靶向蛋白降解（Targeted Protein Degradation， TPD）是通过化学小分子诱导特异性蛋白降解的新技术，可以利用细胞自身的蛋白酶体降解系统，特异的、 高效的降解靶蛋白。 这项技术潜在优势是有效的解决了传统小分子抑制剂因为需要持续和靶点结合，导致用量过大而带来的毒副作用和耐药性。 目前已经发展出非嵌合体的分子胶水（molecular glue） 以及杂合双功能分子 PROTAC 两种类型，两者的功能都是使靶标蛋白和 E3 泛素连接酶结合，并诱导靶标蛋白的泛素化从而被蛋白酶体降解。本课题的出发点是筛选拮抗 NRF2 活性的小分子抑制剂。 NRF2 在近四分之一的肺癌中过表达，是造成癌症细胞增殖和耐药性的关键转录因子。 NRF2 通过结合到抗氧化识别元件（ARE）来激活下游基因的表达。在肺癌细胞系 A549 中利用抗氧化识别元件-荧光素酶报告基因筛选系统 ARE-Luciferase 对 65790 种化合物进行筛选，发现候选化合物 F5950-0461（简称 0461）能降低 NRF2 结合 ARE 序列的活性，进一步实验发现 0461 降低了 NRF2mRNA 和蛋白水平， 从而影响 NRF2 下游基因的表达， 并且发现 0461 具有剂量依赖的细胞杀伤作用。 并且在 A549 细胞中敲除 NRF2 并不影响细胞生长， NRF2 野生型和 NRF2 敲除的细胞对 0461 的敏感性没有显著差异，这说明 NRF2 不是 0461 杀伤癌症细胞的直接靶点。为了找出 0461 在细胞内发挥毒性的功能靶点，首先进行了全基因组 CRISPR-sgRNA的功能遗传学筛选。 细胞感染编码 Cas9 和 sgRNA 文库的病毒后， 在 0461 的药物压力下，筛选出敲除后对 0461 产生耐药性的基因。 实验结果表明在 0461 药物压力下被富集的基因编码 E3 泛素连接酶 Cullin4 RING Ligase（CRL4）（候选基因有 DDB1， Cullin4A， RBX1，DDA1， UBE2G1 等）以及调节 CRL4 活性的 neddylation 相关蛋白（候选基因有 UBE2M，NAA30/35/38， NAE1 等）。 这个实验结果暗示了 0461 通过 CRL4 依赖的蛋白降解系统发挥杀伤癌细胞的功能。为了进一步寻找 0461 降解的功能底物， 接下来使用直肠癌细胞系 HCT116 进行正向遗传学的筛选。 HCT116 的 DNA 错配修复关键基因 MLH1 缺失， 导致其自发产生大量点突变，通过 0461 压力筛选得到 HCT116 抗性克隆。 利用外显子组测序技术和 Sanger 测序发现 0461 抗性克隆中富集了 CDK12 的点突变 G731E/G731R。在 A549 中外源表达编码CDK12 G731E/G731R 的 cDNA 或者使用 CRISPR 基因敲入 CDK12 G731E/G731R 都能显著的使细胞产生 0461 抗性。 CDK12 和 CCNK 形成了异源复合物， 通过磷酸化二型 RNA聚合酶的 C 端结构域（CTD）从而调控转录。 通过免疫印迹实验发现， 0461 处理细胞，并不能显著降低 CDK12 的蛋白水平， 但是 CCNK 的蛋白水平显著降低， 表达 CDK12 G731E或者 G731R 之后， 0461 诱导的 CCNK 降低受阻， 从而证明 CCNK 是 0461 的功能底物。接下来通过生物化学手段研究了 0461 促进 CDK12 和 CCNK 被降解的机理。通过免疫共沉淀发现， 0461 存在情况下，野生型 CDK12-CCNK 复合体与 DDB1 直接结合，但突变型 CDK12 不能与 DDB1 结合。 DDB1 是 CRL4 E3 泛素连接酶上面的接头蛋白，通过结合不同的底物受体蛋白（substrate receptor）实现结合底物的特异性。 深入研究发现， 在 0461催化 CCNK 降解过程中，没有经典的底物受体蛋白存在，相反， CDK12 蛋白充当了底物受体的角色。 这也是首次将非底物受体转变为底物受体的小分子， 从而揭示了一种新的靶向蛋白降解的方式。 进一步通过体内泛素化实验和体外生化重组， 证明了 CDK12 和 DDB1结合之后 CCNK 被泛素化。本课题使用传统小分子高通量筛选的方法，筛选出一个具有抗癌活性的小分子。 通过无偏见的遗传筛选和生物化学重组，揭示了 0461 作为分子胶水，促进 CDK12 和 DDB1 的结合，造成 CDK12 结合蛋白 CCNK 的降解。 0461 可以作为结合 E3 的配体进行改造，为开发新型的 PROTAC 提供了新的功能元件。

Targeted Protein Degradation (TPD) is a new technology that uses small molecules to induce degradation of specific target proteins. TPD relies on the cellular ubiquitin proteasome system (UPS) to specifically and efficiently degrade target proteins. Unlike traditional small molecular inhibitors that need to continuously occupy their target proteins, TPD offers the potential of lowering drug dose to reduce undesirable side effects and emergence of drug resistance. Molecular glues and bifunctional PROTAC are two prominent classes of TPD, both of which function to bind the target protein and an E3 ubiquitin ligase, thereby inducing the polyubiquitination of the target protein for proteasomal degradation. The starting point of this project is to screen small molecule antagonists of overactived NRF2 in cancer cells. NRF2 is overexpressed in nearly a quarter of lung cancers and is a key transcription factor that causes cancer cell proliferation and drug resistance. NRF2 activates downstream gene expression by binding to antioxidant recognition elements (ARE). In the lung cancer cell line A549, we screened 65,790 compounds using the antioxidant recognition element-luciferase (AREluciferase) reporter, and discovered a candidate compound F5950-0461 (abbreviated as 0461) that reduced the activity of ARE-luciferase. Further experiments showed that 0461 reduced NRF2 mRNA and protein levels, thereby affecting the expression of NRF2 downstream genes. Furthermore, 0461 has a dose-dependent cytotoxic effect. However, knocking out NRF2 