptavidin-biotin binding by the adduction of TMP-biotin
conjugates to DNA.
Example 6
Demonstration that Lesion-Bound Streptavidin Hinders Access by a
DNA Repair Enzyme
[0100] Uracil Glycosylase Protection Assay. Double-stranded,
TMP-biotin modified U-17 obtained as described earlier was used as
the probe in this assay. The probe (4000 cpm, .about.0.15 nM for
each reaction) was incubated first in 12 .mu.l of glycosylase
buffer [30 mM Tris.times.HCl (pH 7.4), 50 mM KCl and 5 mM
MgCl.sub.2] at room temperature for 10 min in the presence of
streptavidin (36 ng, .about.50 nM) where indicated. In some
incubations, 0.4 mM free d-biotin was added. After the incubation,
3 .mu.l (0.15 units) of uracil glycosylase (Boehringer-Mannheim)
was added, and the mixtures were then incubated at 37.degree. C.
for 5 min to 40 min. At the end of each incubation. 85 .mu.l
freshly prepared 1.25 M piperidine (Fisher) was added, and the
samples were subsequently heated at 90.degree. C. for 1 hr. Since
an apurinic site in a DNA molecule is labile to alkali cleavage,
Lindahli and Andersson (1972), 11 Biochemistry 3618-3623,
piperidine treatment as stated above would have resulted in DNA
strand breaks if any apurinic sites were generated from the uracil
glycosylase treatment. The samples were vacuum centrifuged to
remove the piperidine and washed by resuspension in distilled water
and followed by vacuum centrifugation again. Washed samples were
finally resuspended in denaturing loading buffer [80% (v/v)
recrystallized formamide, 0.1% (w/v) xylene cyanol and 0.1% (w/v)
bromphenol blue], analyzed on a 20% denaturing polyacrylamide gel.
The gel was exposed (without being dried) to an x-ray film with an
intensifying screen at -80.degree. C.
[0101] Results. As indicated in FIG. 7, streptavidin, when
complexed with the TMP-biotin DNA adducts, inhibited the removal of
a nearby uracil base by the uracil glycosylase (compare lanes 3-6
with lanes 7-10). The inhibition was substantially reversed when
free biotin was added (lanes 11-14). The TMP-biotin DNA adducts
were stable even after being heated at 90.degree. C. for 1 hr (lane
1). A small fraction of the TMP-biotin adducts was removed when the
probe was subjected to piperidine treatment [band (b) in lane 2;
bands (b) and bands (d) in lanes 3-14].
Example 7
Demonstration that Lesion-Bound Streptavidin Acts as a Steric
Shield
[0102] DNase I Protection Assay (Also Called DNase I Footprinting).
Again, .sup.32P end-labeled double-stranded U-17 modified with the
TMP-biotin was used in this assay. Briefly, the lesioned DNA (5000
cpm, .about.0.15 nM) was first incubated with various amounts of
streptavidin (0-50 nM) at room temperature for 10 min in 10 .mu.l
of binding buffer [25 mM Tris.times.HCl (pH 7.4), 100 mM NaCl and
1.5 mM MgC.sub.2]. Where indicated, 0.4 mM free d-biotin was
included in one of the incubations. At the end of each incubation,
2 .mu.l of 2.5 mg/ml freshly diluted DNase I (Worthington Enzymes,
Freehold, N.J.; final concentration, 0.4 mg/ml) was added, and the
digestion was carried out at room temperature for 2 min before
being quenched by the addition of 50 .mu.l of stop solution [20 mM
EDTA (pH 8.0), 1% SDS and 50 ug/ml yeast total RNA]. The samples
were then precipitated by ethanol. After being washed once with 80%
ethanol, the DNA pellets were air dried and then resuspended in
denaturing loading buffer. Finally, the resuspenisions were loaded
onto a 20% denaturing polyacrylamide gel. The gel was dried, and
exposed to an x-ray film with an intensifying screen at -80.degree.
C.
[0103] Results. As shown in FIG. 8, when streptavidin seas added
the modified U-17 became resistant to DNase I cleavage. Enhanced
C.sup.16 and G.sup.17 bands indicated that streptavidin protected
these fragments (full-length or one base less) from being further
cleaved by DNase I. Since the lesioned DNA probe was 5'
end-labeled, and fragments shorter than 5-mer could not be
recovered by the ethanol precipitation used in the experimental
procedures, only two bases (C.sup.5 and G.sup.6) on the 5' side of
the modified thymidine were observed to be covered by streptavidin.
On the 3' side, however, the covered region was more extensive. As
discussed above, the protected region extended at least to the
full-length of the probe, which was ten nucleotides away from the
modified base. It is reasonable to expect that a similar length on
the 3' side of the probe is also protected by streptavidin. These
observations suggested that streptavidin, a protein of about 50 kD,
covered at least twenty nucleotides flanking the thymidine where a
TMP-biotin was monoadducted. DNA excision repair enzymes in
mammalian cells, which are able to repair a variety of DNA damages
(Friedberg (1985), DNA Repair) repair a DNA lesion first by
recognizing it and then excising a "patch" or DNA fragment of 27-
to 29-nucleotides flanking the lesion site on the damaged strand
(Huang et al. (1992), 89 P.N.A.S. USA 3664-3668; Svovoda et al.
(1993), 268 J. Biol. Chem. 1931-1936). It was noted that, even in
the absence of streptavidin, a small region of 4-5 nucleotides on
the 3' side of the modified thymidine resisted DNase I cleavage. It
is possible that this effect was due to the presence of the
TMP-biotin lesion itself. A similar effect would not be observed
had the experiment been conducted with an appropriate repair enzyme
instead of DNase.
Prospective Example 8
Guidelines for Demonstrating that a Heterobifunctional Compound
Forms Genomic Lesions
[0104] The ability of a given programmed genotoxic compound, such
as the nitrogen-mustard-steroid decoy estrogen-chlorambucil, to
damage DNA can be assessed by determining the ability of the
compound to form interstrand crosslinks between opposing strands of
a duplex DNA molecule. Formation of such crosslinks is known to
correlate strongly with the clinical efficacy of Lifunctional
mustards including chloramubucil and melphalen (Ross et al. (1978).
38 Cancer Res. 1502-1506; Zwelling et al. (1981), 41 Cancer Res.
640-649). The assay is both simple and rapid. It is based on the
separation of DNA strands under denaturing conditions of heat and
chaotropic compounds (e.g., urea), or organic solvents (e.g.,
N,N-dimethylforamide). When crosslinked, denatured DNA strands are
unable to separate and consequently migrate more slowly than
uncrosslinked separated strands during electrophoresis in
polyacrylamide gels. After incubating the chosen compound with a
short DNA duplex molecule labeled with .sup.32P, the percentage of
crosslinked DNA molecules can be determined following separation by
gel electrophoresis according to standard methods. Conditions for
crosslinking and gel analysis of both short DNA fragments (Rink et
al. (1993), 115 J. Amer. Chem. Soc. 2551-2557) and longer DNA
fragments (Hartley et al. (1991), 193 Anal. Biochem. 131-134;
Holley et al. (1992), 52 Cancer Res. 4190-4195) have been described
in detail. These methods can be adapted to assess the crosslinking
of DNA fragments ranging in size from, e.g., duplex 17-mer
oligonucleotides such as U-17, to the 166, 235, 540, 1423, and 3199
base pair fragments obtained from Dde I restriction endonuclease
digest of the widely available pGEM plasmid.
