Targeting the E1 Control Point of Protein Ubiquitination in Cancer

项目摘要 泛素-蛋白酶体系统（UPS）是一种体内平衡的酶级联反应， 细胞蛋白与泛素，导致其有效的蛋白酶体降解。蛋白质质量控制至关重要 由于细胞增殖的需求增加和对细胞的持续损害， 癌细胞蛋白质组的变化。多发性骨髓瘤是一种典型的人类癌症 由于抗体蛋白的大量过量产生， 易受硼替佐米抑制蛋白酶体的影响。FDA对硼替佐米的批准预示着 通过靶向E1、E2和E3泛素转移调节UPS以获得治疗益处的多种方法 酶和去泛素化酶。UBE 1是泛素化级联反应中最顶端的酶， 负责E2蛋白99%的泛素充电。开发小分子TAK-243， UBE 1的ATP结合口袋，并与泛素的C-末端形成共价加合物，导致UBE 1的停滞。 酶的活性。TAK-243在体外、小鼠模型和小阶段中表现出抗肿瘤活性 1项试验，强调了阻断UBE 1作为抗癌策略的潜力。然而，点诱变在 TAK-243结合位点可导致耐药性，从而要求采用替代方法靶向癌症中的UBE 1。 对E1/E2复合物结构的检查显示，E1上的表面沟与N-末端α-葡聚糖结合。 1螺旋的E2（E2 h1），这表明E2 h1的a-螺旋模拟物可以被开发为潜在的UBE 1抑制剂。 当从天然蛋白质的背景中取出时，天然基序如E2 h1通常会展开，导致蛋白质的丢失。 生物活性形状和快速蛋白水解降解。肽钉合增强了天然的α-螺旋形状， 生物活性肽并赋予稳定的结构，体内蛋白酶抗性，增强的靶结合亲和力， 和良好的药理学。将这项技术应用于UBE 1靶向，Walensky实验室最近生成了 使用UBE 2A的E2 h1序列的原型抑制剂。与UBE 1表面沟结合的钉合肽 并阻断泛素向E2蛋白的转移，导致体外蛋白泛素化的抑制。这里我 我建议联合收割机我们的钉合肽方法与药物开发和我们最近发现的靶向 UBE 1上的螺旋-凹槽相互作用，以推进人类白血病的独特治疗策略。具体地说， 我的目标是（1）设计，合成和表征新型钉合肽抑制剂E1（SPIEs），靶向螺旋- E1和E2蛋白之间的沟内界面;（2）通过以下方式定义E1靶向的构象结果： SPIE和电极导线SPIE/E1复合体的结构;以及（3）将电极导线SPIE推进到UPS依赖的测试中 白血病细胞，以评估作用机制和抗癌功效。我很高兴能追求提议的 我在达纳大学洛伦·瓦伦斯基博士实验室的化学生物学研究生课程的培训计划- 法伯癌症研究所和哈佛医学院，并期待着发展成为一个创造性的，独立的， 在癌症药物发现和癌症护理的最前沿，创新的医生和科学家。

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.