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US 2010/0248227 A1Published application
Drug-phosphorylating enzyme
AssigneeDaiichi Sankyo Company, Limited
InventorsFutoshi Nara, Kiyoaki Yonesu, Kazuishi Kubota
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Abstract
The objects of the present invention are: elucidation of an enzyme that phosphorylates in vivo a compound such as (2R)-2-amino-2-methyl-4-[5-(5-phenylpentanoyl)thiophen-2-yl]butan-1-ol and provision of a method of phosphorylating the aforementioned compound; provision of a method of screening for a substance phosphorylated by the aforementioned enzyme; provision of a method of determining the ability of a subject to phosphorylate a test compound. Provision of a method of phosphorylating the aforementioned compound using a human fructosamine-3-kinase-related protein and/or fructosamine-3-kinase, and a method of determining the ability of a subject to phosphorylate a test compound.
Claims16
A method of screening for a substance phosphorylated by human FN3KRP and/or human FN3K, which comprises the following steps (1) to (3):
- (1) contacting a test substance with a polypeptide selected from the group consisting of the following (a) to (c):
- (a) a polypeptide having the amino acid sequence of amino acid Nos. 1-309 of SEQ ID NO:
- 2 in the sequence listing;
- (b) a polypeptide having the amino acid sequence of amino acid Nos. 1-309 of SEQ ID NO:
- 4 in the sequence listing;
- and
- (c) a polypeptide having an amino acid sequence comprising a deletion, substitution or addition of one or several amino acids with respect to the amino acid sequence of a polypeptide selected from the above (a) and (b), and having the ability to phosphorylate (2R)-2-amino-2-methyl-445-(5-phenylpentanoyl)thiophen-2-yl]butan-1-ol;
- (2) measuring the amount of phosphoric ester of the test substance generated;
- and
- (3) comparing the amount of the generated phosphoric ester measured in (2) above with the amount of the phosphoric ester of the test substance measured when the test substance is not contacted with the polypeptide selected from the above (a) to (c).
7 more elements
A method of screening for a substance phosphorylated by human FN3KRP and/or human FN3K, which comprises the following steps (1) to (4):
- (1) contacting a test substance with a polypeptide selected from the group consisting of the following (a) to (c):
- (a) a polypeptide having the amino acid sequence of amino acid Nos. 1-309 of SEQ ID NO:
- 2 in the sequence listing;
- (b) a polypeptide having the amino acid sequence of amino acid Nos. 1-309 of SEQ ID NO:
- 4 in the sequence listing;
- and
- (c) a polypeptide having an amino acid sequence comprising a deletion, substitution or addition of one or several amino acids with respect to the amino acid sequence of a polypeptide selected from the above (a) and (b), and having the ability to phosphorylate (2R)-2-amino-2-methyl-445-(5-phenylpentanoyl)thiophen-2-yl]butan-1-ol;
- (2) measuring the amount of phosphoric ester of the test substance generated;
- (3) comparing the amount of the generated phosphoric ester measured in (2) above with the amount of the phosphoric ester of the test substance measured when the test substance is not contacted with the polypeptide selected from the above (a) to (c);
- and
- (4) determining that the test substance has been phosphorylated, when the amount of the phosphoric ester measured in (2) above is greater compared with the amount of the phosphoric ester of the test substance measured when the test substance is not contacted with the polypeptide selected from the above (a) to (c).
8 more elements
A method of screening for a substance having immunosuppressive activity, which comprises the following steps (1) to (3):
- (1) contacting a test substance with a polypeptide selected from the group consisting of the following (a) to (c):
- (a) a polypeptide having the amino acid sequence of amino acid Nos. 1-309 of SEQ ID NO:
- 2 in the sequence listing;
- (b) a polypeptide having the amino acid sequence of amino acid Nos. 1-309 of SEQ ID NO:
- 4 in the sequence listing;
- and
- (c) a polypeptide having an amino acid sequence comprising a deletion, substitution or addition of one or several amino acids with respect to the amino acid sequence of a polypeptide selected from the above (a) and (b), and having the ability to phosphorylate (2R)-2-amino-2-methyl-4-[5-(5-phenylpentanoyl)thiophen-2-yl]butan-1-ol;
- (2) measuring the amount of phosphoric ester of the test substance generated;
- and
- (3) comparing the amount of the generated phosphoric ester measured in (2) above with the amount of the phosphoric ester of the test substance measured when the test substance is not contacted with the polypeptide selected from the above (a) to (c).