[0105] Crosslinking capacity of a particular programmed
heterobifunctional compound should be compared to that of the
parent genotoxic agent (e.g., chlorambucil). The compound under
investigation preferably has the ability to produce interstrand
crosslinks in DNA in vitro that are comparable to those of the
parent compound under similar conditions. If this is not the case,
the reactivity of synthetic intermediates can be examined to
determine what modification(s) to the parent genotoxic agent's
structure is responsible for its reduced crosslinking activity.
With this knowledge in hand, the structure of an optional linker or
other component can be modified, if necessary, to restore
reactivity of the genotoxic agent portion of the heterobifunctional
compound to a desired level.
Prospective Example 9
Guidelines for Demonstrating that a Heterobifunctional Compound
Binds to a Chosen Cell Component
[0106] Tight association between the programmed genotoxic ligand
compound estrogen-chlorambucil and the estrogen receptor protein is
relevant to the compound's intended function. The strength of
association of the ligand decoy receptor complex should be measured
for both the "free" form of the compound, and the form that is
covalently bound to DNA forming a genomic lesion. Knowledge of the
interaction of the free form of the compound with the receptor can
indicate whether the position of chemical attachment of the steroid
ligand to the optional linker has preserved capacity to interact
with the receptor protein. Comparison of the strengths of
association of the free and DNA-bound forms of the compound should
indicate whether or not the DNA molecule sterically impedes the
formation of lesion-shielding complexes.
[0107] One of several routine and widely used assays can be
employed for measuring the ability of the free compound to displace
a natural steroid ligand from the estrogen receptor. Typically,
radiolabeled estradiol is first bound to the receptor protein in a
cell extract prepared from estrogen responsive tissue such as
uterus. Calf uterus is most commonly used for this purpose.
Increasing concentrations of the compound under investigation are
then added. The amount of estradiol remaining tightly associated
with the protein as a function of the increasing concentration of
the other chemical provides a measure of the relative affinities of
the natural and synthetic ligands for the receptor.
[0108] Where the compound under investigation has first been
covalently attached to DNA, its association with the receptor
protein can be investigated by gel electrophoresis using a routine
adaption of mobility shift techniques described fully in Carthew et
al. (1985), 43 Cell 439-448. DNA-receptor complexes can thus be
electrophoretically resolved from lesioned, uncomplexed DNA through
application or routine adaptation of this technique. Furthermore,
the strength of the association can be measured by addition of
competing ligands for the receptor as described in the previous
examples. Increasing amounts of estradiol, for example, would
compete with the DNA bound ligand for the receptor protein and
thereby restrict formation of the DNA-receptor complex. The
effectiveness of estradiol in preventing the formation of the
DNA-receptor complex should provide a useful measure of the
relative strength of association of the heterobifunctional ligand
compounds with the receptor.
[0109] From the results of biomolecular studies such as those
described above, it should be possible to predict the effectiveness
of compounds under investigation for blocking repair of lesions in
living cells.
Prospective Example 10
Guidelines for Demonstrating Efficacy of Ligand Decoy Compounds
[0110] Specificity of heterobifunctional compounds, such as the
estrogen-chlorambucil decoy, for killing tumor cells that express
the estrogen receptor can be tested readily in available cell
culture models for breast cancer. Results derived from these models
will form appropriate grounds for reasonably predicting genotoxic
effectiveness of candidate programmed heterobifunctional compounds
for use in vivo. That is, effectiveness of the compounds in the
present cell culture models will provide an early indication of
genotoxic potential in multicellular organisms, such as mammals,
including humans. For present purposes, breast cancer cell lines
should be chosen for screening protocols because this form of
cancer currently is the principle target for genotoxic uses of
estrogen receptor decoys. Several human breast cancer cell lines
are widely available and have been characterized as to their
estrogen receptor status. The MCF-7 and MDA-MB-231 cell lines are
two such examples. Estrogen receptor status plays a key role in
determining the responses of these cell lines to estrogens and
genotoxic antiestrogens such as tamoxifen. Estrogens stimulate the
growth of the estrogen receptor positive cell line MCF-7, while
having no effect on the growth rate of MDG-MB-231, which lacks the
receptor. Likewise, antiestrogens such as tamoxifen inhibit the
growth of MCF-7 cells, but have no effect on the growth of
MDA-MB-231 cells. These two cell lines therefore allow a
determination of whether compounds such as the
estrogen-chlorambucil decoy are more effective than chlorambucil
itself in killing cells that contain the target receptor. Thus,
cell lines with high levels of estrogen receptor protein should be
much more sensitive to the heterobifunctional decoy.
[0111] Cell sensitivity can be assessed using a growth inhibition
assay. Equal concentrations of chlorambucil and the
chlorambucil-estrogen conjugate can be added to cell cultures, and
the rate of cell proliferation determined by counting the number of
cells in replica cultures up to seven days post treatment. The
increase in cell numbers in both treated and untreated control
cultures can be compared to assess potential antitumor effects.
Favorable results should be confirmed by repeating the test using a
phenotypically different pair of receptor-bearing and receptor
independent cell lines. Drugs that demonstrate a 2-4 fold or
greater ability to inhibit the growth of estrogen receptor positive
cells, as compared to receptor negative cells, should be selected
for further testing in appropriate mammals.
Example 11
In Vitro Genetic Selection of a Pool of Aptamers that Bind
Selectively to Mutant P53
[0112] Two 10-mer peptides (EP240-Cys:
NH.sub.2-Thr-Phe-Arg-His-Ser-Val-Val-Val-Pro-Cys-COOH; and
EP240S-Cys: NH.sub.2-Thr-Phe-Val-His-Val-Ser-Arg-Val-Pro-Cys-COOH)
were synthesized by standard techniques and coupled to a
Thiol-Sepharose supporting matrix through the cysteine residues in
the peptides. Peptide EP240-Cys comprises the five residue epitope
recognized by PAb 240, shown underlined, and thus was used as the
selection target peptide. Peptide EP240S-Cys, in which the epitope
sequence is scrambled, was used to eliminate aptamers that bound
non-sequence-specifically to the target peptide. The C-terminal
cysteine residues, which do not exist in the native protein
sequence, were attached to both peptides to facilitate
immobilization onto thiol-derivitized agarose beads, and elution of
the peptides along with the bound aptamer candidates under reducing
conditions (e.g., 20 mM DTT). A pool of 100-Mer oligonucleotides
containing a central 64-mer totally randomized sequence flanked by
18-mer PCR primer regions at each end was synthesized by standard
techniques. About 90 pmoles of the oligonucleotides, representing
no fewer than 10.sup.13 different molecules, were amplified by
about 100-fold by PCR, using a 5' end-biotinylated primer for one
of the two flanking regions. The unbiotinylated DNA strand was
thereafter isolated by binding the double-stranded PCR products to
a streptavidin column and eluting the column with 0.15 N NaOH. The
amplified pool of single stranded candidate aptamers (about 900
pmoles) was first applied to a pre-selection column containing the
scrambled EP240S-Cys peptides. This step was designed to eliminate
nonspecific binding. The DNA flowthrough from the pre-column was
directly loaded onto the selection column containing EP240-Cys
epitope peptides. After extensive washing with binding buffer, the
selection column was eluted with binding buffer containing 20 mM
DTr. The eluted DNA was subjected to PCR amplification. Rounds of
selection and amplification were repeated to generate a pool rich
in candidate aptamers having the desired binding property. Nine
rounds of selections were completed. Preliminary results indicated
that a population of aptamers has been selected that bind
preferentially to the selection column EP240-Cys and not to the
pre-selection column EP-240S-Cys. Individual aptamers isolated from
this pool can be subjected to assessment of their binding
characteristics for mutant p53, and can be further developed as
heterobifunctional compounds programmed to selectively destroy
cells that express a recognized p53 mutant.
