Multiplexed in vivo drug screening: Inhibitors of metastatic seeding

描述（由申请方提供）：胰腺导管腺癌（PDAC）是一种常见且几乎一致致命的恶性肿瘤。有几个因素导致PDAC患者的不良结局，但癌细胞离开原发肿瘤并建立不可手术转移的能力仍然是成功治疗的主要障碍。转移是一个复杂的多步骤过程，在分子水平上知之甚少。特别是，癌细胞离开血液并进入次级器官所需的分子事件，一个称为转移性播种的过程，仍然难以捉摸。抑制转移性播种的工具化合物的鉴定将不仅阐明实现该过程的基本机制，而且还直接转化为抑制转移性扩散的治疗剂。虽然大的体外化合物筛选已被用于解决各种生物学问题，但仍然难以将这种方法用于复杂的体内过程，如转移，因为仍然不清楚哪些基于细胞的测定和读数准确地反映了体内过程。高含量的体内化学筛选历来具有挑战性，因为并行进行这些测定的成本很高。我们的多学科的建议将建立一个新的方法，在体内多重筛选共价抑制剂，使化学遗传解剖的转移播种过程。为了鉴定抑制转移性接种的初始步骤的化合物，我们将筛选约1，500种水解酶的小分子共价抑制剂的文库。重要的是，这些化合物含有亲电子陷阱，其不可逆地结合其靶标，使得在细胞的体外预处理后能够持续抑制，而不需要继续给药。为了允许多重体内筛选，我们产生了转移来源的PDAC细胞系的96个变体，其中每个变体细胞系含有独特的6-核苷酸条形码（PDACBC）。我们将在体外用一种不可逆抑制剂预处理每个PDACBC细胞系，然后将96个预处理的PDACBC细胞系合并。将使用条形码区域的IlluminaTM测序来确定注射前和转移后接种细胞中的条形码表示。在接种后群体中代表性不足的条形码将鉴定转移性接种的候选抑制剂。为了验证最佳候选物，将用最佳候选物化合物预处理PDAC细胞，并评估其接种转移和形成大转移的能力。为了鉴定这些抑制剂的靶标，我们将产生命中化合物的标记类似物，以使药物-靶标复合物的纯化能够进行基于质谱的蛋白质鉴定。这些靶点的shRNA敲低，随后静脉注射也将用于确认靶点对转移性接种的影响。我们的研究不仅将确定新的药物靶点，而且还将确定足以引起体内转移能力降低的先导化合物。我们的研究将促进我们对PDAC转移的理解，并为其他癌症类型的常见机制提供见解。

DESCRIPTION (provided by applicant): Pancreatic ductal adenocarcinoma (PDAC) is a prevalent and almost uniformly fatal malignancy. Several factors contribute to the poor outcome of PDAC patients, but the ability of cancer cells to leave the primary tumors and establish inoperable metastases remains a major impediment to successful therapy. Metastasis is a complex multistep process that is poorly understood at the molecular level. In particular, the molecular events that are required for a cancer cell to leave the blood and enter a secondary organ, a process called metastatic seeding, have remained elusive. The identification of tool compounds that inhibit metastatic seeding would not only illuminate the fundamental mechanisms that enable this process but also be directly translated into therapeutics to inhibit metastatic spread. While large in vitro compound screens have been used to address a variety of biological questions, it has remained difficult to use this approach for complex in vivo processes like metastasis, as it remains unclear which cell-based assays and readouts accurately reflect the in vivo process. High content in vivo chemical screens have historically been challenging due to the high cost of performing these assays in parallel. Our multi-disciplinary proposal will establish a novel method for in vivo multiplexed screening of covalent inhibitors to allow a chemical genetic dissection of the metastatic seeding process. To identify compounds that inhibit the initial steps of metastatic seeding we will screen a library of ~1,500 small molecule covalent inhibitors of hydrolytic enzymes. Importantly, these compounds contain electrophilic traps that irreversibly bind their targets, enabling sustained inhibition after in viro pretreatment of the cells without the need for continued dosing. To permit a multiplexed in vivo screening, we generated 96 variants of a metastasis-derived PDAC cell line in which each variant cell line contains a unique 6-nucleotide barcode (PDACBC). We will pretreat each PDACBC cell line in vitro with one irreversible inhibitor, then pool the 96 pretreated PDACBC cell lines. Barcode representation in the pre-injection and post-metastatic seeding cells will be determined using IlluminaTM sequencing of the barcode region. Barcodes that are underrepresented the post-seeding population will identify candidate inhibitors of metastatic seeding. To validate top candidates PDAC cells will be pretreated with top candidate compounds and assessed for their ability to seed metastases and form macro-metastasis. To identify the targets of these inhibitors we will generate tagged analogs of hit compounds to enable the purification of drug-target complexes followed by mass spectrometry-based protein identification. shRNA knockdown of these targets followed by intravenous injection will also be used to confirm the effect of the target on metastatic seeding. Our study will identify not only novel drug targets but also lead compounds potent enough to elicit a reduction in metastatic ability in vivo. Our study will advance our understanding of PDAC metastasis and provide insight into common mechanisms used by other cancer types.