Multiplexed in vivo drug screening: Inhibitors of metastatic seeding

描述(申请人提供)：胰腺导管腺癌(PDAC)是一种常见且几乎一致致命的恶性肿瘤。导致PDAC患者预后不良的因素有很多，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，癌细胞离开血液进入次级器官所需的分子事件，即所谓的转移播种过程，仍然难以捉摸。识别抑制转移播撒的工具化合物不仅将阐明实现这一过程的基本机制，而且还将直接转化为抑制转移扩散的治疗药物。虽然大型体外化合物筛选已被用于解决各种生物学问题，但仍难以将这种方法用于体内复杂的过程，如转移，因为目前尚不清楚哪些基于细胞的分析和读数准确地反映了体内的过程。由于并行执行这些分析的高成本，体内高含量化学筛选历来具有挑战性。我们的多学科提案将建立一种新的方法，用于体内多路筛选共价抑制剂，以允许对转移播种过程进行化学遗传解剖。为了确定抑制转移性播种的初始步骤的化合物，我们将筛选一个包含~1,500个水解酶小分子共价抑制剂的文库。重要的是，这些化合物含有不可逆地结合其靶标的亲电陷阱，使细胞在病毒预处理后能够持续抑制，而不需要继续给药。为了能够在体内进行多重筛选，我们产生了96个转移性PDAC细胞株的变体，其中每个变体细胞系包含一个唯一的6核苷酸条形码(PDACBC)。我们将每个PDACBC细胞株在体外用一种不可逆的抑制剂进行预处理，然后汇集96个经预处理的PDACBC细胞系。注射前和转移后种子细胞中的条形码表示将使用条形码区的IlllightaTM测序来确定。未充分代表播种后群体的条形码将识别转移性播种的候选抑制物。为了验证最佳候选者，将用最佳候选者化合物对PDAC细胞进行预处理，并评估其播撒转移和形成宏观转移的能力。为了识别这些抑制剂的靶标，我们将生成HIT化合物的标记类似物，以实现药物靶标复合体的纯化，然后基于质谱学的蛋白质鉴定。将这些靶点的shRNA敲除，然后静脉注射，也将用于确认靶点在转移种植中的作用。我们的研究不仅将确定新的药物靶点，而且还将确定有效的先导化合物，以降低体内转移能力。我们的研究将促进我们对PDAC转移的理解，并为其他癌症类型使用的常见机制提供洞察力。

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.