Example 12
Preparation of a Heterobifunctional Genotoxic Compound Programmed
to Attract the Estrogen Receptor to Genomic Lesion Sites
[0113] 12.1 Overview of the Synthesis of a 2-Phenylindole-Nitrogen
Mustard Compound of the Present Invention:
[0114] A ligand decoy compound designed to attract the estrogen
receptor has been prepared according to the principles disclosed
herein. This compound, which presently is preferred for achieving
the selective killing of transformed human cells that express
estrogen receptor, comprises a nitrogen mustard (chlorambucil)
first agent linked through a protease resistant, hydrophilic
organic linker to a 2-phenylindole second agent. The second agent
ligand has been shown to bind effectively to mammalian estrogen
receptor, even when the heterobifunctional compound is adducted to
double-stranded DNA. The synthesis route employed for the
preparation of this preferred compound, referred to herein as
2-phenylindole-C6NC2, is outlined in FIG. 9, in which like
reference numerals identify the intermediate and product compounds
referred to herein.
[0115] The first step involves alkylation of p-methoxyanisole with
p-methoxy-2-bromopropiophenone (von Angerer et al. (1984), 27 J.
Med. Chem. 1349.) Sequential treatment of 2 with BBr3 and MOM-Cl
furnished the corresponding phenylindole 4. Alkylation of 4 with
1,6-dibromohexane afforded the bromide 5. Treatment of 5 with
1-diphenylphosphinamide-2-TBDMS-ethanolamine (6) produced 7, which
underwent deprotection upon exposure to TBAF, generating the
alcohol 8. Activation of the hydroxy group in the presence of
p-nitrophenyl-carbonate followed by addition of
3-[4-[N,N-bis(2-chloroethyl)amino]-phenyl]-propylamine generated
the carbamate 9. Acid hydrolysis of diphenylphosphinic and
methoxymethyl groups afforded the desired heterobifunctional
mustard-2-phenylindole compound 1. A more detailed discussion of
the synthesis set forth in FIG. 9 follows:
[0116] 12.2 Synthesis of 5-methoxy 2-(4-methoxyphenyl)indole
(2):
[0117] A solution of p-methoxy-2-bromopropiophenone ((1 g, 4.11
mmol) was added to a solution Of p-methoxyanisole in N,N
dimethylaniline (1.7 g, 13.82 mmol)) and heated under refltux for 3
hours. The reaction mixture was cooled to room temperature, poured
into 2N HCl, and extracted with EtOAc. The organic extract was
washed with water and dried over Na.sub.2SO.sub.4. Removal of the
solvent under reduced pressure and flash chromatography on
SiO.sub.2 using CH.sub.2Cl.sub.2 gave the desired intermediate
compound 2.
[0118] 12.3 Synthesis of 5-hydroxy 2-(4-hydroxyphenyl)indole
(3):
[0119] Five g (18.7 mmol) of 5-methoxy 2-(4 methoxyphenyl)indole
was dissolved in 30 mL CH.sub.2Cl.sub.2 under argon, cooled on dry
ice/acetone and 70 mL 1 M BBr.sub.3 were added with stirring. The
solution was warmed to 0.degree. C. for 30 min. It was then stirred
at room temperature overnight. The reaction mixture was suspended
in sat. NaHCO.sub.3 solution and extracted with CH.sub.2Cl.sub.2
(2.times.). The organic extract was washed with brine and dried
over Na.sub.2SO.sub.4. Removal of the solvent in vacuo and flash
chromatography (5% MeOH in CH.sub.2Cl.sub.2) gave 3.33 g of 3.
[0120] 12.4 Synthesis of
5-O-methylmethoxy-2-(4-O-methoxy-methylphenyl)indole (4):
[0121] Three g (12 mmol) of 5-hydroxy-2-(4-hydroxyphenyl)indole was
dissolved in 50 mL dry THF under argon. After cooling the solution
on dry ice/acetone, 1.92 g of MOM Cl and 0.58 g of NaH were added.
The reaction mixture was cooled to room temperature, stirred for 1
hour and washed with water. The organic layer was dried over
Na.sub.2SO.sub.4 and evaporated under reduced pressure. The product
was isolated by flash chromatography which gave 2.5 g (60%) of
4.
[0122] 12.5 Synthesis of
1-(6-bromohexyl)-5-O-methoxymethyl-2-(4-O-methoxymethylphenyl)indole
(5):
[0123] Sodium hydride (0.25 g of a 50% dispersion in oil, 5.14
mmol) was suspended in 50 mL of dry DMF, and cooled to 0.degree. C.
under argon. 5-O-methoxy-methyl-2-(4-O-methoxymethylphenyl)indole
(1.0 g, 3.0 mmol) was added slowly to the reaction mixture. The
mixture was stirred for 1 hour at room temperature and then 1,6
dibromohexane (4.5 g, 18.44 mmol) in 30 mL DMF was added dropwise.
After stirring for 2 hours, the reaction was quenched with water
and extracted with ether. The organic phase was washed with water
(3.times.) and dried (Na.sub.2SO.sub.4). The oily residue fleas
purified by flash chromatography (CH.sub.2Cl.sub.2) affording 5:1.1
g (75%).
[0124] 12.6 Synthesis of
1-diphenylphosphinamide-2-O-TBDNS-ethanolamine (6):
[0125] Five g (21.2 mmol) of diphenylphosphinic chloride was
dissolved in 20 mL of dry pyridine. NH.sub.3 was bubbled through
the stirred solution till saturation occurred. The solution was
stoppered and allowed to stir at room temperature for 2 hours. The
reaction mixture was concentrated under reduced pressure and the
residue dissolved in CH.sub.2Cl.sub.2. The solution was extracted
with sat. NaHCO.sub.3 and brine. The organic layer was dried over
Na.sub.2SO.sub.4 and concentrated to dryness under reduced
pressure. The resulting compound (1.0 g) was suspended in a
biphasic mixture of 15 mL of benzene and 15 mL of 50% NaOH
containing 40 mg tetra-n-butylammonium bromide. To the refluxing
suspension was added 1 g (4.2 mmol) of 1-bromo-2-TBDMS-ethanol
dissolved in 5 mL benzene (1-bromo-2-TBDMS-ethanol was prepared
from 5 g 1-bromoethanol, 10 g of t butyldimethylsilyl chloride and
9.5 g imidazole, which were dissolved in 12 mL DMF and stirred
overnight at room temperature). After refluxing for 4 hours, the
organic layer was removed, extracted with water (3.times.), and
dried over Na.sub.2SO.sub.4. Benzene was removed under reduced
pressure and 0.25 g of the product was isolated from the oily
residue by flash chromatography (3% MeOH in CH.sub.2Cl.sub.2).
[0126] 12.7 Synthesis of
1-[6-(N-diphenylphosphinamide-2-ethanolamine)hexyl]-5-O-methoxymethyl-2-(-
4-O-methoxy-methylphenyl)indole (8):
[0127] A mixture of bromide (6) (0.25 g, 0.5 mmol),
1-diphenylphosphinamide-2-O-TBDMS-ethanolamine (0.25 g, 0.7 mmol),
NaH (24 mg, 1 mmol), and a catalytic amount of tetrabutylammonium
bromide were refluxed in benzene for 3 hours. The reaction mixture
was cooled, extracted with water (2.times.) and dried over
Na.sub.2SO.sub.4. The solvent was removed under reduced pressure
and the resulting clear oil was purified by flash chromatography
giving 0.4 g (0.5 mmol) of 7. Desilylation of the product was
carried out in 5 mL dry THF in the presence of 1 mmol of
tetrabutylammonium fluoride (1.0 mmol) at room temperature for 2
hours. The solvent was removed under reduced pressure, and the
residue was dissolved in CH.sub.2Cl.sub.2 and extracted with water.