in A549 cells did not affect cell growth. The sensitivity of NRF2 WT and NRF2 knockout cells to 0461 did not differ significantly, indicating that NRF2 is not a direct target for 0461’s anti-cancer activity. In order to identify the functional target of 0461, we performed pooled whole genome CRISPR-sgRNA screen. A549 cells were infected with viruses encoding Cas9 and sgRNA libraries and treated with 0461 over a course of two weeks. The experimental results revealed that genes enriched after 0461 selection encode E3 ubiquitin ligase Cullin4 RING Ligase (CRL4) (DDB1, Cullin4A, RBX1, DDA1, UBE2G1, etc.) and neddylation-related proteins that regulate CRL4 activity (UBE2M, NAA30 / 35/38, NAE1, etc.). These experimental results imply that 0461 exerts its cytotoxicity through CRL4-dependent protein degradation. In order to further identify a functional target for 0461, we used the colorectal cancer cell line HCT116 to perform forward genetic screening. HCT116 is deficient in DNA mismatch repair pathway, resulting in a large number of random spontaneous mutations. After selection with 0461, we isolated several independent 0461 resistance clones of HCT116. Using exome sequencing technology and Sanger sequencing, we found that 0461 resistant HCT116 clones were enriched with CDK12 point mutations (G731E, G731R). Exogenous expression of cDNA encoding CDK12 G731E or G731R in A549 or genomic knock-in of CDK12 G731E or G731R using CRISPR all render resistance to 0461. CDK12 and CCNK form a heterodimeric complex, which regulates transcription by phosphorylating the C-terminal domain (CTD) of RNA polymerase II. Through immunoblotting experiments, we found that 0461 treatment did not significantly reduce the protein level of CDK12, instead the protein level of CCNK was significantly reduced. After expressing CDK12 G731E or G731R, 0461 mediated reduction of CCNK was blocked, thus proving evidence that CCNK is a functional substrate of 0461. Next, we use biochemical reconstitution to reveal the mechanism by which 0461 promote CCNK degradation. Through immunoprecipitation, we found that in the presence of 0461, wildtype CDK12-CCNK complex directly binds to DDB1, but the G731E or G731R mutant CDK12 fails to bind to DDB1. DDB1 is an adaptor protein of the CRL4 E3 ubiquitin ligase, which recognizes a variety of substrates via binding different substrate receptor proteins. We found that in the process of 0461 mediated degradation of CCNK, there is no classic substrate receptor protein. On the contrary, CDK12 protein acts as a substrate receptor. To our knowledge, this is the first small molecule that has the ability to convert a non-substrate receptor to a substrate receptor, thus revealing a new possibility of targeting protein degradation. Furthermore, through in vivo ubiquitination experiments and in vitro biochemical reconstitution with purified proteins, we proved that CCNK was polyubiquitinated after CDK12 and DDB1 were induced to interact with each other by 0461. This thesis uses the traditional high-throughput screening method to discovery a small molecule with anti-cancer activity. Through unbiased genetic screening and biochemical reconstitution, a novel mechanism was revealed that 0461 functions as a molecular glue to promote the binding of CDK12 and DDB1, resulting in the degradation of CDK12 binding protein CCNK. 0461 can be potentially modified as a ligand that recruits DDB1, providing a new functional element for the development of a new generation of PROTAC.