Three hybrid assay system
First Claim
Patent Images
1. A hybrid ligand represented by the general formula:
- R1-Y—
R2, wherein;
(i) R1 represents a first ligand selected from;
a steroid, retinoic acid, beta-lactam antibiotic, cannabinoid, nucleic acid, polypeptide, FK506, FK506 derivative, rapamycin, tetracycline, methotrexate, novobiocin, maltose, glutathione, biotin, vitamin D, dexamethasone, estrogen, progesterone, cortisone, testosterone, nickel, 2,4-diaminopteridine or cyclosporin, or a derivative thereof with structural modifications that retain a biological activity thereof;
(ii) Y represents a polyethylene linker having the general formula (CH2—
X—
CH2)n, where X represents O, S, SO, or SO2, and n is an integer from 2 to 25; and
,(iii) R2 represents a user-specified second ligand different from R1 selected from;
a peptide, nucleic acid, carbohydrate, polysaccharide, lipid, prostaglandin, acyl halide, alcohol, aldehyde, alkane, alkene, alkyne, alkyl, alkyl halide, alkaloid, amine, aromatic hydrocarbon, sulfonate ester, carboxylate acid, aryl halide, ester, phenol, ether, nitrile, carboxylic acid anhydride, amide, quaternary ammonium salt, imine, enamine, amine oxide, cyanohydrin, organocadmium, aldol, organometallic, aromatic hydrocarbon, nucleoside, or a nucleotide.
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Abstract
The invention provides compositions and methods for isolating ligand binding polypeptides for a user-specified ligand, and for isolating small molecule ligands for a user-specified target polypeptide using an improved class of hybrid ligand compounds.
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Citations
37 Claims
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1. A hybrid ligand represented by the general formula:
- R1-Y—
R2, wherein;(i) R1 represents a first ligand selected from;
a steroid, retinoic acid, beta-lactam antibiotic, cannabinoid, nucleic acid, polypeptide, FK506, FK506 derivative, rapamycin, tetracycline, methotrexate, novobiocin, maltose, glutathione, biotin, vitamin D, dexamethasone, estrogen, progesterone, cortisone, testosterone, nickel, 2,4-diaminopteridine or cyclosporin, or a derivative thereof with structural modifications that retain a biological activity thereof;(ii) Y represents a polyethylene linker having the general formula (CH2—
X—
CH2)n, where X represents O, S, SO, or SO2, and n is an integer from 2 to 25; and
,(iii) R2 represents a user-specified second ligand different from R1 selected from;
a peptide, nucleic acid, carbohydrate, polysaccharide, lipid, prostaglandin, acyl halide, alcohol, aldehyde, alkane, alkene, alkyne, alkyl, alkyl halide, alkaloid, amine, aromatic hydrocarbon, sulfonate ester, carboxylate acid, aryl halide, ester, phenol, ether, nitrile, carboxylic acid anhydride, amide, quaternary ammonium salt, imine, enamine, amine oxide, cyanohydrin, organocadmium, aldol, organometallic, aromatic hydrocarbon, nucleoside, or a nucleotide. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
- R1-Y—
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12. A hybrid ligand represented by the general formula:
- R1-Y—
R2, wherein;(i) R1 represents a first ligand selected from;
a steroid, retinoic acid, beta-lactam antibiotic, cannabinoid, nucleic acid, polypeptide, FK506, FK506 derivative, rapamycin, tetracycline, methotrexate, novobiocin, maltose, glutathione, biotin, vitamin D, dexamethasone, estrogen, progesterone, cortisone, testosterone, nickel, or 2,4-diaminopteridine or cyclosporin, or a derivative thereof with structural modifications that retain a biological activity thereof;(ii) Y represents a linker; and
,(iii) R2 represents a user-specified second ligand different from R1 selected from;
a peptide, nucleic acid, carbohydrate, polysaccharide, lipid, prostaglandin, acyl halide, alcohol, aldehyde, alkane, alkene, alkyne, alkyl, alkyl halide, alkaloid, amine, aromatic hydrocarbon, sulfonate ester, carboxylate acid, aryl halide, ester, phenol, ether, nitrile, carboxylic acid anhydride, amide, quaternary ammonium salt, imine, enamine, amine oxide, cyanohydrin, organocadmium, aldol, organometallic, aromatic hydrocarbon, nucleoside, or a nucleotide;wherein R2 binds to or inhibits a kinase. - View Dependent Claims (13, 14, 15)
- R1-Y—
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16. A fusion polypeptide, comprising segments P1, Cub-Z, and RM, in an order wherein Cub-Z is closer to the N-terminus of the fusion polypeptide than RM, wherein
(i) P1 is a ligand binding polypeptide that binds to a non-peptide ligand of a hybrid ligand, which has the general formula R1-Y— - R2, where R1 and R2 are ligands, and Y is a linker,
(ii) Cub is a carboxy-terminal subdomain of ubiquitin, (iii) Z is an amino acid residue, (iv) RM is a reporter moiety. - View Dependent Claims (18, 19, 20, 21, 22)
- R2, where R1 and R2 are ligands, and Y is a linker,
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17. A fusion polypeptide, comprising segments P1 and Nux, wherein
(i) Nux is the amino-terminal subdomain of a wild-type ubiquitin or a reduced-associating mutant ubiquitin amino-terminal subdomain, and (ii) P1 is a ligand binding polypeptide that binds to a non-peptide ligand of a hybrid ligand, which has the general formula R1-Y— - R2, where R1 and R2 are ligands, R1 is different from R2, and at least one of R1 and R2 is not a peptide, and Y is a linker.
