In vivo screen using chemical inducers of dimerization

A compound having the formula:

• H1-X-B′
• wherein H1 is capable of binding to a receptor;
• wherein X is a spacer moiety which may be present or absent;
• and
• wherein B′ is a moiety capable of binding to an enzyme.

A method of screening proteins for the ability to catalyze bond cleavage, comprising the steps of:

• a) providing a cell that expresses a pair of fusion proteins which upon dimerization change a cellular readout;
• b) providing a compound which dimerizes the pair of fusion proteins, said compound comprising two portions coupled by a bond that is cleavable by the protein to be screened;
• and
• c) screening for the cellular readout, wherein a change in the cellular readout indicates catalysis of bond cleavage by the protein to be screened.

A method of screening proteins for the ability to catalyze bond formation, comprising the steps of:

• a) providing a cell that expresses a pair of fusion proteins which upon dimerization activate a cellular readout:
• b) providing a first compound and a second compound, each being capable of binding to one of the pair of fusion proteins, said first and second compound comprising a portion through which the first and second compounds are coupled by the action of the bond forming protein to be screened;
• and
• c) screening for the cellular readout, wherein a change in the cellular readout indicates catalysis of bond formation by the protein to be screened.

A compound having the formula:

• H1-Y-H2
• wherein H1 is methorexate or an analog thereof;
• wherein H2 is capable of binding to a receptor, and
• wherein Y is a moiety providing a covalent linkage between H1 and H2, which may be present or absent, and when absent, H1 is covalently linked to H2.

A method for identifying a molecule that binds a known target in a cell from a pool of candidate molecules, comprising:

• (a) covalently bonding each molecule in the pool of candidate molecules to a methotrexate moiety or an analog of methotrexate to form a screening molecule;
• (b) introducing the screening molecule into a cell which expresses a first fusion protein comprising a binding domain capable of binding methotrexate, a second fusion protein comprising the known target, and a reporter gene wherein expression of the reporter gene is conditioned on the proximity of the first fusion protein to the second fusion protein;
• (c) permitting the screening molecule to bind to the first fusion protein and to the second fusion protein so as to activate the expression of the reporter gene;
• (d) selecting which cell expresses the reporter gene;
• and
• (e) identifying the small molecule that binds the known target.

A method for identifying a protein target to which a molecule is capable of binding, comprising:

• (a) providing a screening molecule comprising a methotrexate moiety or an analog of methotrexate covalently bonded to a ligand which has a specificity for an unknown protein target;
• (b) introducing the screening molecule into a cell which expresses a first fusion protein comprising a binding domain capable of binding methotrexate, a second fusion protein comprising the unknown protein target, and a reporter gene wherein expression of the reporter gene is conditioned on the proximity of the first fusion protein to the second fusion protein;
• (c) permitting the screening molecule to bind to the first fusion protein and to the second fusion protein so as to activate the expression of the reporter gene;
• (d) selecting which cell expresses the reporter gene;
• and
• (e) identifying the unknown protein target.

A method for identifying a molecule that binds a known target in a cell from a pool of candidate molecules, comprising:

• (a) covalently bonding each molecule in the pool of candidate molecules to a methotrexate moiety to form a screening molecule;
• (b) introducing the screening molecule into a cell which expresses a first fusion-protein comprising a binding domain capable of binding methotrexate, a second fusion protein comprising the known target, and a reporter gene wherein expression of the reporter gene is conditioned on the proximity of the first fusion protein to the second fusion protein;
• (c) permitting the screening molecule to bind to the first fusion protein and to the second fusion protein so as to activate the expression of the reporter gene;
• (d) selecting which cell expresses the reporter gene;
• and
• (e) identifying the small molecule that binds the known target.