High-throughput screening assays for modulators of nucleic...

Details High-throughput screening assays for modulators of nucleic... High-throughput screening assays for modulators of nucleic...

1999-07-13

2001-03-06

Weber, Jon P. (Department: 1651)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving antigen-antibody binding, specific binding protein...

C435S233000

Reexamination Certificate

active

06197527

ABSTRACT:

FIELD OF THE INVENTION
The field of the invention relates to high-throughput assays for identifying modulators of topoisomerase activity. New solid and liquid phase assays are described, as are related compositions, apparatus and integrated systems.
BACKGROUND OF THE INVENTION
The great advances that have occurred in this century in treating infectious diseases are threatened by the emergence of strains of bacteria and other pathogens that are resistant to all currently known antibiotics. To counteract this threat, therapies that have novel mechanisms of action are needed.
One promising target for novel antipathogenic drugs is the topoisomerase family of enzymes. DNA topoisomerases play multiple roles in the maintenance and propagation of the genomes of both prokaryotes and eukaryotes. Thus, compounds that act as effective cellular inhibitors of topoisomerases are expected to act as cytotoxic agents through perturbation of the normal cell division process. Such agents that are sufficiently potent and selective will be of great use as antibacterial and antifungal pharmaceutical agents. Topoisomerases are also encoded by genomes of certain viruses, so development of topoisomerase inhibitors that are effective against viral topoisomerases may provide effective antiviral agents. In addition, because cell division is an important characteristic of cancers and other proliferative diseases, agents that inhibit topoisomerases will also find use as antineoplastic agents.
Topoisomerase inhibitors are classified into two general types. First, the class designated as “poisons” have in common the property of causing “trapping” of the target topoisomerase in the form of a covalent complex with the nucleic acid substrate. Second, the “non-poison” class inhibits the enzymatic activity of the topoisomerase without specific effects on steps of the catalytic cycle that involve formation or resolution of the enzyme-DNA covalent intermediate. Of the DNA topoisomerase inhibitors currently used as clinical antibiotic or antineoplastic agents, the “poisons” seem to be most effective, probably because such compounds result in the accumulation of irreversible genotoxic damage in target cells.
Shortcomings with previously available assays for topoisomerase inhibitors have hampered the search for novel topoisomerase inhibitors. For example, many previously available assays require the use of radioactive compounds and/or suffer from a lack of sensitivity. Also, previously available topoisomerase inhibitor assays are not always amenable to high throughput screening methods such as are needed to screen large libraries or groups of potential inhibitors. Thus, a need exists for new assay methods for identifying topoisomerase inhibitors. The present invention meets this and other needs.
SUMMARY OF THE INVENTION
High throughput assays for screening of nucleic acid topoisomerase modulators are provided. Inhibitors and activators of topoisomerase activity can both be screened using the assays. Solid and liquid phase high throughput assays are provided, as are related assay compositions, integrated systems for assay screening and other features which will be evident upon review.
In one aspect, high-throughput solid phase assays are provided. In a first embodiment of the solid phase formats, a test mixture that contains a topoisomerase, an oligonucleotide substrate for the topoisomerase, and a potential activity modulator are incubated under conditions suitable for topoisomerase activity. The nucleic acid is bound to a solid support. A denaturant is added to the reaction mixture, which stabilizes any topoisomerase-nucleic acid complexes that are formed due to the presence of the activity modulator. The solid support is washed in a high salt solution, after which the presence or absence of immobilized topoisomerase-nucleic acid complexes is determined by contacting the solid support with a labeling agent that binds to the topoisomerase.
In a second aspect of the solid phase assays, a plasmid is used as the substrate for the topoisomerase. This embodiment involves incubating a test mixture that includes a topoisomerase, a nucleic acid, and a potential activity modulator under conditions suitable for topoisomerase activity. A denaturant is added to the test mixture, as is a moiety that binds to a solid support, and also to the topoisomerase. The solid support is washed in a high salt solution to remove nucleic acid which is not bound to the immobilization moiety. The solid support-bound immobilization moiety is contacted with a labeling agent that binds to the nucleic acid to allow detection of the presence or absence of a topoisomerase-nucleic acid complex on the solid support.
The invention also provides high-throughput liquid phase assays for identification of topoisomerase activity modulators. These assays involve incubating a test mixture that contains a topoisomerase, a nucleic acid comprising a first label and a second label, and a potential activity modulator, under conditions suitable for topoisomerase activity. The presence or absence of a detectable emission, e.g., an emission by the first label, an emission by the second label, and an emission resulting from a combination of the first and second label is detected. The presence or absence of the detectable emission indicates whether the first and second labels remain in close proximity to each other.
Kits, compositions and integrated systems for performing the assays are also provided


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