Screening method and modulators having an improved therapeutic profile

A method to identify compounds that bind to a nuclear receptor and exhibit cell type specific actions, said method comprising:

• a) contacting a first and second modified host cell with a test compound, wherein said first modified host cell comprises:
• i) a first fusion protein, comprising a co-activator fused to a first heterologous DNA binding domain,
• ii) a second fusion protein comprising a ligand binding domain of a nuclear receptor fused to a first transcription activation domain,
• iii) a first reporter gene operably linked to a first transcriptional regulatory sequence specific for said first heterologous DNA binding domain, and
• wherein said second modified host cell comprises,
• i) a third fusion protein, comprising a co-repressor fused to said first heterologous DNA binding domain or a second heterologous DNA binding domain,
• ii) a fourth fusion protein comprising said ligand binding domain of said nuclear receptor fused to said first transcription activation domain or a second transcription activation domain,
• iii) a second reporter gene operably linked to said first transcriptional regulatory sequence specific for said first heterologous DNA binding domain or a second transcriptional regulatory sequence specific for said second heterologous DNA binding domain,
• b)identifying those test compounds which cause altered expression of said first reporter gene product in said first modified host cell compared to a first modified host control cell, and similar or altered expression of said second reporter gene product in said second modified host cell, compared to a second modified host control cell.

A method to identify compounds to identify compounds that bind to a nuclear receptor and exhibit cell type specific actions, said method comprising:

• a) contacting a modified host cell with a test compound, wherein said modified host cell comprises:
• i) a first fusion protein, comprising a co-activator fused to a first heterologous DNA binding domain,
• ii) a second fusion protein comprising a co-repressor fused to a second heterologous DNA binding domain,
• iii) a third fusion protein comprising a ligand binding domain of a nuclear receptor fused to a transcription activation domain,
• iv) a first reporter gene operably linked to a first transcriptional regulatory sequence specific for said first heterologous DNA binding domain,
• v) a relay protein operably linked to a second transcriptional regulatory sequence specific for said second heterologous DNA binding domain,
• vi) a second reporter gene operably linked to a third transcriptional regulatory sequence that is repressed by expression of said relay protein,
• b) identifying those test compounds which caused altered expression of said first reporter gene product compared to a control modified host cell and similar or altered expression of said second reporter gene product compared to said control modified host cell.

A method to identify compounds that bind to a nuclear receptor and exhibit cell type specific actions, said method comprising:

• a) contacting a first and second modified host cell with a test compound,
• wherein said first modified host cell comprises:
• i) a first fusion protein, comprising a co-activator fused to a first heterologous DNA binding domain,
• ii) a second fusion protein comprising a ligand binding domain of a nuclear receptor fused to a first transcription activation domain,
• iii) a first reporter gene operably linked to a first transcriptional regulatory sequence specific for said first heterologous DNA binding domain, and
• wherein said second modified host cell comprises:
• vi) a third fusion protein, comprising a co-repressor fused to said first heterologous binding domain or a second heterologous binding domain,
• vii) a fourth fusion protein, comprising said ligand binding domain fused to said first transcription activation domain or a second transcription activation domain,
• viii) a relay plasmid comprising DNA encoding a relay protein operably linked to said first transcriptional regulatory sequence specific for said first heterologous DNA binding domain or a second transcriptional regulatory sequence specific for said second heterologous DNA binding domain,
• ix) a reporter gene operably linked to a third transcriptional regulatory sequence that is repressed by expression of said relay protein,
• b) identifying those test compounds which cause altered expression of said first reporter gene product in said first modified host cell compared to a first control modified host cell, and similar or altered expression of said second reporter gene product in said second modified host cell compared to a second control modified host cell.

A method to identify compounds that bind to a nuclear receptor and exhibit cell type specific actions, said method comprising:

• a) providing a composition comprising,
• i) an affinity support, comprising a first fusion protein comprising a ligand binding domain of a nuclear receptor fused to an affinity tag that couples said first fusion protein to said affinity support,
• ii) a second fusion protein, comprising a co-activator coupled to a first detectable label,
• iii) a third fusion protein comprising a co-repressor coupled to a second detectable label,
• b) incubating said composition in an aqueous buffer comprising a test compound,
• c) detecting the binding of said co-activator and said co-repressor to said first fusion protein,
• d) selecting compounds that cause disrupted, or substantially disrupted binding of said co-repressor without increasing binding of said co-activator to said ligand binding domain compared to a control composition.

A method to identify compounds that bind to a nuclear receptor and exhibit cell type specific actions, said method comprising,

• a) providing a compositions comprising;
• i) a ligand binding domain of a nuclear receptor, and
• ii) a co-activator coupled to a first detectable label, and
• iii) a co-repressor coupled to a second detectable label,
• b) incubating said composition in an aqueous buffer comprising a test compound,
• c) detecting binding of said co-activator and co-repressor with said ligand binding domain, and
• d) selecting compounds that cause disrupted, or substantially disrupted binding of said co-repressor without increasing binding of said co-activator to said ligand binding domain compared to a control composition.

A method to identify compounds that bind to a nuclear receptor and exhibit cell type specific actions, said method comprising,

• a) providing first and second compositions, wherein said first composition comprises;
• i) a ligand binding domain of a nuclear receptor coupled to a first detectable label, and
• ii) a co-activator coupled to a second detectable label, and
• wherein said second composition comprises;
• iii) said ligand binding domain, coupled to said first detectable label, and
• iv) a co-repressor coupled to said second detectable label,
• b) incubating said first composition and said second composition in an aqueous buffer comprising a test compound,
• c) detecting the binding of said co-activator with said ligand binding domain in said first composition and detecting the binding of said co-repressor with said ligand binding domain in said second composition,
• d) selecting compounds that cause disrupted, or substantially disrupted binding of said co-repressor compared to a first control composition, and failed to increase binding of said co-activator to said ligand binding domain compared to a second control composition.