7 more elements
A method of screening for a substance having immunosuppressive activity, which comprises the following steps (1) to (4):
- (1) contacting a test substance with a polypeptide selected from the group consisting of the following (a) to (c):
- (a) a polypeptide having the amino acid sequence of amino acid Nos. 1-309 of SEQ ID NO:
- 2 in the sequence listing;
- (b) a polypeptide having the amino acid sequence of amino acid Nos. 1-309 of SEQ ID NO:
- 4 in the sequence listing;
- and
- (c) a polypeptide having an amino acid sequence comprising a deletion, substitution or addition of one or several amino acids with respect to the amino acid sequence of a polypeptide selected from the above (a) and (b), and having the ability to phosphorylate (2R)-2-amino-2-methyl-4-[5-(5-phenylpentanoyl)thiophen-2-yl]butan-1-ol;
- (2) measuring the amount of phosphoric ester of the test substance generated;
- (3) comparing the amount of the generated phosphoric ester measured in (2) above with the amount of the phosphoric ester of the test substance measured when the test substance is not contacted with the polypeptide selected from the above (a) to (c);
- and
- (4) determining that the test substance has been phosphorylated, when the amount of the phosphoric ester measured in (2) above is greater compared with the amount of the phosphoric ester of the test substance measured when the test substance is not contacted with the polypeptide selected from the above (a) to (c).
8 more elements
A method of producing a phosphoric ester, which comprises the following steps (1) and (2):
- (1) contacting the compound represented by general formula (I) with a polypeptide selected from the group consisting of the following (a) to (c):
- (a) a polypeptide having the amino acid sequence of amino acid Nos. 1-309 of SEQ ID NO:
- 2 in the sequence listing;
- (b) a polypeptide having the amino acid sequence of amino acid Nos. 1-309 of SEQ ID NO:
- 4 in the sequence listing;
- and
- (c) a polypeptide having an amino acid sequence comprising a deletion, substitution or addition of one or several amino acids with respect to the amino acid sequence of a polypeptide selected from the above (a) and (b), and having the ability to phosphorylate (2R)-2-amino-2-methyl-4-[5-(5-phenylpentanoyl)thiophen-2-yl]butan-1-ol;
- and
- (2) obtaining the phosphoric ester of the compound represented by general formula (I) from the reaction solution in (1) above.
6 more elements
A method of determining the ability of a subject to generate the phosphoric ester of the compound represented by general formula (I), which comprises the following steps (1) to (3):
- (1) extracting total RNA from a sample collected from a subject;
- (2) measuring the expression level of a polynucleotide selected from the group consisting of the following (a) to (c) in the total RNA:
- (a) a polynucleotide having the nucleotide sequence consisting of nucleotide Nos. 6-935 of SEQ ID NO:
- 1 in the sequence listing;
- (b) a polynucleotide having the nucleotide sequence consisting of nucleotide Nos. 27-956 of SEQ ID NO:
- 3 in the sequence listing;
- and
- (c) a polynucleotide hybridizing under stringent conditions with a polynucleotide having a nucleotide sequence complementary to the nucleotide sequence described in (a) or (b) above, and encoding a polypeptide having the ability to phosphorylate in vivo the compound represented by general formula (I);
- and
- (3) comparing the expression level of the polynucleotide measured in (2) above with the expression level of said polynucleotide in a sample that has been confirmed as having the ability to phosphorylate in vivo the compound represented by general formula (I), so as to examine the ability of the subject to phosphorylate the compound represented by general formula (I).
7 more elements
A method of determining the ability of a patient to generate the phosphoric ester of the compound represented by general formula (I), which comprises the following steps (1) and (2):
- (1) measuring the expression level of a polypeptide selected from the group consisting of the following (a) to (c) in a sample collected from a subject:
- (a) a polypeptide having the amino acid sequence of amino acid Nos. 1-309 of SEQ ID NO:
- 2 in the sequence listing;
- (b) a polypeptide having the amino acid sequence of amino acid Nos. 1-309 of SEQ ID NO:
- 4 in the sequence listing;
- and
- (c) a polypeptide having an amino acid sequence comprising a deletion, substitution or addition of one or several amino acids with respect to the amino acid sequence of a polypeptide selected from the above (a) and (b), and having the ability to phosphorylate (2R)-2-amino-2-methyl-4-[5-(5-phenylpentanoyl)thiophen-2-yl]butan-1-ol;
- and
- (2) comparing the expression level of the polypeptide measured in (1) above with the expression level of said polypeptide in a sample that has been confirmed as having the ability to phosphorylate in vivo the compound represented by general formula (I), so as to examine the ability of the subject to phosphorylate the compound represented by general formula (I).
6 more elements
A method of determining the ability of a patient to generate the phosphoric ester of the compound represented by general formula (I), which comprises the following steps (1) and (2):
- (1) measuring the enzyme activity of a polypeptide selected from the group consisting of the following (a) to (c) in a sample collected from a subject:
- (a) a polypeptide having the amino acid sequence of amino acid Nos. 1-309 of SEQ ID NO:
- 2 in the sequence listing;
- (b) a polypeptide having the amino acid sequence of amino acid Nos. 1-309 of SEQ ID NO:
- 4 in the sequence listing;
- and
- (c) a polypeptide having an amino acid sequence comprising a deletion, substitution or addition of one or several amino acids with respect to the amino acid sequence of a polypeptide selected from the above (a) and (b), and having the ability to phosphorylate (2R)-2-amino-2-methyl-4-[5-(5-phenylpentanoyl)thiophen-2-yl]butan-1-ol;
- and
- (2) comparing the enzyme activity of the polypeptide measured in (1) above with the enzyme activity of said polypeptide in a sample that has been confirmed as having the ability to phosphorylate in vivo the compound represented by general formula (I), so as to examine the ability of the subject to phosphorylate the compound represented by general formula (I).