After drying over Na.sub.2SO.sub.4, 0.3 g (0.44 mmol) of the
product (8) was obtained as a viscous liquid.
[0128] 12.8 Synthesis of 1 [6 (N diphenylpliosphlilnamide 2
ethoxy-(O-(3-(4-(N,N-bis(2-chloroethyl)aminophenyl)propylamine)carbamoyl)-
amine)-hexyl]-5-O-methoxymethyl-2 (40 methoxymethylphenyl)indole
(9):
[0129] The alcohol (8) (0.3 g, 0.44 mmol) in 5 mL of dry pyridine
was slowly added to a stirred solution of
p-nitrophenylchloroformate (0.25 g, 1.2 mmol) in 2 mL of
CH.sub.2Cl.sub.2. After stirring at room temperature for 1 hour,
the reaction was diluted with CH.sub.2Cl.sub.2 and washed with sat.
NaHCO.sub.3 (3.times.) and brine. The resulting oily residue was
purified by flash chromatography (2% MeOH in CH.sub.2Cl.sub.2).
This activated alcohol (0.4 g) was added to a solution of
3-[4-[N,N-bis(2-chloroethyl)amino]-phenyl]propylamine (0.25 g, 1.0
mmol) in THF containing TEA (0.18 mL, 1.3 mmol) and refluxed for 45
min. The reaction mixture was cooled and the solvent was removed
under reduced pressure. The residue was dissolved in
CH.sub.2Cl.sub.2 and extracted with NaHCO.sub.3 (3.times.) and
brine. After drying over Na.sub.2SO.sub.4 and concentration under
reduced pressure, 0.35 g of the product was isolated by flash
SiO.sub.2 chromatography (4% MeOH in CH.sub.2Cl.sub.2) as a viscous
liquid.
[0130] 12.9 Synthesis of
1-[6-(N-2-ethoxy-(O-(3-(4-(N,N-bis(2-chloroethyl)-aminophenyl)propylamine-
)carbamoyl)amine)hexyl]-5-O-hydroxy-2-(4-hydroxyphenyl)indole
(1):
[0131] To a solution of 0.35 g of 9 in 5 mL of MeOH was added
concentrated HCl (0.3 mL). After stirring 4 hours at room
temperature, the solvent was removed under reduced pressure and the
resulting residue was dissolved in 2% MeOH in CH.sub.2Cl.sub.2 and
washed with sat. NaHCO.sub.3 (2.times.) and brine. Drying over
Na.sub.2SO.sub.4 and purification by flash chromatography gave 200
mg of 1, the desired programmed heterobifunctional genotoxic
compound.
Example 13
Demonstration that the 2-phenylindole-C6NC2-Mustard Ligand Decoy
Compound 1 Binds Effectively to Estrogen Receptor (ER)
[0132] An in vitro competition assay utilizing calf uterine
extracts as the source of estrogen receptor (ER) was used according
to the guidelines set forth in Example 9 (above), to determine the
relative affinity of 2-phenylindole-C6NC2-mustard (I) and related
compounds for the mammalian ER. The receptor-protein assay seas
performed as described by Korenman (1969), 13 Steroids 163.
Briefly, uterine cytosol was prepared by homogenizing calf uteri in
0.01 M Tris-HCL pH 8.0, 1 mM EDTA, 0.25 M sucrose and centrifuging
the homogenate at 105,000 g for 1 hr. The resulting supernatant was
stored in liquid N.sub.2. Assays were performed by premixing a
range of concentrations of the test compound with a fixed amount of
tritium labeled estradiol ([.sup.3H]E.sub.2). Calf uterine cytosol
was then added. After incubation overnight at 4.degree. C.,
activated charcoal/dextran was added to remove unbound
[.sup.3H]E.sub.2. Following centrifugation to remove the
charcoal/dextran, levels of bound (i.e., non-competed)
[.sup.3H]E.sub.2 were determined by liquid scintillation
spectrometry. Low concentrations of test compound required to
abolish [.sup.3H]E.sub.2 binding in this assay indicate a tight
association between the test compound and the ER.
[0133] This competition assay was used to determine the binding
affinities for the estrogen receptor of the
2-phenylindole-C6NC2-mustard and its congeners (comprising the
first and second agents of 1, linked through organic linkers of
varying length), as well as DNA lesions containing the
2-phenylindole second agent formed by reaction of these compounds
with purified and isolated DNA. In the latter case, various levels
of 2-phenylindole-C6NC2-mustard were used to modify plasmid DNA,
and residual unbound test compound was removed from the modified
DNA by ethanol precipitation and multiple ethanol/water washes. The
purified plasmid DNA containing 2-phenylindole-C6NC2-mustard
lesions then was combined with [.sup.3H]E.sub.2 and used in the
competition assay as described above.
[0134] Results with the free 2-phenylindole compounds are shown in
FIG. 10. The 2-phenylindole-C6NC2-mustard compound has the highest
affinity for the ER. The affinity of this ligand decoy for the ER
is within about 20 fold that of the natural ligand, estradiol (E2).
The homologous 2-phenylindole-C5NC3-mustard compound has a lower
affinity for the ER, about 200 times less than E2, while the
homologous 2-phenylindole-C3NC3-mustard has little, if any,
affinity for the ER. As noted previously, these compounds differ
from the preferred 2-phenylindole-C6NC2-mustard, compound 1 in FIG.
9, by the number of CH.sub.2 groups disposed on either side the
central NH group of the linker. These homologous compounds were
prepared by routine modifications of the synthesis scheme discussed
in Example 12.
[0135] FIG. 11 shows results of receptor competition assays using
isolated DNAs lesioned with the preferred
2-phenylindole-C6NC2-mustard (1), 2-phenylindole-C5NC3-mustard, or
the underivatized chorambucil mustard. These data show that DNA
lesions formed by chorambucil have no affinity for ER, while those
formed by compounds of the present invention compete effectively
with E.sub.2 for ER. These results demonstrate that the
2-phenylindole first agent can attract ER to genomic lesions,
providing reasonable basis for the expectation that compounds of
the present invention, including particularly the preferred
compound 1, can localize the sterically large ER at genomic lesions
in vivo, thereby hindering access by cellular repair enzymes.
[0136] The results set forth in FIGS. 10 and 11 further demonstrate
optimization of the optional linker disposed between the first and
second agents of ligand decoy compounds programmed to attract the
mammalian ER. Similar optimization studies can be carried out with
other heterobifunctional compounds designed according to the
principles disclosed herein to attract other cell components. It
should be expected that the characteristics of the linker will vary
depending in part on the particular cell component attracted by a
chosen heterobifunctional genotoxic compound.
Example 14
Demonstration that the 2-phenylindole-C6NC2-mustard ligand decoy
Compound 1 is Toxic Selectively to Mammalian Cells Expressing
Estrogen Receptor (R)
[0137] The toxicities of the preferred 2-phenylindole-C6NC2-mustard
and homologous ligand decoy compounds for selected cells
distinguished phenotypically from nonselected cells by expression
of ER were tested using the human breast tumor cell lines, MCF-7
and MDA-MB-231, according to the guidelines set forth in Example 10
(above). The MCF-7 cell line expresses the estrogen receptor
protein, while no ER protein can be detected in the MDA-MB-231 cell
line.
