Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1998-10-27
2001-01-30
Slobodyansky, Elizabeth (Department: 1652)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C514S039000, C514S041000
Reexamination Certificate
active
06180612
ABSTRACT:
TABLE OF CONTENTS
1. INTRODUCTION
2. BACKGROUND OF THE INVENTION
3. SUMMARY OF THE INVENTION
4. BRIEF DESCRIPTION OF THE FIGURES
5. DETAILED DESCRIPTION OF THE INVENTION
5.1. THE TARGETED ROLE OF NUCLEIC ACID-DEPENDENT ATPASES IN NUCLEIC ACID METABOLISM
5.2. THE NUCLEIC ACID-DEPENDENT ATPASE A POLYNUCLEOTIDES
5.3. NUCLEIC ACID-DEPENDENT ATPASE POLYPEPTIDE TARGETS
5.3.1. DNA-DEPENDENT ATPASE A POLYPEPTIDES
5.3.2. OTHER TARGET ATPASE
5.4. INHIBITORS OF DNA-dependent ATPASE ACTIVITY
5.4.1. PHOSPHOAMINOGLYCOSIDES AND DERIVATIVES
5.4.2. PRODUCTION OF PHOSPHOAMINOGLYCOSIDES
5.4.3. PRODUCTION OF TOXICITY-FREE ANTIBIOTICS
5.5. SCREENING ASSAYS
5.5.1. ASSAYS FOR INHIBITORS OF DNA-dependent ATPASE ACTIVITY
5.5.1.1. BIOCHEMICAL ASSAYS
5.5.1.2. CELL AND ANIMAL BASED ASSAYS
5.5.2. EFFECTOR PREFERENCE OF DNA-DEPENDENT ATPase A
5.5.3. ASSAYS FOR EFFECTORS AND INHIBITORY EFFECTOR ANALOGS
5.6. METHODS OF TREATMENT
5.6.1. CANCER
5.6.2. INFECTIOUS DISEASE
5.6.2.1. Fungal Infections
5.6.2.2. Bacterial Infections
5.6.2.3. Viral Targeting
5.6.2.4. Protozoan Targets
5.7.1. EFFECTIVE DOSE
5.7.2. FORMULATIONS AND USE
5.8. USE OF PHOSPHOAMINOGLYCOSIDES AS DELIVERY SYSTEM FOR OTHER THERAPEUTIC AGENTS
6. EXAMPLE: ISOLATION OF THE DNA-DEPENDENT ATPASE A GENE
6.1. AMINO ACID ANALYSIS OF NATIVE DNA-DEPENDENT ATPASE A
6.1.1. Cyanogen Bromide Digestion
6.1.2. Tricine Gel Electrophoresis
6.1.3. Peptide Transfer from Gel to Membrane
6.1.4. Edman Degradation Peptide Sequencing
6.2 CLONING AND ANALYSIS OF BOVINE DNA-DEPENDENT ATPASE A cDNA
6.2.1. Determining the Encoding Nucleic Acid Sequence for DNA-dependent Adenosine Triphosphatase A
6.2.1.1. Primer preparation for cloning
6.2.1.2. DNA Templates for PCR Cloning
6.2.1.3. mRNA Extraction
6.2.1.4. cDNA Generation from mRNA
6.2.1.5. Polymerase Chain Reaction (PCR) Techniques and Cloning
6.3. NORTHERN ANALYSIS OF BOVINE DNA-DEPENDENT ATPASE A mRNA
6.4. SOUTHERN ANALYSIS OF HUMAN, MURINE, AND BOVINE DNA-DEPENDENT ATPASE A GENE
6.5. ISOLATION OF HUMAN DNA-DEPENDENT ATPASE A cDNA
7. EXAMPLE: PREPARATION AND ANALYSIS OF THE 82 kDa ACTIVE DNA-DEPENDENT ADENOSINE TRIPHOSPHATASE A DOMAIN (ADAAD)
7.1. Bacterial Expression of DNA-dependent ATPase A
7.2. Purification of the 82 kDa polypeptide (ADAAD)
7.3. DNA-dependent ATPase A Assays
7.3.1. Colorimetric assay
7.3.2. NADH oxidation assay
7.3.3. Radioactive assay
7.4. DNA Effector Specificity for DNA-dependent ATPase A
8.1. SYNTHESIS OF PHOSPHORYLATED AMINOGLYCOSIDES
8.1.1. Preparation of Aminoglycoside Phosphotransferase
8.1.1.1. Bacterial growth
8.1.1.2. APH(3′)-IIIa activity assay
8.1.2. Synthesis of phosphorylated aminoglycosides
8.1.2.1. 3′-phosphokanamycin
8.1.2.2. 3′-phosphoneomycin
8.1.2.3. 3′-phosphogeneticin
8.1.3. Purification of phosphorylated aminoglycosides:
8.1.3.1. Bio-Rex 70 column protocol
8.1.3.2. Thin Layer Chromatography (TLC) Analysis
8.2 Characterization of Phosphoaminoglycoside Inhibitory Effects
9. EXAMPLE: ISOLATION OF FULL-LENGTH DNA-DEPENDENT ATPASE A
10. EXAMPLE: INHIBITION OF CELLULAR DNA SYNTHESIS
11. EXAMPLE: INHIBITION OF PROSTATE TUMOR CELL GROWTH
12. EXAMPLE: INHIBITION OF BREAST CANCER CELL GROWTH
13. EXAMPLE: TREATMENT OF TUMORS
14. EXAMPLE: INHIBITION OF AMEBIC GROWTH
15. EXAMPLE: INHIBITION OF LEISHMANIA GROWTH
16. EXAMPLE: INHIBITION OF DNA REPAIR THROUGH INHIBITION OF DNA-DEPENDENT ATPASE A
17. DEPOSIT OF PLASMID-CONTAINING MICROORGANISMS
WHAT IS CLAIM IS:
ABSTRACT OF THE DISCLOSURE
1. INTRODUCTION
The invention provides protein targets for disease intervention through inhibition of nucleic acid metabolism. Novel polypeptides for one such target, DNA-dependent ATPase A, and novel polynucleotides encoding DNA-dependent ATPase A are disclosed. The invention also provides compounds, including phosphoaminoglycosides, which act on such protein targets to inhibit nucleic acid metabolism. In addition, the invention provides screening assays for identifying compounds that inhibit nucleic acid-dependent ATPase activity, including, but not limited to, DNA-dependent ATPase A. Such compounds are useful in the treatment of diseases, including but not limited to cancer and infectious disease, thruogh disruption of nucleic acid metabolism and induction of apoptosis. Moreover, the invention provides methods for prevention and treatment of diseases including, but not limited to cancer and infectious disease.
