Satori Pharmaceuticals, 281 Albany Street, Cambridge, MA 02139, USA.
J Neurosci Methods. 2013 Feb 15;213(1):14-21. doi: 10.1016/j.jneumeth.2012.11.011. Epub 2012 Dec 7.
γ-Secretase modulators (GSM), which reduce amyloidogenic Aβ(42) production while maintaining total Aβ levels, and Notch-sparing γ-secretase inhibitors (GSIs) are promising therapies for the treatment of Alzheimer's Disease (AD). To have a safety margin for therapeutic use, GSMs and GSIs need to allow Notch intracellular domain (NICD) production, while preventing neurotoxic Aβ peptide production. Typically, GSI and GSM effects on these substrates are determined using two different cell lines, one for the measurement of enzyme activity against each substrate. However, predicting selectivity for different substrates across cell systems may reduce the reliability of such ratios such that the in vitro data are not useful for predicting in vivo safety margins. This is especially concerning since the IC(50)'s of some GSIs vary depending upon the level of APP expression in a cell line. To circumvent this problem, we utilized the SUP-T1 cell line which expresses a truncated Notch receptor fragment that does not need sheddase cleavage to be a γ-secretase substrate. When combined with a sensitive method of measuring Aβ production, this assay system allows both substrates to be measured simultaneously, reducing the potential to calculate imprecise selectivity margins. To demonstrate the value of this system, known GSIs and GSMs were examined in the SUP-T1 dual substrate assay. IC(50)'s were determined for both substrates and the in vitro selectivity margin was calculated. These data suggest using a single cell line is a more accurate prediction of the fold difference between NICD inhibition and Aβ(42) lowering for therapeutically promising GSIs and GSMs.
γ-分泌酶调节剂（GSM）可降低淀粉样蛋白 Aβ（42）的产生，同时保持总 Aβ 水平，而 Notch 节约 γ-分泌酶抑制剂（GSI）是治疗阿尔茨海默病（AD）的有前途的疗法。为了具有治疗用途的安全裕度，GSM 和 GSI 需要允许 Notch 细胞内结构域（NICD）的产生，同时防止神经毒性 Aβ 肽的产生。通常，使用两种不同的细胞系来确定 GSI 和 GSM 对这些底物的作用，一种用于测量每种底物的酶活性。然而，预测不同细胞系统中不同底物的选择性可能会降低此类比率的可靠性，使得体外数据对于预测体内安全裕度没有用处。这尤其令人担忧，因为一些 GSI 的 IC（50）取决于细胞系中 APP 表达的水平。为了规避这个问题，我们利用表达不依赖于剪切酶切割的截断 Notch 受体片段的 SUP-T1 细胞系作为 γ-分泌酶的底物。当与一种测量 Aβ 产生的灵敏方法结合使用时，该测定系统允许同时测量两种底物，从而减少计算不精确的选择性裕度的可能性。为了证明该系统的价值，在 SUP-T1 双底物测定中检查了已知的 GSI 和 GSM。确定了两种底物的 IC（50），并计算了体外选择性裕度。这些数据表明，使用单个细胞系是预测具有治疗潜力的 GSI 和 GSM 之间 NICD 抑制和 Aβ（42）降低的折叠差异的更准确方法。
