# US 5,705,334 A · Uses for DNA structure-specific recognition protein > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 5,705,334 A - Document ID: US5705334A - Status: Granted - Kind: A - Application number: 08/328,809 - Filed: 1994-10-25 - Granted: 1998-01-06 - Earliest claimed: 1988-09-22 - Assignees: Massachusetts Institute of Technology - Inventors: Stephen J. Lippard; John M. Essigmann; Brian Donahue; Jeffrey H. Toney; Suzanne L. Bruhn; Pieter M. Pil; Steven J. Brown; Patti J. Kellett - Web page: https://patentgrep.com/patent/US5705334A ## CPC - C07K 14/435: from animals; from humans - C07K 14/43581: {from Drosophila} - C07K 14/47: from mammals - C07K 16/18: against material from animals or humans - C12Q 1/68: involving nucleic acids - C12Q 1/6876: Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes - C12Q 1/6883: for diseases caused by alterations of genetic material - A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) - C12Q 2600/142: Toxicological screening, e.g. expression profiles which identify toxicity - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) ## Abstract Methods disclosed herein capitalize on the ability of DNA Structure Specific Recognition Proteins (SSRPs) to bind to genomic lesions formed by chemotherapeutic agents, particularly cisplatin-type agents. Methods are provided for predicting whether an agent that damages DNA will also be cytotoxic, and for predicting whether particular eukaryotic cells will be susceptible to killing by a genotoxic drug. A screening method is provided for identifying new genotoxic drugs that produce SSRP-recognized lesions in DNA. Methods also are provided for sensitizing particular eukaryotic cells to killing by chemotherapeutic agents, particularly cisplatin-type drugs. ## Claims ### Claim 1 A method for predicting cytotoxicity of an agent that binds to DNA to form a covalent 1,2-intrastrand d(ApG) or d(GpG) dinucleotide adduct, comprising the steps of: (a) providing a sample of double-stranded DNA beating a lesion formed by the binding of said agent to DNA; (b) contacting said sample with a DNA structure-specific recognition protein having at least one HMG domain and being large enough to sterically obscure at least 5 base pairs of DNA adjacent to said lesion, under conditions such that a lesioned DNA/protein complex forms; and (c) detecting said complex, wherein formation of said complex indicates that said agent will be cytotoxic. ### Claim 2 A method of identifying a cytotoxic agent that binds to DNA to form a genomic lesion comprising a covalent 1,2-intrastrand d(ApG) or d(GpG) dinucleotide adduct, comprising the steps of: (a) contacting a sample of DNA with a candidate cytotoxic agent; (b) incubating said DNA with said candidate under conditions sufficient for binding of cytotoxic agents to DNA; (c) separating DNA beating said lesion formed by the binding thereto of said cytotoxic agent from unlesioned DNA and unbound candidate; (d) contacting DNA beating said lesion with a DNA structure-specific recognition protein having at least one HMG domain and being large enough to sterically obscure at least 5 base pairs of DNA adjacent to said lesion, under conditions such that a lesioned DNA/protein complex forms; and (c) detecting said complex, wherein formation of said complex indicates that said agent will be cytotoxic. ### Claim 3 (depends on 1, 2) A method of claim 1 or 2 wherein said complex is detected by nitrocellulose filter retention assay, electrophoretic mobility shift assay or Southwestern blotting. ### Claim 4 (depends on 1, 2) A method of claim 1 or 2 wherein, in the absence of said protein, said lesion is excisable and wherein formation of said complex shields said lesion from excision in an excinuclease protection assay. ### Claim 5 (depends on 4) A method of claim 4 wherein said excinuclease protection assay comprises the steps of: (i) contacting said complex with excinuclease; (ii) incubating said complex with excinuclease under conditions sufficient for the removal of lesions from DNA; and (iii) detecting whether said lesion has been removed from DNA, wherein failure to remove said lesion indicates that said agent will be cytotoxic due to the formation of persistent genomic lesions in eukaryotic cellular DNA. ### Claim 6 (depends on 1, 2) A method of claim 1 or 2 wherein said agent is a metal coordination compound. ### Claim 7 (depends on 6) A method of claim 6 wherein agent is a platinum compound. ### Claim 8 (depends on 7) A method of claim 7 wherein said agent is a platinum(II) or platinum(IV) compound comprising a platinum atom linked to a pair of cis configured substitutionally labile moieties and a pair of cis configured electron donor moieties. ### Claim 9 (depends on 1) A method of claim 1 wherein said structure-specific recognition protein is encoded by nucleic acid that hybridizes specifically to a nucleic acid probe encoding amino acid residues 539-614 of SEQ ID No. 2. ### Claim 10 (depends on 9) A method of claim 9 wherein said structure-specific recognition protein is of human, rodent, Xenopus, Drosophila or yeast origin. ### Claim 11 (depends on 9) A method of claim 9 wherein said structure-specific recognition protein comprises at least one domain having an amino acid sequence selected from residues 539-614 of SEQ ID No. 2, residues 547-620 of SEQ ID No. 6, and amino acid sequence variants thereof that retain HMG domain DNA binding properties. ### Claim 12 (depends on 11) A method of claim 11 wherein said structure-specific recognition protein has an amino acid sequence selected from SEQ ID No. 2, SEQ ID No. 6, and amino acid sequence variants thereof that retain HMG domain DNA binding properties. ### Claim 13 (depends on 1) A method of claim 1 wherein said structure-specific recognition protein is selected from the group consisting of HMG1, HMG2, UBF, LEF-1, SRY, mtTFA, ABF2, IXR1 and SSRP.