Organic compounds -- part of the class 532-570 series – Organic compounds – Heterocyclic carbon compounds containing a hetero ring...
Reexamination Certificate
2000-12-05
2004-02-10
Rotman, Alan L. (Department: 1625)
Organic compounds -- part of the class 532-570 series
Organic compounds
Heterocyclic carbon compounds containing a hetero ring...
C546S036000
Reexamination Certificate
active
06689887
ABSTRACT:
BACKGROUND OF THE INVENTION
I. Field of the Invention
This invention relates to the field of cancer therapy. The invention also relates to screening methods for identifying pharmacologically active compounds that may be useful for treating proliferative diseases. More particularly, the inventors have identified non-nucleoside molecule compounds that interact with specific DNA structures and which inhibit human telomerase.
II. Description of Related Art
Cancer, which is a cell proliferative disorder, is one of the leading causes of disease, being responsible for 526,000 deaths in the United States each year (Boring et al, 1993). For example, breast cancer is the most common form of malignant disease among women in Western countries and, in the United States, is the most common cause of death among women between 40 and 55 years of age (Forrest, 1990). The incidence of breast cancer is increasing, especially in older women, but the cause of this increase is unknown. Malignant melanoma is another form of cancer whose incidence is increasing at a frightening rate, at least sixfold in the United States since 1945, and is the single most deadly of all skin diseases (Fitzpatrick, 1986).
One of the devastating aspects of cancer is the propensity of cells from malignant neoplasms which disseminate from their primary site to distant organs and develop into metastatic cancers. Animal tests indicate that about 0.01% of circulating cancer cells from solid tumors establish successful metastatic colonies (Fidler, 1993). Despite advances in surgical treatment of primary neoplasms and aggressive therapies, most cancer patients die as a result of metastatic disease. Hence, there is a need for new and more efficacious cures for cancer.
The ends of chromosomes have specialized sequences, termed telomeres, comprising tandem repeats of simple DNA sequences. Human telomeres consist of the sequence 5′-TTAGGG (SEQ ID No. 1) (Blackburn, 1991; Blackburn et al., 1995). Telomeres have several functions apart from protecting the ends of chromosomes, the most important of which appear to be associated with senescence, replication, and the cell cycle clock (Counter et al., 1992). Progressive rounds of cell division result in a shortening of the telomeres by some 50-200 nucleotides per round. Almost all tumor cells have shortened telomeres, which are maintained at a constant length (Allshire et al., 1988; Harley et al., 1990; Harley et al., 1994) and are associated with chromosome instability and cell immortalization.
The enzyme telomerase adds the telomeric repeat sequences onto telomere ends, ensuring the net maintenance of telomere length in tumor cells commensurate with successive rounds of cell division. Telomerase is a DNA polymerase with an endogenous RNA template (Feng et al., 1995), on which the nascent telomeric repeats are synthesized. A significant recent finding has been that approximately 85-90% of all human cancers are positive for telomerase, both in cultured tumor cells and primary tumor tissue, whereas most somatic cells appear to lack detectable levels of telomerase (Kim et al., 1994; Hiyama et al., 1995a). This finding has been extended to a wide range of human tumors (see, for example, references Broccoli, 1994 and Hiyama et al., 1995b) and is likely to be of use in diagnosis.
Human telomerase has been proposed as a novel and potentially highly selective target for antitumor drug design (Feng et al., 1995; Rhyu et al., 1995; Parkinson, 1996). Studies with antisense constructs against telomerase RNA in HeLa cells show that telomere shortening is produced, together with the death of these otherwise immortal cells (Feng et al., 1995). Sequence-specific peptide-nucleic acids directed against telomerase RNA have also been found to exert an inhibitory effect on the enzyme (Norton et al., 1996).
Among chemical agents, 2,6-diamido-anthraquinones have been reported as DNA-interactive agents (Collier and Neidle, 1988; 1992; Agbandje et al., 1992). These compounds have been shown to act as selective DNA triplex interactive compounds (Fox et al., 1995; Haq et al., 1996), with reduced affinity for duplex DNA and only moderate conventional cytotoxicity in a range of tumor cell lines. A carbocyanine dye, 3,3′-diethyloxadicarbocyanine (DODC,), has been reported to bind dimeric hairpin G-quadruplex structures (Chen et al., 1996).
This invention describes a novel class of non-nucleoside molecules that are telomerase inhibitors. These compounds have demonstrated their ability to interact with telomeres which form structures called the G-quadruplex structures. As telomeres are involved in controlling the cell cycle, cell replication and aging, these inhibitors of telomerase prevent uncontrolled cell growth and the immortality of tumor cells.
SUMMARY OF THE INVENTION
The present invention has demonstrated for the first time that a non-nucleoside, small molecule can target the G-quadruplex structure and can act as a telomerase inhibitor. Accordingly, methods have been developed that identify these classes of compounds and several inhibitors identified.
Compounds such as those described here, which interact selectively with G-quadruplex structures and inhibit telomerase, are expected to be useful as inhibitors of the proliferation of cells that require telomerase to maintain telomere length for continued growth. The invention thus relates to novel methods for identifying compounds that will be useful in this regard, and also includes new classes of telomerase inhibitors. In this regard, several perylene compounds and carbocyanines have been shown to interact with G-quadruplex structures. Since telomerase appears to be found almost exclusively in tumor cells, these agents are contemplated to be useful as antitumor agents.
In one aspect of the invention, compounds that act as telomerase inhibitors have been identified. It has been found that compounds that bind to the human G-quadruplex structure inhibit the human telomerase. The identification of such G-quadruplex interactive agents is an efficient approach for identifying human telomerase inhibitors. Methods for identifying these G-quadruplex interactive agents include identifying compounds whose three-dimensional structure is complementary to that of the G-quadruplex structure. Another method for identifying G-quadruplex interactive compounds is to identify compounds that interact with G-quadruplexes using such methods as dye displacement or melting points of G-quadruplex/compound hybrids.
More particularly, candidate compounds that inhibit telomerase activity are identified by first obtaining the three-dimensional structure of a compound that might interact with the G-quadruplex.selected compound. The complementarity of the compound to human telomere DNA G-quadruplex is then determined. If there is a high degree of complementarity, telomerase inhibition activity is indicated.
Alternatively, one can contact a telomerase inhibitor candidate compound with human DNA G-quadruplex; and then determine the melting point of the human DNA G-quadruplex. The inventors have found that an increase in melting point of the quadruplex indicates telomerase inhibitory activity of the compound.
Additionally, telomerase inhibitors may be identified by first preparing a DNA G-quadruplex/dye complex with a dye intercalated into the G-quadruplex; then contacting complex with a telomerase inhibitor candidate. Displacement of the dye in the complex identifies the candidate as a telomerase inhibitor.
Yet another aspect of this invention is to provide non-nucleoside inhibitors of telomerase. Using the disclosed screening methods, compounds have been identified that bind to human G-quadruplex structures. The invention includes perylene compounds, exemplified by N,N′-bis(2-dimethylaminoethyl)-3,4,9,10-perylenetetracarboxylic acid diimide that are useful telomerase inhibitors. Novel compounds such as N,N′-bis(2-piperdinoethyl)-3,4,9,10-perylenetetracarboxylic acid diimide are also within the scope of the invention.
A preferred G-quadruplex structure is formed from t
Fedoroff Oleg Y.
Hurley Laurence H.
Kerwin Sean M.
Salazar Miguel
Board of Regents , The University of Texas System
Fulbright & Jaworski L.L.P.
Robinson Binta
Rotman Alan L.
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