# US 8,541,236 B2 · Mutant apolipoprotein A-1 polypeptide with increased resistance to oxidation and reactive carbonyls > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 8,541,236 B2 - Document ID: US8541236B2 - Status: Granted - Kind: B2 - Application number: 11/952,044 - Filed: 2007-12-06 - Published: 2008-09-25 - Granted: 2013-09-24 - Earliest claimed: 2006-12-08 - Assignees: University of Washington; Children's Hospital & Research Center at Oakland - Applicants: Jay W. Heinecke; John F. Oram; Michael N. Oda - Inventors: Jay W. Heinecke; John F. Oram; Michael N. Oda - Web page: https://patentgrep.com/patent/US8541236B2 ## CPC - C07K 14/775: Apolipopeptides - 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) - 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) - 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 In one aspect, the present invention provides isolated oxidation resistant mutant apoA-I polypeptides comprising an amino acid sequence substantially homologous to SEQ ID NO:4, the mutant apoA-I polypeptide comprising a combination of: (1) a conservative amino acid substitution at residue Tyr192; and (2) at least one conservative amino acid substitution at residue Met86, Met112, or Met148, wherein the mutant apoA-I polypeptide is resistant to modification by an oxidizing agent. In another aspect, the invention provides a method of promoting cholesterol efflux activity in a mammalian subject in need thereof, the method comprising the step of administering an effective amount of an oxidation resistant apoA-I agonist to the subject to promote cholesterol efflux. ## Claims ### Claim 1 An isolated oxidation resistant mutant apolipoprotein A-I (apoA-I) polypeptide comprising: an amino acid sequence having at least 85% amino acid sequence identity to the amino acid sequence of SEQ ID NO:4, the mutant apoA-I polypeptide comprising a combination of: (1) a conservative amino acid substitution at the residue corresponding to Tyr192 of the amino acid sequence of SEQ ID NO:3; and (2) at least one conservative amino acid substitution at the residue corresponding to Met86, Met112, or Met148 of the amino acid sequence of SEQ ID NO:3, wherein the oxidation resistant mutant apoA-I polypeptide is more resistant to modification by an oxidizing agent as compared to an apoA-I polypeptide comprising the amino acid sequence of SEQ ID NO:3 and interacts with ATP-binding cassette transporter A1 (ABCA1) and promotes ABCA1-mediated cholesterol efflux activity. ### Claim 2 (depends on 1) The isolated oxidation resistant mutant apoA-I polypeptide of claim 1, wherein the polypeptide has at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO:4. ### Claim 3 (depends on 1) The isolated oxidation resistant mutant apoA-I polypeptide of claim 1, wherein the polypeptide has at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO:3. ### Claim 4 (depends on 1) The isolated oxidation resistant mutant apoA-I polypeptide of claim 1, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:5, wherein at least one of Xaa at positions 86, 112, and 148 of the amino acid sequence of SEQ ID NO:5 is Leu. ### Claim 5 (depends on 1) The isolated oxidation resistant mutant apoA-I polypeptide of claim 1, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:6, wherein at least one of Xaa at positions 43, 69, and 104 of the amino acid sequence of SEQ ID NO:6 is Leu. ### Claim 6 (depends on 1) The isolated oxidation resistant mutant apoA-I polypeptide of claim 1, wherein the polypeptide comprises at least two conservative amino acid substitutions at the residues corresponding to Met86, Met112, and Met148 of the amino acid sequence of SEQ ID NO:3. ### Claim 7 (depends on 1) The isolated oxidation resistant mutant apoA-I polypeptide of claim 1, wherein the at least one conservative amino acid substitution at the residue corresponding to Met86, Met112, or Met148 of the amino acid sequence of SEQ ID NO:3 is with Leu. ### Claim 8 (depends on 6) The isolated oxidation resistant mutant apoA-I polypeptide of claim 6, wherein the conservative amino acid substitution at the residue corresponding to Tyr192 of the amino acid sequence of SEQ ID NO:3 is with Phe and the conservative amino acid substitution at the residue corresponding to Met86 of the amino acid sequence of SEQ ID NO:3 is with Leu. ### Claim 9 (depends on 6) The isolated oxidation resistant mutant apoA-I polypeptide of claim 6, wherein the conservative amino acid substitution at the residue corresponding to Tyr192 of the amino acid sequence of SEQ ID NO:3 is with Phe and the conservative amino acid substitution at the residue corresponding to Met112 of the amino acid sequence of SEQ ID NO:3 is with Leu. ### Claim 10 (depends on 6) The isolated oxidation resistant mutant apoA-I polypeptide of claim 6, wherein the conservative amino acid substitution at the residue corresponding to Tyr192 of the amino acid sequence of SEQ ID NO:3 is with Phe and the conservative amino acid substitution at the residue corresponding to Met148 of the amino acid sequence of SEQ ID NO:3 is with Leu. ### Claim 11 (depends on 6) The isolated oxidation resistant mutant apoA-I polypeptide of claim 6, wherein the conservative amino acid substitution at the residue corresponding to Tyr192 of the amino acid sequence of SEQ ID NO:3 is with Phe and the conservative amino acid substitutions at the residues corresponding to Met86 and Met112 of the amino acid sequence of SEQ ID NO:3 are with Leu. ### Claim 12 (depends on 6) The isolated oxidation resistant mutant apoA-I polypeptide of claim 6, wherein the conservative amino acid substitution at the residue corresponding to Tyr192 of the amino acid sequence of SEQ ID NO:3 is with Phe and the conservative amino acid substitutions at the residues corresponding to Met86 and Met148 of the amino acid sequence of SEQ ID NO:3 are with Leu. ### Claim 13 (depends on 6) The isolated oxidation resistant mutant apoA-I polypeptide of claim 6, wherein the conservative amino acid substitution at the residue corresponding to Tyr192 of the amino acid sequence of SEQ ID NO:3 is with Phe and the conservative amino acid substitutions at the residues corresponding to Met112 and Met148 of the amino acid sequence of SEQ ID NO:3 are with Leu. ### Claim 14 (depends on 6) The isolated oxidation resistant mutant apoA-I polypeptide of claim 6, wherein the conservative amino acid substitution at the residue corresponding to Tyr192 of the amino acid sequence of SEQ ID NO:3 is with Phe and the conservative amino acid substitutions at the residues corresponding to Met86, Met112, and Met148 of the amino acid sequence of SEQ ID NO:3 are with Leu. ### Claim 15 (depends on 1) The isolated oxidation resistant mutant apoA-I polypeptide of claim 1, further comprising a conservative amino acid substitution at the residue corresponding to Lys226 of the amino acid sequence of SEQ ID NO:3. ### Claim 16 (depends on 1) A method of producing the oxidation resistant mutant apoA-I polypeptide of claim 1, the method comprising: (1) introducing a conservative amino acid substitution at the residue corresponding to Tyr192 of the amino acid sequence of SEQ ID NO:3 in an amino acid sequence having at least 85% amino acid sequence identity to the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4; and (2) introducing at least one conservative amino acid substitution at the residue corresponding to Met86, Met112, or Met148 of the amino acid sequence of SEQ ID NO:3 in the amino acid sequence having at least 85% amino acid sequence identity to the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4, thereby producing the oxidation resistant mutant apoA-I polypeptide of claim 1, wherein the oxidation resistant mutant apoA-I polypeptide is more resistant to modification by an oxidizing agent as compared to an apoA-I polypeptide comprising the amino acid sequence of SEQ ID NO:3 and interacts ABCA1 and promotes ABCA1-mediated cholesterol efflux activity.