[0138] 14.1 Cell Culture Conditions
[0139] Both cell lines were routinely cultured in Minimal Essential
Medium (MEM) supplemented with 2 mM gluatamine, 1 mM sodium
pyruvate and 10% fetal calf serum. To determine the toxicity of
test compounds, cells were trypsinized and plated in 100 .mu.l of
media in 96-well microtiter plates at the following densities:
MCF-7, 2000 cells/well; MDA-MB-231, 1000 cells/well. Cells were
allowed to grow for 24 hours in the 96-well plates prior to
treatment with the compounds.
[0140] 14.2 Treatment of Cells
[0141] All ligand decoy compounds were prepared as 10 mM stock
solutions in dimethyl sulfoxide (DMSO). Compounds were initially
diluted to 2 mM in DMSO. Subsequent dilutions were conducted in the
appropriate tissue culture medium. The final concentration of DMSO
under the testing conditions was 0.25%. In a control study, the
mustard portion of the test compound was first inactivated by
hydrolysis. This was carried out by heating the compound at
70.degree. C. for 6 hrs. in a solution containing 50% DMSO and 50%
20 mM Hepes, pH 8.0. Hydrolysis of the mustard groups was verified
by analysis by HPLC. The final concentration of DMSO in the media
of cells treated with hydrolyzed compounds was 0.1%.
[0142] Each treatment condition was conducted in replicates of 8
wells. Treatment of the cells was carried out for either 4 hrs or 4
days, as indicated below. For the 4 hr treatment, media containing
the test compound was aspirated from the wells and replaced with
fresh media. For the 4 day treatment, cells remained in the
original media containing the compound for the duration of the
assay. On day 4, cell growth in treated and control cultures was
determined using the methylene blue dye binding assay.
[0143] 14.3 Methylene Blue Dye Binding Assay
[0144] A dye-binding assay as described by Finlay et al. (1984),
139 Anal. Biochem. 272 was used to assess the cytotoxic effects of
2-phenylindole-C6NC2-mustard and homologous ligand decoy compounds.
For this assay, the media was aspirated from each of the wells, and
replaced with 100 .mu.l of 0.5% methylene blue in 50% ethanol. The
dye solution remained on the cells for 30 min., after which the
excess unbound dye was removed by several successive washes with
water. The stained cells were allowed to air dry for 1-2 hours,
then the bound dye was solubilized by adding 100 .mu.l of 1%
sarkosyl in phosphate buffered saline. The absorbance of the
solubilized dye was read at 620 nm in a Ceres 9000 plate reader
(Biotek).
[0145] Results of a typical study are shown in FIG. 12, and
establish that the ED.sub.50 (the concentration that inhibits cell
growth by 50%) of the preferred 2-phenylindole-C6NC2-mustard in
MCF-7 ER positive breast cancer cells two- to three-fold lower than
the ED.sub.50 of the same compound in MDA-MB-231 ER negative breast
cancer cells. This demonstrates that the presently preferred ligand
decoy compound, prepared according to the principles of the present
invention, is toxic selectively to breast cancer cells that are
phenotypically distinguishable from nonselected cells by their
expression of the ER.
Example 15
Demonstration that Selective Toxicity of the
2-phenylindole-C6NC2-mustard Ligand Decoy Compound 1 is not due to
antiestrogenic activity
[0146] The anticancer drug tamoxifen is toxic selectively to ER
expressing breast cancer cells due to its antiestrogenic activity.
A novel assay system accordingly was developed, using genetic
engineering techniques, to distinguish among several possible
mechanisms that could be responsible for the selective toxicity
observed in FIGS. 12 and 13.
[0147] 15.1 Construction of Novel HeLa-Derived Cell Lines
[0148] Isogenic cell lines, expressing functional estrogen receptor
(ER) protein or a control therefor, were established to determine
whether the selective toxicity of the preferred
2-phenylindole-C6NC2-mustard was attributable to an antiestrogenic
activity or another mechanism of action, such as selective
genotoxicity, e.g., according to the steric shielding model of FIG.
1. HeLa cells were stably transfected by the technique of
lipofection (as described by Felgner et al. (1987), 85 PNAS 7413)
with a eukaryotic expression vector containing the wild type ER
gene (Tora et al. (1989), 8 EMBO J. 1981). The ER gene was in
either the sense or antisense orientation under transcriptional
control of the cytomegalovirus (CMV) promoter. Three days after
lipofection, cells were replated at low density and selected for
neomycin resistance in media containing 500 .mu.g/ml G418 for 9
days. Resistant colonies were isolated and tested for the presence
of functional ER protein by using a tritium-labeled estradiol
([.sup.3H]E.sub.2) binding assay as described by Olea-Serrano et
al. (1985), 21 Eur. J. Cancer Clin. Oncol. 965. Briefly, cells were
derived from single G418-resistant clones were seeded into separate
wells of tissue culture plates and incubated for 3 days with
estrogen free media to remove endogenous estrogens.
[.sup.3H]E.sub.2 was added for 1 hr., after which the cells were
washed with PBS several times to remove unbound [.sup.3H]E.sub.2.
The remaining [.sup.3H]E.sub.2 was solubilized in ethanol and the
amount of .sup.3H radioactivity associated with the cells was
determined by liquid scintillation spectrometry.
[0149] Several clones with high levels of [.sup.3H]E.sub.2 binding
activity were selected. The highest levels of binding, observed in
clone HeLa 36, were comparable to level of [.sup.3H]E.sub.2 binding
found in the ER-positive human breast cancer cell line. MCF-7. The
ability to bind [.sup.3H]E, established the capacity of these cells
to express stably functional ER protein.
[0150] Similar analysis of control cells transfected with the CMV
expression vector in which the ER gene was in the antisense
orientation revealed no detectable [.sup.3H]E.sub.2 binding
activity in G418 resistant cells. This established that the stable
transfectants lacked functional ER protein. One such ER negative
control clone used in the following toxicity studies was designated
HeLa 7.
[0151] 15.2 Selective Toxicity of 2-phenylindole-C6NC2-mustard 1 in
HeLa 36 Cells, Expressing ER but Insensitive to Antiestrogenic
Activity
[0152] Stable HeLa transformants expressing functional ER (HeLa 36)
or control therefor (HeLa 7) were cultured in Dulbecco's Modified
Eagles Medium (DMEM) supplemented with 2 mM glutamine, 1 mM sodium
pyruvate and 10% fetal calf serum. All cells were grown in 75
cm.sup.2 flasks and subcultured every third day by
trypsinization.
[0153] Selective toxicity of the 2-phenylindole-C6NC2-mustard and
homologous ligand decoy compounds in the HeLa ER transformants was
assessed essentially as described above in Example 14, except that
cells initially were seeded at 1500 cells/well for the HeLa 7 and
2000 cells/well for the HeLa 36 cell lines.
[0154] Cytotoxicity and growth inhibitory results for the preferred
2-phenylindole-C6NC2-mustard in HeLa 7 cells and HeLa 36 cells are
shown in FIGS. 13 and 14, respectively. FIG. 13 sets forth results
establishing that a 4 hr. exposure to a 5 .mu.M dose of the
preferred heterobifunctional ligand decoy compound produces a four-
to five-fold greater cytotoxicity in HeLa 36 cells than in HeLa 7
cells, which lack functional ER. A similar exposure period to a 2.5
.mu.M dose of the same compound produced approximately a two-fold
differential in toxicity between these cell HeLa cell lines. FIG.
14 sets forth results demonstrating that a longer (4 day) exposure
to the presently preferred ligand decoy compound consistently
produced a selectively toxic and growth inhibitory effect in HeLa
36 cells, expressing the recognized ER cell component, relative to
the control HeLa 7 cells, lacking the recognized cell
component.