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23. A composition, comprising:
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(i) a hybrid ligand of the general formula R1-Y—
R2, where R1 and R2 are ligands, R1 is different from R2 and at least one of R1 and R2 is not a peptide, Y is a linker; and
,(ii) at least one of two fusion polypeptides comprising; (a) a first fusion polypeptide comprising segments P2, Cub-Z, and RM, in an order wherein Cub-Z is closer to the N-terminus of the first fusion polypeptide than RM, wherein P2 is a ligand binding polypeptide that may bind to ligand R1 or R2 of the hybrid ligand, Cub is a carboxy-terminal subdomain of ubiquitin and RM is a reporter moiety, and Z is an amino acid residue; (b) a second fusion polypeptide comprising segments Nux and P1, wherein Nux is the amino-terminal subdomain of a wild-type ubiquitin or a reduced-associating mutant ubiquitin amino-terminal subdomain, and P1 is a ligand binding polypeptide that may bind to ligand R1 or R2 of the hybrid ligand. - View Dependent Claims (26, 27)
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24. A composition, comprising:
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(i) a hybrid ligand represented by the general formula;
R1-Y—
R2, wherein;(a) R1 represents a first ligand selected from;
a steroid, retinoic acid, beta-lactam antibiotic, cannabinoid, nucleic acid, polypeptide, FK506, FK506 derivative, rapamycin, tetracycline, methotrexate, 2,4-diaminopteridine derivative, novobiocin, maltose, glutathione, biotin, vitamin D, dexamethasone, estrogen, progesterone, cortisone, testosterone, nickel, or cyclosporin, or a derivative thereof with minor structural modifications that retain a biological activity thereof;(b) Y represents a polyethylene linker having the general formula (CH2—
X—
CH2)n, where X represents O, S, SO, or SO2, and n is an integer from 2 to 25;(c) R2 represents a user-specified second ligand different from R1 selected from;
a peptide, nucleic acid, carbohydrate, polysaccharide, lipid, prostaglandin, acyl halide, alcohol, aldehyde, alkane, alkene, alkyne, alkyl, alkyl halide, alkaloid, amine, aromatic hydrocarbon, sulfonate ester, carboxylate acid, aryl halide, ester, phenol, ether, nitrile, carboxylic acid anhydride, amide, quaternary ammonium salt, imine, enamine, amine oxide, cyanohydrin, organocadmium, aldol, organometallic, aromatic hydrocarbon, nucleoside, or a nucleotide;(ii) at least one fusion polypeptide selected from; (a) a first fusion polypeptide comprising;
a ligand binding domain P1 and a domain selected from the group consisting of;
a DNA binding domain and a transcriptional activation domain, wherein the ligand binding domain binds the first ligand R1; and
,(b) a second fusion polypeptide comprising;
a candidate ligand-binding domain P2 for the user-specified ligand R2 and a domain selected from the group consisting of;
a DNA binding domain and a transcriptional activation domain.wherein one of the first and second fusion polypeptides contains a DNA binding domain and the other fusion polypeptide contains a transcription activation domain.
