PO.CH01.02 · 化学
用于发现SMARCAL1 ATP酶抑制剂的HTS检测方法的开发与验证
Development and validation of HTS assays for discovery of SMARCAL1 ATPase inhibitors
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
SWI/SNF相关的基质相关肌动蛋白依赖性染色质调节因子亚家族A样蛋白（SMARCAL1）是一种ATP依赖性SNF2马达蛋白家族的退火解旋酶，通过催化复制叉倒退和Holliday连接体迁移，在DNA复制期间维持基因组稳定性。SMARCAL1的缺失及其伴随的DNA损伤已被证明可通过刺激cGAS/STING通路和抑制PDL-1表达来增强肿瘤免疫，使其成为癌症免疫治疗的一个有吸引力的靶点。在此，我们描述了使用Transcreener ADP 2检测法来测量DNA依赖性ATP酶活性，以开发用于筛选和分析SMARCAL1抑制剂的HTS兼容检测方法。Transcreener ADP 2检测法在竞争性免疫测定中直接检测ADP，具有远红色荧光偏振（FP）、荧光强度（FI）或时间分辨福斯特共振能量转移（TR-FRET）读出。该格式为均相（混合即读）、与多种DNA底物兼容，并可抵抗筛选化合物的干扰。一种由核心催化结构域加HARP1、HARP2结构域组成的人SMARCAL1构建体，在BaV感染的昆虫细胞中生产，在使用合成分叉DNA（fDNA）和剪切的鲑鱼精子DNA（sssDNA）的初步测试中显示出强大的ATP酶活性；ATP的水解完全依赖于DNA的存在。ATP在两种底物下的Km均为110 μM，sssDNA和fDNA的Km值分别为30 ng/ml和38 pM。该酶在两种相应DNA底物下分别显示出58.6 s-1和81.1 s-1的显著催化速率（kcat），使得在初速度条件下使用少于100 pM的酶即可产生强大的信号。使用三种不同检测读出（FP、FI和TR-FRET）测定的动力学参数彼此一致。FP检测法通过筛选1280种生物活性物质的集合进行了HTS验证，得到Z' > 0.9，并鉴定出在剂量反应实验中得到确认的命中物。
查看英文原文 English abstract
SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A-like protein (SMARCAL1) is an ATP-dependent SNF2 motor protein family annealing helicase that maintains genomic stability during DNA replication by catalyzing fork regression and Holliday junction migration. Loss of SMARCAL1 and the attendant DNA damage has been shown to increase tumor immunity via stimulation of the cGAS/STING pathway and suppression of PDL-1 expression, making it an attractive target for cancer immunotherapy. Here we describe development of HTS-compatible assays for screening and profiling SMARCAL1 inhibitors using the Transcreener ADP 2 Assay to measure DNA-dependent ATPase activity. The Transcreener ADP 2 Assay directly detects ADP in a competitive immunoassay with far-red, fluorescence polarization (FP), fluorescence intensity (FI) or time-resolved Förster-resonance-energy-transfer (TR-FRET) readouts. This format is homogenous (mix-and-read), compatible with diverse DNA substrates, and resistant to interference from screening compounds. A human SMARCAL1 construct comprising the core catalytic domain plus the HARP1, HARP2 domains, produced in BaV-infected insect cells, showed robust ATPase activity in initial testing with synthetic forked DNA (fDNA) and sheared salmon sperm DNA (sssDNA); hydrolysis of ATP was completely dependent on the presence of DNA. The K m for ATP with either substrate was 110 μM, and the K m values for sssDNA and fDNA were 30 ng/ml and 38 pM, respectively. The enzyme showed remarkable catalytic rates (kcat) of 58.6 s -1 and 81.1 s -1 with the two respective DNA substrates, enabling the use of less than 100 pM enzyme to produce a robust signal under initial velocity conditions. The kinetic parameters determined using the three different assay readouts (FP, FI and TR-FRET) were in concordance. The FP assay was validated for HTS by screening a collection of 1280 bioactives, yielding a Z' > 0.9 and identification of hits that were confirmed in dose response experiments.
利益披露 Disclosure
M. Blackburn, None..
R. Lowery, None.