School of Veterinary Medicine, Department of Molecular Biosciences, University of California-Davis, Davis, CA 95616, United States of America; School of Pharmacy, Department of BioMolecular Sciences, University of Mississippi, MS 38677, United States of America.
School of Pharmacy and Pharmaceutical Sciences, Department of Pharmaceutical Sciences, Binghamton University, Binghamton, NY 13902, United States of America.
Biochim Biophys Acta Gene Regul Mech. 2020 Jan;1863(1):194478. doi: 10.1016/j.bbagrm.2019.194478. Epub 2019 Dec 28.
Non-B-DNA G-quadruplex (G4) structures have shown promise as molecular targets. Modulating G4 stability for oncogenic transcriptional control is a promising avenue for the development of novel therapeutics. Extracellularly, G4 stabilization can be mediated by alkali cations, modifying water content, or with molecular crowding. Intracellularly, G4 formation is mediated by negative superhelicity and transcriptional activity, and can be stabilized with small molecules or oligonucleotides. Numerous G4-stabilizing compounds have been identified that impact promoter activity in plasmids. These compounds, however, infrequently show activity in cells, are found to have non-G4-mediated mechanisms of action, or do not demonstrate activity in vivo. The G4 field requires enhanced predictive screening methods to identify compounds with G4-mediated in vitro activity and in vivo efficacy. Using the best characterized promoter G4 to date, MYC, we examined the effects of varying annealing conditions (rate of cool down and number of heat/cool cycles), co-solvents (glucose, acetonitrile, polyethylene glycol, dextran sulfate, sucrose, ficoll-70, glycerol) and nucleoplasm on G4 formation and compound screening. We observed a marked decrease in hit rates when shifting from simple buffer conditions to include potassium and glycerol, and utilizing two or more rapid annealing cycles; the difference in hit compounds coincides with previous findings of active, inactive, and non-G4-mediated activity, including NSC338258, Quindoline i, and TMPyP4; with these changes, we describe a modification of the primary FRET Melt screening assay - the FRET Melt. This understanding of physiological principles governing the above G4 formation will better inform future drug discovery efforts for this and other oncogenic promoters.
非 B-DNA 四链体 (G4) 结构已显示出作为分子靶标的潜力。调节 G4 稳定性以控制致癌转录是开发新型治疗方法的有前途的途径。在细胞外，G4 的稳定性可以通过碱金属阳离子、改变水含量或分子拥挤来介导。在细胞内，G4 的形成由负超螺旋和转录活性介导，并可以用小分子或寡核苷酸稳定。已经鉴定出许多稳定 G4 的化合物，这些化合物会影响质粒中的启动子活性。然而，这些化合物在细胞中很少表现出活性，被发现具有非 G4 介导的作用机制，或者在体内没有表现出活性。G4 领域需要增强预测筛选方法，以识别具有 G4 介导的体外活性和体内疗效的化合物。使用迄今为止最好表征的启动子 G4-MYC，我们研究了改变退火条件（冷却速度和热/冷循环次数）、共溶剂（葡萄糖、乙腈、聚乙二醇、硫酸葡聚糖、蔗糖、菲科尔-70、甘油）和核质对 G4 形成和化合物筛选的影响。我们观察到，当从简单的缓冲条件转变为包括钾和甘油，并利用两个或更多快速退火循环时，命中率显著下降；命中化合物的差异与先前的发现一致，即活性、非活性和非 G4 介导的活性化合物，包括 NSC338258、Quindoline i 和 TMPyP4；通过这些变化，我们描述了对主要 FRET 融解筛选测定法 - FRET 融解的修改。对控制上述 G4 形成的生理原理的理解将更好地为针对该和其他致癌启动子的未来药物发现工作提供信息。
