PO.CH01.02 · 化学
高通量筛选鉴定用于胰腺导管腺癌钙网蛋白表面转位的新型药物
High throughput screening identifies novel drugs for calreticulin surface translocation in pancreatic ductal adenocarcinoma
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：胰腺导管腺癌（PDAC）目前是美国癌症相关死亡的第四大原因，由于其预后不良和发病率上升，预计在未来十年内还将增加。亟需开发更有效的治疗方案和检测策略，以改善PDAC患者的生存结局。钙网蛋白（CALR）是一种内质网蛋白，已知在细胞应激后会转位至细胞表面。表面表达的CALR在癌症检测或治疗递送方面具有前景。为探索这一潜力，我们进行了高通量筛选，以鉴定能够诱导表面CALR表达的化合物。
方法：使用Nano-Glo HiBiT细胞外检测系统对两种人PDAC细胞系（Panc1、MIA PaCa-2）进行工程改造，将HiBiT稳定整合入CALR基因中。在美国国家推进转化科学中心（NCATS）对诱导表面CALR的化合物进行了高通量筛选。将表达HiBiT-CALR的细胞系用来自机制探查平板（MIPE）库的约3,000种药物处理24或48小时，随后使用CellTiter-Glo评估细胞活力，并通过发光检测评估表面CALR水平。表面HiBiT-CALR和细胞活力信号使用ViewLux微孔板成像仪以及利用XTS Staubli UniVAL机器人系统的自动化流程进行定量。对表现出高CALR信号（AUC）且毒性最小的前十种药物，通过流式细胞术验证其对表面CALR的诱导。还使用MTT法分析细胞活力，以P < 0.05为具有统计学意义。
结果：在所评估的化合物中，102种化合物显示出CALR表面表达增加，被选入二次筛选，从中进一步选出十种进行实验室内验证。其中，bisindolylmaleimide IV和AEM1被鉴定为能够可靠地增加CALR。流式细胞术分析证实活的表面CALR+细胞显著增加，其中bisindolylmaleimide IV（1.1μM）使MIA PaCa-2中的CALR增加1.7倍，使Panc1中增加1.32倍，而AEM1（10μM）使MIA PaCa-2中的CALR增加7倍，使PANC-1中增加3.7倍。活力检测证实bisindolylmaleimide IV（1.1μM）的细胞毒性较低，在MIA PaCa-2和PANC-1细胞中分别观察到77%和84%的细胞活力。同样，AEM1（10μM）在MIA PaCa-2中显示81%的活力，在PANC-1中显示76%。
结论：我们的结果鉴定了两种新型药物bisindolylmaleimide IV和AEM1的潜力，作为增强表面CALR表达的有前景候选物，为PDAC检测和肿瘤特异性疗法递送提供了新途径。
关键词：钙网蛋白、胰腺导管腺癌、药物发现、治疗递送、HiBiT-CALR。
查看英文原文 English abstract
Background: Pancreatic ductal adenocarcinoma (PDAC) is currently the fourth leading cause of cancer-related deaths in the United States and is projected to increase within the next decade due to its poor prognosis and increasing incidence. There is an urgent need to develop more effective therapeutic options and detection strategies to improve survival outcomes for PDAC patients. Calreticulin (CALR), an endoplasmic reticulum protein, is known to translocate to the cell surface following cellular stress. Surface-expressed CALR holds promise for cancer detection or therapeutic delivery. To explore this potential, we performed high-throughput screening to identify compounds which induce surface CALR expression.
Methods: Two human PDAC cell lines (Panc1, MIA Pa Ca-2) were engineered using the Nano-Glo HiBiT Extracellular Detection System to stably integrate HiBiT into the CALR gene. A high throughput screen for surface CALR-inducing compounds was performed at the National Center for Advancing Translational Sciences (NCATS). Cell lines expressing HiBiT-CALR were treated with approximately 3,000 drugs from the Mechanism interrogation PlatEs (MIPE) library for 24 or 48 hours and then assessed for cell viability using CellTiter-Glo and surface CALR levels by luminescence. Surface HiBiT-CALR and cell viability signals were quantified using a ViewLux microplate imager and automated processes utilizing the XTS Staubli UniVAL robotic system. The top ten drugs demonstrating high CALR signal (AUC) with minimal toxicity were validated for surface CALR induction by flowcytometry. Cells were also analyzed for viability using MTT assay, with P < 0.05 considered significant.
Results: Out of the compounds evaluated, 102 compounds showed increased CALR surface expression and were selected for secondary screening, from which ten were further chosen for in-lab validation. Among these, bisindolylmaleimide IV and AEM1 were identified to reliably increase CALR. Flow cytometry analysis confirmed significant increases in live surface CALR+ cells, with bisindolylmaleimide IV (1.1µM) increasing CALR by 1.7-fold in MIA Pa Ca-2 and 1.32-fold in Panc1, while AEM1 (10µM) increased CALR by 7-fold in MIA PaCa-2 and 3.7-fold in PANC-1. Viability assays confirmed low cytotoxicity for bisindolylmaleimide IV (1.1 µM), with 77% and 84% cell viability observed in MIA PaCa-2 and PANC-1 cells, respectively. Similarly, AEM1 (10 µM) showed 81% viability in MIA PaCa-2 and 76% in PANC-1 cells.
Conclusion: Our results identified the potential of two novel drugs, bisindolylmaleimide IV and AEM1, as promising candidates for enhancing surface CALR expression, offering new avenues for PDAC detection and tumor specific delivery of therapies.
Keywords: Calreticulin, pancreatic ductal adenocarcinoma, drug discovery, therapeutic delivery, HiBiT-CALR.
利益披露 Disclosure
Y. Golivi, None..
S. D. Forsythe, None..
R. Guenter, None..
F. Zaibaq, None..
C. Thomas, None..
J. Rose, None.