[0155] A control study, results of which are set forth in FIG. 15,
established that the above-observed selective cytotoxicity is
dependent on the structural integrity of the genotoxic first agent
(chlorambucil) of the presently preferred compound. No significant
cytotoxic effect was observed in either HeLa 7 or HeLa 36 cells
following 4 hr. exposures to up to 5 .mu.M doses of a chemically
hydrolyzed preparation of 2-phenylindole-C6NC2-mustard. These
results establish that the observed selective cytotoxicity of a
preferred ligand decoy compound (1) is not due to antiestrogenic
activity.
Example 16
Synthesis of Heterobifunctional Genotoxin with Estradiol as a
Second Agent
[0156] Another exemplary improved compound has been prepared which
employs estradiol as the second agent 9. This compound, referred to
herein as 7.alpha.-estradiol-C6NC2-mustard, includes an estradiol
moiety linked at the 7.alpha. position through a protease
resistant, hydrophilic, organic linker to an aromatic nitrogen
mustard.
[0157] The 7.alpha.-estradiol-C6NC2-mustard was synthesized, and
its selective cytotoxicity confirmed in target cells that express
an estrogen receptor. The 7.alpha.-estradiol-C6NC2-mustard is an
analog of a 2-phenylindole-linked nitrogen mustard disclosed in
co-pending U.S. patent application Ser. No. 08/434,664, filed May
4, 1995, Attorney Docket No. MIT-018CP, which is now allowed, the
teachings of which are herein incorporated by reference. The
improved estradiol-linked mustard described herein has a greater
affinity for the estrogen receptor and shows greater specificity in
its cytotoxic effects toward cells that express the estrogen
receptor. These results corroborate earlier teachings regarding the
design, construction and testing of compounds within the amended
generic claims.
[0158] For example, estradiols with substituents at 17.alpha., and
11.beta. positions have been prepared and evaluated as diagnostic
reagents for imaging receptor positive tumors. Pomper et al.
(1990), 33 J. Med. Chem., 3143-3155; Salman, et al. (1991), 56
Steroids 375; Napolitano et al. (1995), 38 J. Med. Chem. 429-434.
Small lipophilic groups at the 11.beta. position of estrogens have
been shown to increase affinities for the receptor up to 30-fold as
compared with estradiol. Bindal et al. (1987) 28 J. Steroid
Biochem. 361-370.
[0159] FIG. 16 illustrates the steps used to synthesize the
7.alpha.-estradiol-C6NC2-mustard. The reference numbers in FIG. 16
refer to specific reagents or separation steps. The key to the
reference numbers in FIG. 16 is as follows:
TABLE-US-00001 Ref. No. Reagant or Separation Step 1
AcCl/Ac.sub.2O/DMAP/pyridine 2 NBS/Li.sub.2CO.sub.3/LiBr/DMF 3
MgBr(CH.sub.2).sub.6OTBDMS/CuI/THF 4 Separation of .alpha. and
.beta. isomers by flash chromatography 5 Bu.sub.4N.sup.+F.sup.-/THF
6 Ac.sub.2O/DMAP/pyridine 7 CuBr.sub.2/LiBr/CH.sub.3CN 8
(i-Pr).sub.2NEt/CH.sub.3OCH.sub.2Br 9 K.sub.2CO.sub.2/aq.
CH.sub.3OH 10 CH.sub.3SO.sub.2Cl/LiBr/DMF 11
Ph.sub.2P(O)NH(CH.sub.2).sub.2OTBDMS/NaH/DMF 12
Bu.sub.4N.sup.+F.sup.-/THF 13
p-ClCO.sub.2C.sub.6H.sub.4NO.sub.2/(i-Pr).sub.2NEt/CH.sub.2Cl.sub.2
14
p-((ClCH.sub.2CH.sub.2).sub.2N)C.sub.6H.sub.4(CH.sub.2).sub.3NH.sub.2/(-
i-Pr).sub.2NEt/THF 15 HCl/CH.sub.3OH
[0160] 16.1 Preparation of
19-nortestosteroneacetate-3-enolacetate.
[0161] A solution of 19-nortestosterone in acetic anhydride
(Ac.sub.2O), pyridine, and acetyl chloride (AcCl) was heated at
reflux under an argon atmosphere for 2.5 hr, then concentrated to
near dryness. The residue was dissolved in methylene chloride
(CH.sub.2Cl.sub.2) and washed with water and brine. The organic
phase was dried over sodium sulfate (Na.sub.2SO.sub.4) and the
solvent removed under reduced pressure. The product was isolated by
flash chromatography on SiO.sub.2 gel using CH.sub.2Cl.sub.2.
[0162] 16.2 Preparation of
estra-4,6-dien-3-one-17.beta.-acetate.
[0163] Three grams (3 g) of 19-nortestosteroneacetate-3-enolacetate
was suspended in 10 mL of N,N-dimethylformamide (DMF), then 200
.mu.L of water was added and the suspension cooled to 0.degree. C.
under an argon atmosphere. Over 45 min, 1.56 g of N
bromosuccinimide (NBS) was added in small portions. Following the
last addition, the solution was warmed to room temperature and 1.47
g of lithium carbonate (Li.sub.2CO.sub.3) and 0.75 g of lithium
bromide (LiBr) were added. After heating at 95.degree. C. for 3 hr,
200 mL of water and 10 mL of acetic acid (HOAc) were added. The
resulting suspension was extracted with CH.sub.2Cl.sub.2, the
organic phase washed with brine, dried over Na.sub.2SO.sub.4 and
the solvent removed under reduced pressure. The product (2.15 g)
was isolated by flash chromatography on SiO.sub.2 gel using
CH.sub.2Cl.sub.2 containing 0.75% methanol (CH.sub.3OH).
[0164] 16.3 Preparation of
7.alpha.-(t-butyldimethylsilyloxyhexyl)-estra-4-ene-3-one-17.beta.-acetat-
e.
[0165] The appropriate Grignard reagent. i.e.,
t-butyldimethylsilyloxy hexyl magnesium bromide
(TBDMSO(CH.sub.2).sub.6MgBr), was formed by the addition of 10 g of
6-bromohexyl t-butyldimethylsilyl ether to 0.9 g of magnesium
turnings in 10 mL of tetrahydrofuran (THF). The suspension was
heated at reflux for 20-30 min. After cooling to -35.degree. C.
2.65 g of copper iodide (CuI) was added and the suspension kept at
-35.degree. C. for 45 min. A solution of 3 g of
estra-4,6-dien-3-one-17b-acetate in 8 mL of THF was added slowly
over 1 hr and the temperature maintained at -35.degree. C. for an
additional 2.5 hr. Three milliliters (3 mL) of HOAc was then added
and the resulting suspension warmed to room temperature. Subsequent
to addition of the suspension to water and extraction with ether,
the organic phase was washed with brine, dried over
Na.sub.2SO.sub.4, and the solvent removed under reduced pressure.
The desired 7.alpha.-product (1.0 g) was purified and separated
from its 7.beta.-isomer by flash chromatography on SiO.sub.2 gel
using hexanes containing 20% ethyl acetate (EtOAc).
[0166] 16.4 Preparation of
7.alpha.-(acetyloxyhexyl)-17.beta.-hydroxy-estra-4-ene-3-one.
[0167] One gram of
7.alpha.-(t-butyldimethylsilylhexyl)-estra-4-ene-3-one-17-acetate
was dissolved in 10 mL of THF, then 5 mL of a 1 M solution of
tetrabutylammonium fluoride (Bu.sub.4N.sup.+F--) in THF was added.
After 1 hr, the solvent was removed under reduced pressure and the
residue dissolved in CH.sub.2Cl.sub.2. The organic phase was washed
with a saturated bicarbonate solution and brine, dried over
Na.sub.2SO.sub.4 and the solvent removed under reduced pressure.
