Regulation of cell survival by HSP90 and IP6K2
Anti-cancer drugs are identified by screening for agents and compounds which inhibit the binding of HSP90 and IP6K2. In vitro and in vivo assays can be used. Any phenomenon associated with the binding or inhibition can be monitored, including cell death, subcellular localization, catalytic activity of IP6K2, and IP7 formation.
1. A method of identifying compounds which interfere with the binding of human IP6K2 protein to human HSP90 protein, comprising:
contacting a cell with a test compound, wherein the cell comprises three recombinant DNA constructs,
said first construct encoding a first polypeptide fused to a sequence-specific DNA-binding domain,
said second construct encoding a second polypeptide fused to a transcriptional activation domain,
said third construct comprising a reporter gene downstream from a DNA element which is recognized by said sequence-specific DNA-binding domain,
wherein said first polypeptide is a IP6K2 polypeptide and said second polypeptide is a HSP90 polypeptide, or said first polypeptide is a HSP90 polypeptide and said second polypeptide is a IP6K2 polypeptide; wherein said HSP90 polypeptide comprises amino acids 751-854 of SEQ ID NO: 2 or amino acids 630-731 of SEQ ID NO: 3 and said IP6K2 polypeptide comprises amino acids 131-140 of SEQ ID NO: 1;
determining quantity of expression of the reporter gene in the presence of said compound, wherein a decrease in the expression of the reporter gene in the presence of the compound indicates that the compound interferes with the binding of human IP6K2 protein to human HSP90.
2. An isolated cell which comprises three recombinant DNA constructs,
said first construct encoding a first polypeptide fused to a sequence-specific DNA-binding domain,
said second construct encoding a second polypeptide fused to a transcriptional activation domain,
said third construct comprising a reporter gene downstream from a DNA element which is recognized by said sequence-specific DNA-binding domain,
wherein said first polypeptide is an IP6K2 polypeptide and said second polypeptide is a HSP90 polypeptide, or said first polypeptide is a HSP90 polypeptide and said second polypeptide is an IP6K2 polypeptide; wherein said HSP90 polypeptide comprises amino acids 751-854 of SEQ ID NO: 2 or amino acids 630-731 of SEQ ID NO: 3 and said IP6K2 polypeptide comprises amino acids 131-140 of SEQ ID NO: 1.