Three hybrid screening assay
First Claim
1. A method for identifying which if any small molecule from a pool of candidate small molecules binds a known first target in a population of cells, comprising:
- (a) with respect to each candidate small molecule, forming a hybrid ligand by chemical linkage of the small molecule to a known molecule, the known molecule binding a known second target;
(b) introducing each hybrid ligand into the cells, each cell containing;
(i) a first expression vector, including a DNA encoding the known first target, linked to a coding sequence for a first transcriptional module for expression as a first hybrid protein;
(ii) a second expression vector including DNA encoding the known second target, linked to a coding sequence for a second transcriptional module for expression as a second hybrid protein; and
(iii) a third vector including a reporter gene wherein expressing the reporter gene is conditioned on the proximity of the first and second hybrid proteins;
(c) permitting the hybrid ligand to bind to the first hybrid protein and the second hybrid protein so as to activate the expression of the reporter gene;
(d) selecting which sample expresses the reporter gene; and
(e) identifying the small molecule that binds the first target.
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Abstract
Methods and a kit are provided for characterizing small molecules from a library of small molecules or alternatively identifying protein targets to which known small molecules bind. The methods include forming hybrid ligand in which at least one ligand is a small molecule. The hybrid ligand is introduced into cells that in turn contain a first and a second expression vector. Each expression vector includes DNA for expressing a hybrid protein that encodes a target protein linked to a coding sequence for a transcriptional module. The cells further contains a reporter gene, the expression of which is conditioned on the proximity of the first and second hybrid proteins to each other, an event that occurs only if the hybrid ligand binds to target sites on both hybrid proteins. Those cell which express the reporter gene are selected and the unknown small molecule or the unknown hybrid protein is identified.
65 Citations
33 Claims
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1. A method for identifying which if any small molecule from a pool of candidate small molecules binds a known first target in a population of cells, comprising:
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(a) with respect to each candidate small molecule, forming a hybrid ligand by chemical linkage of the small molecule to a known molecule, the known molecule binding a known second target; (b) introducing each hybrid ligand into the cells, each cell containing; (i) a first expression vector, including a DNA encoding the known first target, linked to a coding sequence for a first transcriptional module for expression as a first hybrid protein; (ii) a second expression vector including DNA encoding the known second target, linked to a coding sequence for a second transcriptional module for expression as a second hybrid protein; and (iii) a third vector including a reporter gene wherein expressing the reporter gene is conditioned on the proximity of the first and second hybrid proteins; (c) permitting the hybrid ligand to bind to the first hybrid protein and the second hybrid protein so as to activate the expression of the reporter gene; (d) selecting which sample expresses the reporter gene; and (e) identifying the small molecule that binds the first target. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)
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17. A method for identifying a protein target to which a small molecule is capable of binding, comprising:
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(a) providing a hybrid ligand consisting essentially of a first small molecule ligand, identified as ligand A and a second molecule identified as ligand B, that are covalently linked by chemical synthesis, wherein ligand A has a specificity for a first preselected protein target and ligand B has a specificity for a second unknown protein target; (b) introducing the hybrid ligand into a population of cells, each cell containing; (i) a first expression vector, including DNA encoding the first protein target for ligand A, linked to a coding sequence for a first transcriptional module for expression as a first hybrid protein; (ii) a second expression vector including a random DNA fragment encoding the second protein target linked to a second transcriptional module for expression as a second hybrid protein; and (iii) a third vector including a reporter gene wherein the expression of the reporter gene is conditioned on the proximity of the first and second hybrid proteins; (c) permitting the hybrid ligand to bind the first hybrid protein through ligand A and the second hybrid protein through ligand B so as to activate the expression of the reporter gene; (d) selecting the cells expressing the reporter gene; and (e) identifying the second protein target in the samples selected in (d). - View Dependent Claims (19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33)
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18. A kit for detecting interactions between pharmacologically relevant small molecules and proteins, comprising;
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(a) a preactivated small molecule ligand A and reagents for forming a hybrid molecule with at least one type of small molecule ligand B; (b) a first expression vector including DNA encoding the binding protein for Ligand A linked to a coding sequence for a first transcriptional module for expression as a first hybrid protein; (c) a second expression vector including a random DNA fragment encoding a polypeptide linked to a coding sequence for a second transcriptional module for expression as a second hybrid protein; (d) a third vector including a reporter gene wherein transcription of the reporter gene is conditioned on the proximity of the first and second hybrid proteins; (e) an environment for transcription and translation of the hybrid proteins and the reporter gene; and (f) a signal to detect the expression of the reporter gene following the formation of a trimeric complex between the hybrid ligand and the hybrid proteins.
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Specification