2. BACKGROUND OF THE INVENTION
The interactions of proteins with nucleic acids involve a host of mechanisms for nucleic acid binding. Many nucleic acid-binding proteins (transcriptional repressors, transcriptional activators, restriction endonucleases, etc.) interact with a primary recognition sequence in a polynucleotide. These proteins: i) are generally classified as “sequence specific binding proteins”; ii) tend to bind double-stranded nucleic acids; and iii) tend to have significant numbers of contacts between their amino acid side chains and the edges of the bases which are exposed in either the minor or the major groove of a double-stranded nucleic acid. Proteins in this class have been the subject of extensive biochemical characterization and a significant number of protein-DNA co-crystal structures are now available (Steitz. Q.
Rev. Biophys
. 23, 205-280 (1990); Pabo and Sauer.
Annu. Rev. Biochem
. 61, 1053-1059 (1992)).
A second class of proteins, “nonspecific binding proteins” (single-stranded DNA binding protein, DNA polymerases, etc.) are generally found to interact with single-stranded nucleic acids. The non-specific proteins are commonly considered to bind to a nucleic acid through predominately electrostatic interactions with the phosphodiester backbone of the nucleic acid and the favorable binding can be enhanced through protein-protein interactions (cooperativity). Biochemical analysis has been extensive for many of these proteins but unlike the sequence specific binding proteins, the information about protein-DNA contacts from crystallographic structures is very limited (Lohman and Ferrari.
Annu. Rev. Biochem
. 63, 527-570 (1994)).
Finally, there are a number of proteins that are not readily classified according to the specific or nonspecific categories. This third group of proteins is not generally grouped as a class but have the common feature of recognizing and binding to specific nucleic acid structures with neither the sequence specificity nor the electrostatic interactions of either group of proteins described above. This latter group would include proteins such as: i)
E. coli
RuvA and RuvB, which bind Holliday junctions and promote branch migration (Parsons et al.,
Proc. Natl. Acad. Sci. U. S. A
. 89, 5452-5456 (1992); Muller et al.,
J. Biol. Chem
. 268, 17185-17189 (1993)); ii)
E. coli
ribosomal protein L11, which recognizes the three-dimensional conformation of an RNA backbone and thus may regulate conformational changes during the ribosome elongation cycle (Ryan et al.,
J. Mol. Biol
. 221, 1257-1268 (1991); Ryan and Draper.
Biochemistry
. 28, 9949-9956 (1989)); iii) topoisomerase II, which can yield cleavage of DNA following secondary structure-specific DNA recognition (Froelich-Ammon et al.,
J. Biol. Chem
. 269, 7719-7725 (1994)); iv) DNA-dependent protein kinase, which phosphorylates proteins when activated by the presence of DNA double-stranded to single-stranded transitions (Morozov et al.,
Journal of Biological Chemistry
. 269, 16684-16688 (1994); Chan and Lees-Miller.
Journal of Biological Chemistry
. 271, 8936-8941 (1996)); and v) transcription factor EBP-80, which also recognizes double- to single-stranded transitions in DNA (Falzon et al.,
Journal of Biological Chemistry
. 268, 10546-10552 (1993)). The sequence specific binding proteins described above utilize a host of motifs for interacting with nucleic acids (zinc fingers, helix-turn-helix, “saddle”, etc.). Different potential motifs for this latter group of proteins have not yet been elucidated.
Nucleic acid-dependent ATPases are proteins that previously have not been generally classified as either specific or nonspecific binding proteins. Assays of helicases (molecula
Hockensmith Joel W.
Muthuswami Rohini
Pennie & Edmonds LLP
Slobodyansky Elizabeth
The University of Virginia Patent Foundation
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