Targeting the E1 Control Point of Protein Ubiquitination in Cancer - PROJECT SUMMARY The ubiquitin-proteasome system (UPS) is a homeostatic enzymatic cascade that tags defective or unwanted cellular proteins with ubiquitin, resulting in their efficient proteasomal degradation. Protein quality control is critical to cancer cell survival due to the heightened demands of cell proliferation and the damage sustained to the cancer cell proteome from a host of stresses. Multiple myeloma is a classic example of a human cancer exquisitely dependent on the proteasome due to massive overproduction of antibody proteins and is thus susceptible to proteasome inhibition by bortezomib. FDA approval of bortezomib heralded the development of multiple approaches to modulate the UPS for therapeutic benefit by targeting the E1, E2, and E3 ubiquitin transfer enzymes and deubiquitinases. UBE1 is the most apical enzyme of the ubiquitination cascade and is singularly responsible for 99% of ubiquitin charging of E2 proteins. The small molecule TAK-243 was developed to target the ATP binding pocket of UBE1 and forms a covalent adduct with the C-terminus of ubiquitin, resulting in arrest of enzymatic activity. TAK-243 demonstrated anti-tumor activity in vitro, in mouse models, and in a small phase 1 trial, underscoring the potential of blocking UBE1 as an anti-cancer strategy. However, point mutagenesis at the TAK-243 binding site can cause resistance, mandating alternative approaches to targeting UBE1 in cancer. Examination of the structures of E1/E2 complexes revealed a surface groove on E1 bound by the N-terminal a- 1 helix of E2 (E2h1), suggesting that an a-helical mimic of E2h1 could be developed as a potential UBE1 inhibitor. Natural motifs such as E2h1 often unfold when taken out of context of the native protein, resulting in loss of bioactive shape and rapid proteolytic degradation. Peptide stapling reinforces the natural a-helical shape of bioactive peptides and confers stabilized structure, protease resistance in vivo, enhanced target binding affinity, and favorable pharmacology. Applying this technology to UBE1 targeting, the Walensky lab recently generated a prototype inhibitor using the E2h1 sequence of UBE2A. The stapled peptide bound to the UBE1 surface groove and blocked ubiquitin transfer to E2 proteins, resulting in suppression of protein ubiquitination in vitro. Here, I propose to combine our stapled peptide approach to drug development and our recent discovery of a targetable helix-in-groove interaction on UBE1 to advance a unique treatment strategy for human leukemias. Specifically, I aim to (1) design, synthesize, and characterize novel stapled peptide inhibitors of E1 (SPIEs) that target a helix- in-groove interface between E1 and E2 proteins; (2) define the conformational consequences of E1 targeting by SPIEs and the structure of a lead SPIE/E1 complex; and (3) advance lead SPIEs to testing in UPS-dependent leukemia cells to evaluate mechanism of action and anti-cancer efficacy. I am excited to pursue the proposed training program for my chemical biology graduate studies in the laboratory of Dr. Loren Walensky at the Dana- Farber Cancer Institute and Harvard Medical School, and look forward to developing as a creative, independent, and innovative physician-scientist at the forefront of cancer drug discovery and cancer care.
