PO.CH01.01 · 化学
高通量毛细管免疫分析实现肿瘤学研发中蛋白降解剂效力与动力学的精确定量
High-throughput capillary immunoassays enable precise quantitation of protein degrader potency and kinetics in oncology discovery
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
诸如蛋白水解靶向嵌合体（PROTAC降解剂）之类的蛋白降解剂，通过实现对疾病相关蛋白的催化性清除（而非基于占位的抑制），已彻底改变了肿瘤学药物研发。为了有效地对降解剂候选物进行排序和优化，研发团队需要定量、可重复的分析方法，以在大量化合物、浓度和时间点范围内同时解析效力（如DC₅₀、Dmax）和降解动力学。传统的Western印迹缺乏在筛选漏斗早期担当此角色所需的通量和一致性。
在此，我们展示了如何利用Leo系统上的Simple Western毛细管免疫分析，以96样本通量实现精确、定量的降解剂评估，用于上游初筛和二筛。以AR表达的MDA-MB-453乳腺癌细胞中的雄激素受体（AR）降解剂作为模型系统，该方法提供了稳健的、剂量和时间依赖性的AR降解谱，变异系数为个位数，且在生物学重复和技术重复之间的DC₅₀、Dmax和降解速率（kdeg）估算值高度一致。
Leo的开放抗体平台灵活性可提供内源性蛋白表达测量，用于对模型系统进行正交验证，例如抗HiBiT抗体。通过按分子大小分辨蛋白质，Leo能够将真正的靶标降解与基于板的分析中的假象（如发光或荧光报告基团丢失）区分开来，从而验证基于板的筛选阳性结果，并揭示那些在靶标或报告基团中缺乏相应大小分辨变化的假读数。
这些结果确立了Leo上的高通量Simple Western分析作为一种切合用途的解决方案，可实现大规模的精确降解剂定量。开放的、不依赖抗体的检测化学、大小分辨的读数以及96样本通量相结合，可在肿瘤学筛选漏斗的更早阶段提供更丰富的药理学洞见，并在推进至更复杂、资源密集的模型之前，支持对蛋白降解剂候选物进行数据驱动的优先级排序和正交验证。
查看英文原文 English abstract
Protein degraders such as PROteolysis TArgeting Chimeras (PROTAC degraders) have transformed oncology drug discovery by enabling catalytic removal of disease-relevant proteins rather than occupancy-based inhibition. To effectively rank and optimize degrader candidates, discovery teams need quantitative, reproducible assays that resolve both potency (e.g., DC₅₀, D max) and degradation kinetics across large panels of compounds, concentrations, and time points. Conventional Western blotting lacks the throughput and consistency required for this role early in the screening funnel.
Here, we demonstrate how Simple Western capillary immunoassays with the Leo system enables precise, quantitative degrader assessment with 96-sample throughput for upstream primary and secondary screening. Using androgen receptor (AR) degraders in AR-expressing MDA-MB-453 breast cancer cells as a model system, this method delivered robust, dose- and time-dependent AR degradation profiles with single-digit coefficients of variation and highly consistent DC₅₀, D max, and degradation rate (kdeg) estimates between biological and technical replicates.
The open antibody platform flexibility of Leo provides endogenous protein expression measurements for orthogonal validation of model systems such as anti-HiBiT antibodies. By resolving proteins by size, Leo can distinguish true target degradation from artifacts in plate-based assays (e.g., luminescent or fluorescence reporter loss), thereby validating plate-based screening hits and revealing false readouts that lack corresponding size-resolved changes in the target or reporter.
These results establish high-throughput Simple Western assays on Leo as a fit-for-purpose solution for precise degrader quantitation at scale. The combination of open, antibody-agnostic detection chemistry, size-resolved readouts, and 96-sample throughput enables richer pharmacologic insight earlier in oncology screening funnels and supports data-driven prioritization and orthogonal validation of protein degrader candidates before advancing into more complex, resource-intensive models.
利益披露 Disclosure
C. Haitjema, None..
P. Joshi, None.