Targeting the E1 Control Point of Protein Ubiquitination in Cancer

项目总结 泛素-蛋白酶体系统(UPS)是一种动态平衡的酶级联反应，标记有缺陷或不需要的 与泛素结合的细胞蛋白，导致其有效的蛋白酶体降解。蛋白质质量控制至关重要 由于对细胞增殖的更高要求以及对癌细胞的损害 来自一系列压力的癌细胞蛋白质组。多发性骨髓瘤是人类癌症的典型例子。 由于抗体蛋白的大量过量生产而精致地依赖于蛋白酶体，因此 波特佐米对蛋白酶体抑制敏感。FDA批准Bortezomib预示着 通过靶向E1、E2和E3泛素转移来调节UPS以获得治疗益处的多种方法 酶和去泛素酶。UBE1是泛素化级联反应中最顶端的酶，它的独特之处在于 负责99%的泛素充电的E2蛋白。小分子TAK-243是为靶向而开发的 UBE1的ATP结合口袋，并与泛素的C-端形成共价加合物，导致停滞 酶的活性。Tak-243在体外、小鼠模型和小阶段显示了抗肿瘤活性 1项试验，强调了阻断UBE1作为抗癌策略的潜力。然而，点突变发生在 TAK-243结合位点可以引起耐药性，从而授权了在癌症中靶向UBE1的替代方法。 对E_1/E_2复合体结构的研究表明，E_1表面有一条沟槽，它与N-末端的a-结合。 1螺旋的E2(E2h1)，表明模拟E2h1的α-螺旋结构可能成为一种潜在的UBE1抑制剂。 像E2h1这样的自然基序通常在脱离天然蛋白质的上下文时展开，导致 具有生物活性的外形和快速的蛋白质降解能力。多肽装订加强了天然的α-螺旋形状 生物活性多肽和赋予其稳定的结构，体内抗蛋白酶，增强靶向结合亲和力， 以及良好的药理作用。将这项技术应用于UBE1目标定位，Walensky实验室最近生成了 使用UBE2A的E2h1序列的原型抑制剂。结合在UBE1表面凹槽上的装订多肽 并阻断泛素向E2蛋白的转移，导致体外抑制蛋白质泛素化。在这里，我 建议将我们的装订多肽方法与我们最近发现的靶向多肽相结合 UBE1上的螺旋沟槽相互作用，以推进人类白血病的独特治疗策略。具体来说， 我的目标是(1)设计、合成和表征以螺旋为靶点的新型E1(SPIES)钉接肽抑制剂。 E1和E2蛋白之间的沟槽内接口；(2)通过以下方式定义E1靶向的构象结果 间谍和铅SPIE/E1复合体的结构；以及(3)将铅间谍推进到依赖UPS的测试中 评价白血病细胞的作用机制和抗癌效果。我很高兴能继续推行建议的 我在达纳大学洛伦·沃伦斯基博士的实验室学习化学生物学研究生的培训计划- 法伯癌症研究所和哈佛医学院，并期待发展成为一个有创造力的，独立的， 以及处于抗癌药物发现和癌症治疗前沿的创新内科科学家。

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.