Methods to identify compounds useful for the treatment of proliferative and differentiative disorders

Abstract

The present invention relates to the discovery, identification and characterization of nucleotide sequences that encode novel substrate-targeting subunits of ubiquitin ligases. The invention encompasses nucleotides encoding novel substrate-targeting subunits of ubiquitin ligases: FBP1, FBP2, FBP3, FBP4, FBP5, FBP6, FBP7, FBP8, FBP9, FBP10, FBP11, FBP12, FBP13, FBP14, FBP15, FBP16, FBP17, FBP18, FBP19, FBP20, FBP21, FBP22, FBP23, FBP24, and FBP25, transgenic mice, knock-out mice, host-cell expression systems and proteins encoded by the nucleotides of the novel substrate-targeting subunits. The present invention relates to screening assays that use novel and known substrate-targeting subunits of ubiquitin ligases to identify potential therapeutic agents such as small molecules, compounds or derivatives and analogues of the novel and known ubiquitin ligases which modulate activity of the novel and known ubiquitin ligases for the treatment of proliferative and differentiative disorders, such as cancer, major opportunistic infections, immune disorders, certain cardiovascular diseases, and inflammatory disorders. The invention further encompasses therapeutic protocols and pharmaceutical compositions designed to target ubiquitin ligases and their substrates for the treatment of proliferative and differentiative disorders.

Description

1. CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation-in-part of Ser. No. 10 / 632,150, filed Jul. 30, 2003, which is a division of Ser. No. 09 / 385,219, filed Aug. 27, 1999. This application is also a continuation-in-part of Ser. No. 10 / 042,417, filed Jan. 7, 2002.2. INTRODUCTION [0002] The present invention relates to the discovery, identification and characterization of nucleotide sequences that encode novel substrate-targeting subunits of ubiquitin ligases. The invention encompasses nucleic acid molecules comprising nucleotide sequences encoding novel substrate-targeting subunits of ubiquitin ligases: FBP1, FBP2, FBP3a, FBP3b, FBP4, FBP5, FBP6, FBP7, FBP8, FBP11, FBP12, FBP13, FBP14, FBP15, FBP17, FBP18, FBP20, FBP21, FBP22, FBP23, and FBP25, transgenic mice, knock-out mice, host cell expression systems and proteins encoded by the nucleotides of the present invention. The present invention relates to screening assays to identify potential therapeutic ...

AI Technical Summary

Benefits of technology

[0033] The invention further encompasses a method for screening compounds useful for the treatment of proliferative and differentiative disorders, comprising contacting a compound with a cell or a cell extract expressing both Fbp1 and β-Trcp2, and an Fbp1 target substrate, and detecting a change in the activity of Fbp1 or β-Trcp2. In a specific embodiment, the target substrate is β-catenin. In another specific embodiment, the target substrate is IkBα. In another specific embodiment, the change in the activity of Fbp1 or β-Trcp2 is detected by detecting a change in the interaction of Fbp1 or β-Trcp2 with β-catenin. In a further specific embodiment, the change in the activity of Fbp1 or βTrcp2 is detected by detecting a change in the interaction of Fbp1 or β-Trcp2 with IkBα. In another specific embodiment, the change in the activity of Fbp1 or β-Trcp2 is detected by detecting a change in the levels of protein of β-catenin. In an additional specific embodiment, the change in the activity of Fbp1 or β-Trcp2 is detected by detecting a change in the levels of protein of IkBα.

[0034] The invention also encompasses a method for screening compounds useful for the treatment of proliferative and differentiative disorders comprising (a) contacting a compound with a cell or a cell extract expressing Fbp1 and a test compound, and detecting a change in the activity of Fbp1, (b) contacting a compound with a cell or a cell extract expressing β-Trcp2 , and a test compound, and detecting a change in the activity of β-Trcp2, and (c) contacting a compound with a cell or a cell extract expressing Fbp1 and β-Trcp2, and the test compound or compounds identified as changing the activity of Fbp1 or β-Trcp2, and detecting a change in the activity of Fbp1 or β-Trcp2. In a specific embodiment, the change in the activity of Fbp1 or β-Trcp2 is detected by detecting a change in the levels of protein of β-catenin. In another specific embodiment, the change in the activity of Fbp1 or β-Trcp2 is detected by detecting a change in the levels of protein of IkBα.

[0035] The invention further encompasses a method for screening compounds useful for the treatment of proliferative and differentiative disorders or for inducing or inhibiting apoptosis in a cell, comprising contacting a compound with a cell or a cell extract expressing both Fbp24 and an Fbp24 target substrate, and detecting a change in the activity of Fbp24. In a specific embodiment, the change in activity of FBP24 is detected by detecting a change in the interaction of FPB24 with the target substrate. In a specific embodiment, the target substrate is DNA. In another specific embodiment, the target substrate is Elongation Factor 2. In a further specific embodiment, the target substrate is endostatin-like protein. In an additional specific embodiment, the target substrate is BAF57. In another specific embodiment, the target substrate is splice isoform SP100-HMG (P23497 nuclear autoantigen SP100). In a further specific embodiment, the target substrate is H326 Protein.

[0036] The invention further encompasses a method for screening compounds useful for the treatment of proliferative and differentiative disorders or for modulating cell proliferation comprising contacting a compound with a cell or a cell extract expressing both Fbl11 and an Fbl11 target substrate, and detecting a change in the activity of Fbl11. In a specific embodiment, the target substrate is DNA. In another specific embodiment, the target substrate is MCM7. In an additional specific embodiment, the change in the activity of Fbl11 is detected by detecting a change in the interaction of Fbl11 with DNA. In an additional specific embodiment, the change in the activity of Fbl11 is detected by detecting a change in the interaction of Fbl11 with MCM7.

[0037] The invention also encompasses a method for screening compounds useful for the treatment of proliferative and differentiative disorders, for modulating cell proliferation, or for sensitizing cells to chemotherapy, comprising contacting a compound with a cell or a cell extract expressing both Fbl12 and an Fbl2 target substrate, and detecting a change in the activity of Fbl12.

Problems solved by technology

When positive signals overcome or when negative signals are absent, the cells multiply too quickly and cancer develops.

This mechanism goes awry in tumors, leading to the excessive accumulation of positive signals (oncogenic proteins), or resulting in the abnormal degradation of negative regulators (tumor suppressor proteins).

F box proteins in yeast are known to regulate genomic stability and senescence, and recent data has shown that F box inhibition in mammalian cells can lead to the loss of DNA damage checkpoints.

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