6 more elements
A method of determining the ability of a subject to generate the phosphoric ester of the compound represented by general formula (I), which comprises the following steps (1) to (3):
- (1) examining the nucleotide sequence of a polynucleotide selected from the group consisting of the following (a) to (c) in a sample collected from a subject:
- (a) a polynucleotide having the nucleotide sequence consisting of nucleotide Nos. 1-1466 of SEQ ID NO:
- 1 in the sequence listing;
- (b) a polynucleotide having the nucleotide sequence consisting of nucleotide Nos. 1-1781 of SEQ ID NO:
- 3 in the sequence listing;
- and
- (c) a polynucleotide hybridizing under stringent conditions with a polynucleotide having a nucleotide sequence complementary to the nucleotide sequence described in (a) or (b) above, and encoding a polypeptide having the ability to phosphorylate in vivo the compound represented by general formula (I);
- (2) examining the presence or absence of a mutation in the nucleotide sequence of said polynucleotide that influences enzyme activity;
- and
- (3) determining that the subject has only a low ability to phosphorylate the compound represented by general formula (I) when the subject has, in the nucleotide sequence, a mutation that decreases the phosphorylating activity of the polypeptide encoded by said polynucleotide, and determining that the subject has the ability to phosphorylate the compound represented by general formula (I) when the subject does not have, in the nucleotide sequence, a mutation that decreases the phosphorylating activity of said polypeptide.
7 more elements
A method of identifying, in a nucleotide sequence, a mutation that influences the phosphorylating activity of a polypeptide encoded by a polynucleotide selected from the group consisting of the following (a) to (c):
- (a) a polynucleotide having the nucleotide sequence consisting of nucleotide Nos. 1-1466 of SEQ ID NO:
- 1 in the sequence listing;
- (b) a polynucleotide having the nucleotide sequence consisting of nucleotide Nos. 1-1781 of SEQ ID NO:
- 3 in the sequence listing;
- and
- (c) a polynucleotide hybridizing under stringent conditions with a polynucleotide having a nucleotide sequence complementary to the nucleotide sequence described in (a) or (b) above, and encoding a polypeptide having the ability to phosphorylate in vivo the compound represented by general formula (I),
- said method comprising the following steps (1) to (3):
- (1) examining the nucleotide sequence of a polynucleotide selected from the group consisting of the above (a) to (c) in a sample collected from a subject;
- (2) examining the presence or absence of a mutation in the nucleotide sequence of said polynucleotide;
- and
- (3) examining the relationship between said mutation in the nucleotide sequence of said polynucleotide and the phosphorylating activity of the polypeptide encoded by said polynucleotide, so as to identify in the nucleotide sequence of the polynucleotide a mutation that influences the phosphorylating activity.
8 more elements
A kit for diagnosing ability to metabolize a drug, which comprises at least one selected from the group consisting of the following (1) to (5):
- (1) an oligonucleotide primer comprising 15 to 30 contiguous nucleotides that is used for specifically amplifying a part of or the entire polynucleotide having the nucleotide sequence as shown in SEQ ID NO:
- 1 or 3 in the sequence listing;
- (2) a polynucleotide probe comprising 15 or more contiguous nucleotides that hybridizes under stringent conditions with the polynucleotide having the nucleotide sequence as shown in SEQ ID NO:
- 1 or 3 in the sequence listing, for detecting said polynucleotide;
- (3) a solid-phase sample having a polynucleotide selected from either the oligonucleotide primer described in (1) above or the polynucleotide probe described in (2) above immobilized thereon;
- (4) an antibody that specifically binds to a polypeptide selected from the following (a) to (c), so as to detect the protein:
- (a) a polypeptide having the amino acid sequence of amino acid Nos. 1-309 of SEQ ID NO:
- 2 in the sequence listing;
- (b) a polypeptide having the amino acid sequence of amino acid Nos. 1-309 of SEQ ID NO:
- 4 in the sequence listing;
- and
- (c) a polypeptide having an amino acid sequence comprising a deletion, substitution or addition of one or several amino acids with respect to the amino acid sequence of a polypeptide selected from the above (a) and (b), and having the ability to phosphorylate (2R)-2-amino-2-methyl-445-(5-phenylpentanoyl)thiophen-2-yl]butan-1-ol;
- and
- (5) a secondary antibody that binds to the antibody described in (4) above.
11 more elements
Dependencies are inferred from the claim text.