Ubiquitin (Ub) signaling regulates not only proteasomal protein degradation, but also protein trafficking, receptor- mediated signal transduction, and control of mitophagy and autophagy. Ub conjugating (E3 ligases) and deconjugating (deubiquitinases) enzymes and the proteasome have been targets of drug discovery focusing on degradation or preservation of disease-associated proteins, with some success in the approval of proteasome inhibitors and three drugs (thalidomide, pomalidomide, and lenalidomide) that target the E3 ligase Cereblon for the treatment of multiple myeloma or mantle cell lymphoma. The non-degradative events of Ub conjugation and deconjugation have not been widely exploited for therapeutic application, although therapeutic hypotheses lend themselves to these functions. Currently, a novel approach to target and dispose of disease-associated proteins is gaining interest and known as targeted protein degradation (TPD). This emerging technology uses bifunctional molecules known as PROTACs (Proteolysis targeting chimeras) that bind to a protein of interest while simultaneously tagging it for degradation via an E3 Ub ligase. Whats unique about this technology is that the target protein doesnt necessarily need to by a physiological substrate for the E3 ubiquitin ligase. This opens the door for the undruggable proteome including signaling adaptors and transcription factors. Although the therapeutic application of PROTACs has heretofore been considered protein degradation, the chemistry involved could have non-degradative applications, including subcellular localization or altering the activity of candidate substrates to generate a desired pharmacologic effect. The goal of this proposal is to develop a new application of PROTAC technology which we term LOTAC for localization targeting chimeras that hijacks the E3 Ub ligase TRAF6, which is known to catalyze K63-linked polyubiquitination. This goal will be addressed by achieving milestones including: 1) reconstituting active TRAF6 from E. coli and showing that specific TRAF6-binding compounds can function as LOTAC ligands; and 2) demonstrating that TRAF6 mediated polyubiquitination of FOXP3 causes its cellular localization from the cytosol to the nucleus. In Phase II, TRAF6 LOTACs will be designed using selective, high affinity ligands that bind to TRAF6 and alter the subcellular location of various substrates via K63-linked ubiquitination. This technology will assist medicinal chemists in developing LOTAC molecules that work by changing the subcellular location and/or activity of therapeutic target proteins rather than by eliminating them by proteasomal degradation. Successful development of such LOTACs will expand the utility of this novel form of ubiquitin-based therapy.
Public Health Relevance Statement: Ubiquitin ligases have been used as therapeutic targets for numerous diseases; inhibiting or activating these enzymes produces pharmacological effects based on protein degradation (or inhibition of degradation). Proteolysis-targeted chimeras or PROTACs for short, are bifunctional molecules developed to link ubiquitin ligases to target proteins for their proteasomal destruction with an increased efficiency. In addition to degradation, the process of ubiquitination also regulates the biological function or subcellular localization of proteins; and thus, we propose herein to develop a novel PROTAC strategy we term LOTAC (localization- targeted chimeras) using TRAF6, an E3 ubiquitin ligase that decorates substrates with non-degradative polyubiquitin chains that affects their cellular location and thereby expands the utility of PROTAC technology in treating various diseases.
Project Terms: Address; Affect; Affinity; amino group; Autoimmune Diseases; Autophagocytosis; base; Binding; Biological Assay; Biological Process; C-terminal; cell growth regulation; Cell Nucleus; Cells; Chemistry; Chimera organism; Clinical Trials; Cytosol; design; Development; Disease; drug discovery; Drug Targeting; Emerging Technologies; Enzymes; Escherichia coli; Event; FOXP3 gene; G-Protein-Coupled Receptors; Gene Expression Regulation; Glycine; Goals; Half-Life; human disease; Human Genome; interest; Investments; Lead; lenalidomide; Ligand Binding; Ligands; Ligase; Link; Location; Lysine; Malignant Neoplasms; Mantle Cell Lymphoma; Mediating; Modality; Modeling; multicatalytic endopeptidase complex; Multiple Myeloma; mutant; Neurodegenerative Disorders; new technology; novel; novel strategies; novel therapeutics; Nuclear Receptors; overexpression; Pharmaceutical Preparations; Pharmacologic Substance; Pharmacology; Phase; Phosphotransferases; Physiological; Play; Polyubiquitin; Polyubiquitination; pomalidomide; preservation; Process; Protac; Proteasome Inhibitor; protein degradation; protein transport; Proteins; Proteome; receptor function; Receptor Mediated Signal Transduction; reconstitution; Regulation; Regulatory T-Lymphocyte; Revlimid; Role; Signal Transduction; Site; Specificity; Structure; success; System; targeted treatment; Technology; Thalidomide; Therapeutic; Therapeutic Intervention; therapeutic target; Tissues; TRAF6 gene; transcription factor; Ubiquitin; ubiquitin ligase; ubiquitin-protein ligase; Ubiquitination; Work