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25. A composition comprising:
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(i) A hybrid ligand represented by the general formula;
R1-Y—
R2, wherein;(a) R1 represents a first ligand selected from;
a steroid, retinoic acid, beta-lactam antibiotic, cannabinoid, nucleic acid, polypeptide, FK506, FK506 derivative, rapamycin, tetracycline, methotrexate, 2,4-diaminopteridine derivative, novobiocin, maltose, glutathione, biotin, vitamin D, dexamethasone, estrogen, progesterone, cortisone, testosterone, nickel, or cyclosporin or a derivative thereof with structural modifications that retain a biological activity thereof.(b) Y represents a polyethylene linker having the general formula (CH2—
X—
CH2)n, where X represents O, S, SO, or SO2, and n is an integer from 2 to 25;(c) R2 represents a user-specified second ligand different from R1 selected from;
a peptide, nucleic acid, carbohydrate, polysaccharide, lipid, prostaglandin, acyl halide, alcohol, aldehyde, alkane, alkene, alkyne, alkyl, alkyl halide, alkaloid, amine, aromatic hydrocarbon, sulfonate ester, carboxylate acid, aryl halide, ester, phenol, ether, nitrile, carboxylic acid anhydride, amide, quaternary ammonium salt, imine, enamine, amine oxide, cyanohydrin, organocadmium, aldol, organometallic, aromatic hydrocarbon, nucleoside, or a nucleotide; and(ii) a fusion polypeptide that includes; (a) at least one ligand binding domain; and
,(b) a functional domain heterologous to the ligand binding domain which by itself is not capable of inducing or allowing the detection of a detectable event, but which is capable of inducing or allowing the detection of a detectable event when brought into proximity of a second functional domain.
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28. A kit comprising:
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(i) a compound of general structure R1-Y-L, wherein; (a) Y is of the general structure (CH2—
X—
CH2)n, wherein X represents O, S, SO, or SO2, and n is an integer from 2 to 25;(b) R1 is selected from;
steroid, retinoic acid, beta-lactam antibiotic, cannabinoid, nucleic acid, polypeptide, FK506, FK506 derivative, rapamycin, tetracycline, methotrexate, novobiocin, maltose, glutathione, biotin, vitamin D, dexamethasone, estrogen, progesterone, cortisone, testosterone, nickel, 2,4-diaminopteridine or cyclosporin, or a derivative thereof with structural modifications that retain a biological activity thereof, and,(c) L is a chemical group that can be substituted by a different chemical group, and (ii) instructions to use the compound for the synthesis of a hybrid ligand R1-Y—
R2 where R1 is different from R2, and at least one of R1 and R2 is not a peptide.
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29. A composition comprising:
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(i) a hybrid ligand of general structure R1-Y—
R2, wherein R1 represents a first ligand selected from;
steroid, retinoic acid, beta-lactam antibiotic, cannabinoid, nucleic acid, polypeptide, FK506, FK506 derivative, rapamycin, tetracycline, methotrexate, novobiocin, maltose, glutathione, biotin, vitamin D, dexamethasone, estrogen, progesterone, cortisone, testosterone, nickel, 2,4-diaminopteridine or cyclosporin, or a derivative thereof with structural modifications that retain a biological activity thereof;
Y is a linker having the general formula (CH2—
X—
CH2)2; and
R2 represents a user-specified second ligand different from R1 selected from;
a peptide, nucleic acid, carbohydrate, polysaccharide, lipid, prostaglandin, acyl halide, alcohol, aldehyde, alkane, alkene, alkyne, alkyl, alkyl halide, alkaloid, amine, aromatic hydrocarbon, sulfonate ester, carboxylate acid, aryl halide, ester, phenol, ether, nitrile, carboxylic acid anhydride, amide, quaternary ammonium salt, imine, enamine, amine oxide, cyanohydrin, organocadmium, aldol, organometallic, aromatic hydrocarbon, nucleoside, or a nucleotide, and(ii) a fusion polypeptide, comprising at least two domains which are not found in combination in nature, wherein (a) one domain is a user specified polypeptide, and (b) a second domain is a DNA binding domain. - View Dependent Claims (30, 31, 32)
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33. A composition comprising
(i) a polypeptide comprising a fragment of a β - -lactamase and a user specified polypeptide, and
(ii) a second polypeptide comprising a second fragment of a β
-lactamase and a domain chosen from the group consisting of;
β
-lactamase, a steroid receptor, retinoic acid receptor, cannabinoid receptor, FKB12, Tet-R, DHFR, GyrB, maltose binding protein, glutathione-S-transferase, vitamin D receptor, glucocorticoid receptor, estrogen receptor, progesterone receptor, testosterone receptor, or a fragment of one of the above, which fragment retains the binding capacity to its respective ligand,wherein said first and second fragments of a β
-lactamase individually possess no β
-lactamase activity, and wherein the enzymatic activity of a β
-lactamase is reconstituted when P1 and P2 are brought into close spatial proximity. - View Dependent Claims (34, 35, 36, 37)
- -lactamase and a user specified polypeptide, and
Specification