Lysine-free ubiquibody variants for long-lived intracellular protein silencing

A method of screening agents for therapeutic efficacy against a disease, said method comprising:

• providing a biomolecule whose presence is mediated by a disease state;
• providing a test agent comprising (i) a degradation domain comprising a E3 ubiquitin ligase motif without lysine residues, (ii) a targeting domain comprising a substrate-binding motif which is heterologous to the E3 ubiquitin ligase motif, and (iii) a linker coupling said degradation domain to said targeting domain;
• contacting said biomolecule and said test agent under conditions effective for the test agent to facilitate degradation of the biomolecule;
• determining the level of said biomolecule as a result of said contacting;
• and
• identifying said test agent which, based on said determining, decreases the level of said biomolecule as being a candidate for therapeutic efficacy against said disease.

A method of screening for disease biomarkers, said method comprising:

• providing a sample of diseased cells expressing one or more ligands;
• providing a plurality of chimeric protein molecules comprising (i) a degradation domain comprising an E3 ubiquitin ligase motif without lysine residues, (ii) a targeting domain comprising a substrate-binding motif which is heterologous to the E3 ubiquitin ligase motif, and (iii) a linker coupling said degradation domain to said targeting domain;
• contacting said sample with said plurality of chimeric protein molecules under conditions effective for the diseased cells to fail to proliferate in the absence of the chimeric protein molecule;
• determining which of said chimeric protein molecules permit the diseased cells to proliferate;
• and
• identifying, as biomarkers for the disease, based on said determining the ligands which bind to the chimeric protein molecules and permit diseased cells to proliferate.