The free hydroxyl group of the resulting compound was acetylated
with acetic anhydride in pyridine in the presence of a catalytic
amount of 4-N,N-dimethylaminopyridine (DMAP). After removal of the
solvents under reduced pressure, the product was isolated by flash
chromatography on SiO.sub.2 gel using hexanes containing 15%
EtOAc.
[0168] 16.5 Synthesis of
7.alpha.-(acetyloxyhexyl)-17.beta.-hydroxy-estra-1,3,5(10)-trien-3-ol.
[0169] One gram (1 g) of
7.alpha.-(acetyloxyhexyl)-17.beta.-hydroxy-estra-4-ene-3-one was
added to a suspension of 190 mg of LiBr and 1 g of copper bromide
(CuBr.sub.2) in 10 mL of acetonitrile (CH.sub.3CN). The suspension
was heated at reflux for 30 min, cooled, and 20 nit of a saturated
bicarbonate solution was added. The mixture was extracted twice
with EtOAc and the combined organic extracts washed with water,
dried over Na.sub.2SO.sub.4 and the solvent removed under reduced
pressure. The product (200 mg) was isolated by flash chromatography
on SiO.sub.2 gel using toluene containing 10% EtOAc.
[0170] 16.6 Synthesis of
7.alpha.-(6-bromohexyl)-17.beta.-hydroxy-3-O-methoxymethyl-estra-1,3,5(10-
)-triene.
[0171] Two hundred milligrams (200 mg) of
7.alpha.-(acetyloxyhexyl)-17.beta.-hydroxy-estra-1,3,5-trien-3-ol
was dissolved in diisopropylethylamine ((i-Pr).sub.2NEt), 0.2 mL of
bromomethyl methyl ether (CH.sub.3OCH.sub.2Br) was added and the
reaction stirred for 2 hr at room temperature. Thirty milliliters
(30 mL) of CH.sub.2CH.sub.2 was added and the solution washed with
water and brine. Following removal of the solvents under reduced
pressure, the product was dissolved in 3 mL of CH.sub.3OH and
treated with 0.3 mL of a 1 M aqueous potassium carbonate
(K.sub.2CO.sub.3) solution. After 1 hr, the solution was added to
50 mL of water. The organics were extracted with EtOAc, washed with
water and dried over Na.sub.2SO.sub.4. Removal of the EtOAc
produced 100 mg of crude
7.alpha.-(6-hydroxyhexyl)-17.beta.-hydroxy-3-O-methoxymethyl-estra-1,3,5(-
10)-triene which was dissolved in 5 mL of THF containing 0.1 mL of
(i-Pr).sub.2NEt. The THF solution was cooled to 0.degree. C. and
0.02 mL of methanesulfonyl chloride (CH.sub.3SO.sub.2Cl) was added.
After 3 hr, 50 mL water was added and the product isolated by
extraction with EtOAc. Removal of solvent produced 100 mg of an
intermediate which was treated with 200 mg of LiBr in DMF at
60.degree. C. for 1 hr. The organics were isolated by extraction
with ether which was washed with water, dried over Na.sub.2SO.sub.4
and the solvent removed under reduced pressure to yield the product
(110 mg).
[0172] 16.7 Synthesis of
1-t-butyldimethylsilyloxy-2-diphenylphosphinamide Ethane.
[0173] Nine grams (9 g) of potassium phthalimide was suspended in
100 mL of DMF to which 10 g of
1-bromo-2-t-butyldimethylsilyloxyethane (BrCH.sub.2CH.sub.2OTBDMS)
was added and the mixture heated at 75.degree. C. overnight. The
mixture was added to 300 mL of a 5% sodium hydroxide (NaOH)
solution and extracted with 300 mL of ether. The ether phase was
washed twice with water, dried over Na.sub.2SO.sub.4 and the
solvent removed under reduced pressure. The resulting solid was
dissolved in 100 mL of CH.sub.3OH, then 3 mL of hydrazine
(H.sub.2NNH.sub.2) was added. After 5 hr, 300 mL ether was added
and the organic phase separated from the white precipitate by
filtration. Removal of the solvent under reduced pressure yielded
11 g of O-(t-butyldimethylsilyl)ethanolamine. Five grams (5 g) of O
(t butyldimethylsilyl)ethanolamine was dissolved in 40 mL of THF
and 8 mL of (i-Pr).sub.2NEt. Five milliliters (5 mL) of
diphenylphosphinic chloride (Ph.sub.2P(O)Cl) was slowly added which
produced a white precipitate. After 2 hr, the THF was removed under
reduced pressure and the residue dissolved in 200 mL of
CH.sub.2Cl.sub.2. The CH.sub.2Cl.sub.2 solution was washed with a
saturated bicarbonate solution and brine. Removal of
CH.sub.2Cl.sub.2 under reduced pressure followed by flash
chromatography on SiO.sub.2 gel using CH.sub.2Cl.sub.2 produced the
product (9 g).
[0174] 16.8 Synthesis of
7.alpha.-((N-diphenylphosphino)-hydroxyethylaminohexyl)-17.beta.-hydroxy--
3-O-methoxymethyl-estra-1,3,5(10)-triene.
[0175] One hundred fifteen milligrams (115 mg) of
1-t-butyldimethylsilyloxy-2-diphenylphosphinamide ethane
(Ph.sub.2P(O)NHCH.sub.2CH.sub.2OTBDMS) was dissolved in 1 mL of DMF
and cooled in an ice bath. Twenty five milligrams (25 mg) of sodium
hydride (NaH) was added. After 40 min, 2 mL of a DMF solution
containing 250 mg of
7.alpha.-(6-bromohexyl)-17.beta.-hydroxy-3-O-methoxymethyl-estra-1,3,5-
(10)-triene was added and the solution warmed to room temperature.
After 3 hr, the mixture was added to 50 mL of water and the
organics extracted with EtOAc, washed with water and dried over
Na.sub.2SO.sub.4. After removal of the solvent under reduced
pressure, the product was dissolved in 2 mL of THF and treated for
1 hr with 0.5 mL of a 1 M solution of Bu.sub.4N.sup.+F-- in THF.
Removal of the solvent and flash chromatography on SiO.sub.2 gel
using 3% CH.sub.3OH in CH.sub.2Cl.sub.2 yielded the product (100
mg).
[0176] 16.9 Synthesis of
7.alpha.-{N-(diphenylphosphino)-[2-(N--((N,N-bis-2-chloroethylaminophenyl-
)propyl)-carbamoyloxy)ethyl]aminohexyl}-17.beta.-hydroxy-3-O-methoxymethyl-
-estra-1,3,5(10)-triene.
[0177] One hundred milligrams (100 mg)
7.alpha.-((N-diphenylphosphino)-hydroxyethylaminohexyl)-17.beta.-hydroxy--
3-O-methoxymethyl-estra-1,3,5(10)-triene was dissolved in 2 mL of
CH.sub.2Cl.sub.2 and 0.1 mL of (i-Pr).sub.2NEt. Sixty milligrams
(60 mg) of p-nitrophenylchloroformate
(p-ClCO.sub.2C.sub.6H.sub.4NO.sub.2) was added to the solution and
after 2 hr, the reaction was diluted with 30 mL of
CH.sub.2Cl.sub.2, washed four times with a saturated bicarbonate
solution and once with brine. Removal of the solvent under reduced
pressure yielded a yellow oil to which was added a THF solution
containing 100 mg of
3-(4-(N,N-bis-2-chloroethyl)amino)phenyl)propylamine(p((ClCH.sub.2CH.sub.-
2).sub.2N)C.sub.6H.sub.4(CH.sub.2).sub.2NH.sub.2) and 0.1 mL of (i
Pr).sub.2NEt. This solution was heated and maintained at 85.degree.
