Li Quan, Henry Eric R, Hofrichter James, Smith Jeffrey F, Cellmer Troy, Dunkelberger Emily B, Metaferia Belhu B, Jones-Straehle Stacy, Boutom Sarah, Christoph Garrott W, Wakefield Terri H, Link Mary E, Staton Dwayne, Vass Erica R, Miller Jeffery L, Hsieh Matthew M, Tisdale John F, Eaton William A
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520.
Office of the Clinical Director, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520.
Proc Natl Acad Sci U S A. 2017 Jan 31;114(5):E689-E696. doi: 10.1073/pnas.1619054114. Epub 2017 Jan 17.
Although it has been known for more than 60 years that the cause of sickle cell disease is polymerization of a hemoglobin mutant, hydroxyurea is the only drug approved for treatment by the US Food and Drug Administration. This drug, however, is only partially successful, and the discovery of additional drugs that inhibit fiber formation has been hampered by the lack of a sensitive and quantitative cellular assay. Here, we describe such a method in a 96-well plate format that is based on laser-induced polymerization in sickle trait cells and robust, automated image analysis to detect the precise time at which fibers distort ("sickle") the cells. With this kinetic method, we show that small increases in cell volume to reduce the hemoglobin concentration can result in therapeutic increases in the delay time prior to fiber formation. We also show that, of the two drugs (AES103 and GBT440) in clinical trials that inhibit polymerization by increasing oxygen affinity, one of them (GBT440) also inhibits sickling in the absence of oxygen by two additional mechanisms.
尽管60多年前就已知道镰状细胞病的病因是血红蛋白突变体的聚合，但羟基脲是美国食品药品监督管理局批准用于治疗该病的唯一药物。然而，这种药物仅取得了部分成功，由于缺乏灵敏且定量的细胞检测方法，阻碍了其他抑制纤维形成药物的发现。在此，我们描述了一种基于镰状性状细胞中激光诱导聚合以及强大的自动图像分析的96孔板形式的方法，以检测纤维扭曲（“镰变”）细胞的确切时间。通过这种动力学方法，我们表明细胞体积的小幅增加以降低血红蛋白浓度可导致纤维形成前延迟时间的治疗性增加。我们还表明，在临床试验中通过增加氧亲和力来抑制聚合的两种药物（AES103和GBT440）中，其中一种（GBT440）还通过另外两种机制在无氧情况下抑制镰变。