C. for 2 hr. The solvent seas removed and the residue dissolved in
EtOAc, which was washed with a saturated bicarbonate solution and
brine. The product (50 mg) was isolated by flash chromatography on
SiO.sub.2 gel using 2% CH.sub.3OH in CH.sub.2Cl.sub.2.
[0178] 16.10 Synthesis of
7.alpha.-{N-[2-(N-((N,N-bis-2-Chloroethylaminophenyl)propyl)-carbamoyloxy-
)ethyl]aminohexyl}-3,17.beta.-dihydroxyestra-1,3,5(10)-triene.
[0179] Removal of the methoxymethyl and diphenylphosphinamido
groups was accomplished by dissolving 50 mg of
7.alpha.-{N-(diphenylphosphino)-[2-(N-((N,N-bis-2-Chloroethylaminophenyl)-
propyl)-carbamoyloxy)ethyl]aminohexyl}-17.beta.-hydroxy-3-O-methoxymethyl--
estra-1,3,5(10)-triene in 1 mL of CH.sub.3OH and adding 0.025 mL of
concentrated hydrochloric acid (HCl). After 3 hr, solid sodium
bicarbonate (NaHCO.sub.3) was added along with 25 mL of water. The
product was extracted with EtOAc which was washed with water and
brine. Following removal of the solvent, the product (20 mg) was
isolated by flash chromatography on SiO.sub.2 gel using a
CH.sub.2Cl.sub.2 solution containing 2% CH.sub.3OH and 2%
triethylamine (Et.sub.3N).
Example 17
Demonstration that the 7.alpha.-estradiol-C6NC2-mustard Compound
has High Affinity for the Estrogen Receptor
[0180] A receptor binding assay was carried out essentially as
described in Example 13. Calf uterine extract was used as a source
of estrogen receptor in these studies, results of which are set
forth in FIG. 17. The 7 .alpha.-estradiol-C6NC2 compound
competitively displaced radiolabelled estradiol from the estrogen
receptor.
Example 18
Demonstration of Selective Toxicity of
7.alpha.-estradiol-C6NC2-mustard Compound to Mammalian Cells that
Express the Estrogen Receptor
[0181] Cells lines, culture conditions, and protocols used to
assess the selective toxicity of 7.alpha.-estradiol-C6NC2-mustard
were essentially described in Example 14. FIG. 18 shows the results
of clonogenic survival assays in which estrogen receptor positive
(MCF-7) and estrogen receptor negative (MDA-MB231) breast cancer
cell lines were exposed to the indicated concentrations of
7.alpha.-estradiol-C6NC2-mustard for 2 hr. FIG. 18 demonstrates
that the 7.alpha.-estradiol-C6NC2-mustard has greater cytotoxic
potency in the MCF-7 cell line, which expresses high levels of
estrogen receptor, than in the MDA-MB231 cell line, which does not
substantially express this receptor.
Example 19
Synthesis of a Heterobifunctional Genotoxin with a Second Agent
that Mediates Binding of the Androgen Receptor
[0182] A ligand decoy compound designed to attract the androgen
receptor can be prepared according to the principles disclosed
herein. Such a compound is preferred for achieving the selective
killing of transformed human cells that express androgen receptor.
In one embodiment, an androgen-chlorambucil decoy would comprise a
nitrogen mustard (chlorambucil) first agent linked through a
protease resistant, hydrophilic organic linker to an androgen,
androgen-analog, androgen-agonist or androgen-antagonist second
agent. In a further embodiment, the compound would comprise
(N,N-bis-2-chloroethylaminophenyl)propylamine as its genotoxic
first agent.
[0183] In another embodiment, the compound would comprise
19-nor-17.beta.-hydroxyandrost-4-ene-3-one-7.alpha.-hexanol
("7.alpha.-19NT-C6(OH)") as a second agent.
[0184] In another embodiment, the compound would comprise a
derivative of a 1,2-dihydropyridono[5,6-g]quinolone as a second
agent.
[0185] In another embodiment, the compound would comprise a
derivative of the antiprogestin RU486 as a second agent. In a
further embodiment, the compound would comprise
17.beta.-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one as a second
agent. In yet a further embodiment, the compound would comprise a
17.beta.-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one second agent
linked at the 11.beta. position to a first agent through a protease
resistant, hydrophilic organic linker.
[0186] Sources of guidance for the synthesis of second agents
designed to attract the androgen receptor include Hackenberg et al.
(1996) 32A Euro. J. Cancer 696-701, which discusses the
androgen-like and anti-androgen-like effects of antiprogestins, and
Hamann et al. (1998) J. Med. Chem. 623-639, which discusses a
series of androgen receptor antagonists. The teachings of these
papers are herein incorporated by reference.
[0187] The methods of synthesis of these compounds would be similar
to those used in Examples 12 and 16, and should employ techniques
swell known to one of ordinary skill in the art.
Example 20
Use of a Heterobifunctional Genotoxin with a Second Agent that
Mediates Binding the Androgen Receptor
[0188] Specificity of an androgen receptor decoy for killing tumor
cells that express the androgen receptor can be tested readily in
available cell culture models for prostate cancer. A
heterobifunctional genotoxin with an androgen-analog second agent
can be demonstrated to selectively target cells expressing the
androgen receptor in a method analogous to Examples 10, 14 and
18.
[0189] Similar techniques can be applied or adapted with no more
than routine experimentation, to demonstrate functional properties
of heterobifunctional compounds programmed to attract the androgen
receptor. Electrophoretic mobility shift and DNase I protection
analysis are suitable techniques generally for demonstrating
whether a particular heterobifunctional compound forms genotoxic
lesions, whether a chosen cell component is bound by a suitably
programmed heterobifunctional compound, and whether the resulting
complex is effective for shielding genomic lesions from the action
of enzymes that act on cellular DNA.
[0190] Cell sensitivity can be assessed using a growth inhibition
assay. Equal concentrations of chlorambucil and the
androgen-chlorambucil decoy can be added to cell cultures, and the
rate of cell proliferation determined by counting the number of
cells in replica cultures up to seven days post treatment. The
increase in cell numbers in both treated and untreated control
cultures can be compared to assess potential antitumor effects.
Favorable results should be confirmed by repeating the test using a
phenotypically different pair of receptor-bearing and receptor
independent cell lines. Androgen receptor-bearing cell lines
include, but are not limited to, LNCaP.FGC, PC-3, and DU 145, all
available from the American Type Culture Collection (Rockville,
Md.). Drugs that demonstrate a 2-4 fold or greater ability to
inhibit the growth of androgen receptor positive cells, as compared
to receptor negative cells, should be selected for further testing
in appropriate mammals.
EQUIVALENTS
[0191] The invention may be embodied in other specific forms
Without departing from the spirit or essential characteristics
thereof. The foregoing embodiments are therefore to be considered
in all respects illustrative rather than limiting on the invention
described herein. Scope of the invention is thus indicated by the
appended claims rather than by the foregoing description, and all
changes which come within the meaning and range of equivalency of
the claims are therefore intended to be embraced therein.
Sequence CWU 1
1
4110PRTArtificialEP240-Cys peptide 1Thr Phe Arg His Ser Val Val Val
Pro Cys1 5 10210PRTArtificialEP240S-Cys peptide 2Thr Phe Val His
Val Ser Arg Val Pro Cys1 5 10326DNAArtificialhUBF binding DNA
sequence 3cagtctcctt ctggtctctt ctcagt 26417DNAArtificialU17 probe
4cggccgtacg ugcgccg 17
* * * * *