阿尔兹海默症人工智能药物设计
本项目中我们将从分子结构入手,设计开发BODIPY使其不仅可以诊断早期AD,并能干预抑制AD发展,开发出基于BODIPY的阿尔兹海默症人工智能药物,达到AD早期诊断和干预治疗的目的,为临床AD早期诊疗提供理论基础和技术支持。整个研究工作具备以下特点:(1)设计开发近红外BODIPY荧光探针对细胞和活体进行成像可避免生物背景荧光的干扰;(2)BODIPY对与AD早期相关的Aβ寡聚体具有特异响应,为临床前AD早期诊断提供科学依据;(3)BODIPY通过与Aβ聚集的作用点结合,呈现荧光,到达有效诊断的目的,在此基础上Aβ聚集缠结的作用点被BODIOY占据从而达到一定程度上抑制AD发展的目的;(4)将抑制Aβ聚集的天然小分子药物山柰酚与BODIPY有效结合,可进一步提高AD早期诊疗的效果。
Scheme 1. Aβ derives from the proteolytic cleavage of a larger glycoprotein named amyloid precursor protein. (A) A near-infrared BODIPY probe (NB-K) was synthesized which detected and drove self-assembly of FF. (B) NB-K designed according to the structure of FF and the two aromatic rings of FF overlap well with the two aromatic rings of NB-K. When NB-K binds to Aβ oligomers, free rotation of three benzene rings of NB-K is restricted resulting in 1650% increasing of NB-K fluorescence. (C) Overview of the amino acid sequences of the Aβ-related peptides Aβ1–40 and Aβ1–42. (D) Aβ produces β-folds and then aggregates to form tetrad oligomers. NB-K could be potentially useful in the early diagnosis (via imaging) of AD via binding to the FF of oligomeric Aβ. On the other hand, the tetramer could rotate 90° along the β-fold axis to form fibrils.
Aβ源自β-和γ-分泌酶对糖蛋白(称为淀粉样前体蛋白(APP))的蛋白水解切割(Scheme 1C)。二苯丙氨酸二肽(FF)是Aβ折叠起始作用点,对Aβ聚集过程起着关键作用。四个β折叠的Aβ通过FF的π-π堆积作用和其它氨基酸之间的氢键作用以面对面的方式排列形成Aβ寡聚物,这是AD早期的重要生理标志,严重损害了大脑的健康。当β折叠的Aβ形成四聚体Aβ寡聚物时,FF几乎被完全暴露,这为近红外BODIPY荧光探针(NB-K)与FF有意组合提供了极好的机会(Scheme 1D),并能够通过荧光信号传输有效地诊测早期AD。Aβ寡聚物沿β折叠链方向逐渐以90°旋转,变成Aβ原纤维,其比Aβ八聚体更大,且与中期/晚期AD有关。当β折叠的Aβ形成原纤维时,疏水性片段(包括FF)聚集在球形结构的核心,大多数FF参与Aβ的自组装并形成球形结构,导致NB-K与Aβ原纤维的结合不良(Scheme 1D)。而且,Aβ单体表现出更大的自由弹性,这可能导致NB-K对Aβ单体的不良反应。总的来说,NB-K可以有效地分化以响应寡聚体和单体/原纤维,从而达到AD早期诊断的目的。如Scheme 1B所示,FF的两个芳环与NB-K的两个芳环很好地重叠,形成稳定的π-π结构。FF的羧基和氨基进一步促进了NB-K-FF的结合。NB-K和ThS在染色Aβ方面的主要区别如下:1)NB-K的分子量约为ThS的三倍。由于更大的空间位阻,NB-K不能进入由芳香环形成的浅槽,因此NB-K不能染色结合Aβ原纤维。 2)Aβ中的NB-K结合基段为FF。当Aβ形成β折叠时,折叠点恰好在FF,然后Aβ形成Aβ寡聚体。如Scheme 1所示,Aβ寡聚物中的FF几乎完全暴露,结果是NB-K会牢固结合识别响应Aβ寡聚物。
Figure 1. (A) Aβ aggregation assay: in vitro study to detect Aβ aggregation over time. ThT was used to detect formation of fibrillary Aβ species. Total fluorescence (%) was plotted as the fluorescence intensity divided by the maximum fluorescence intensity obtained during the plateau; (B) and (C) Fluorescence emission of NB-K and ThT response to buffer (background fluorescence, black line), oligomer and fibrils; (D) △I refers to the increased fluorescence intensity, I0 corresponds to background fluorescence of NB-K or ThT; Aβ morphology was evaluated by SEM after 160 hours incubation with NB-K (E) or ThT (F).
单体Aβ可以在24小时内衍变形成Aβ寡聚物,在72小时后开始有Aβ纤维形成。硫黄素-T(ThT)是市售检测Aβ原纤维的绿色荧光探针,以它为参照对比NB-K,以实时监测单体Aβ随时间的衍变聚集。在72小时后,ThT荧光强度略有增加,表明Aβ原纤维的形成(Figure 1A, )。而对于NB-K,荧光强度在10小时后迅速增加,仅在40小时后才达到平稳状态,这表明NB-K缩短了Aβ衍变聚集成核相时间(Figure 1A, )。 在24小时NB-K荧光强度急剧升高,这应与NB-K阳性Aβ物种有关,即Aβ寡聚体。换句话说,NB-K抑制寡聚体转变为原纤维。此外,使用荧光光谱法评价了NB-K在Aβ寡聚物和原纤维的溶液中区分识别Aβ寡聚物与Aβ原纤维的能力。对于Aβ寡聚物和Aβ原纤维,NB-K荧光分别增强了1650%±15%和450%±10%(Figure 1B, 1D)。相比之下,ThT荧光强度并未随Aβ寡聚物而增加,而随Aβ原纤维而增加了460%±10%(Figure 1C, 1D)。这说明ThT只对Aβ原纤维有荧光响应信号,而NB-K对Aβ寡聚物有很好的荧光响应信号,相比之下,NB-K对Aβ寡聚物的荧光响应性能高于ThT对Aβ原纤维荧光响应。此外,分别在ThT和NB-K存在下,Aβ单体衍变聚集160小时后,通过SEM观察Aβ单体最终衍变聚集形态。我们发现,在NB-K存在下,Aβ显示出六边形结构(Figure 1E),而在ThT存在下,Aβ显示出复杂的如斑块状的聚集体结构(Figure 1F)。这表明NB-K可能影响Aβ的构象聚集,从而产生有序排列的结构,而ThT对Aβ单体衍变聚集没有良性影响。
Figure 2. Epifluorescence microscopy of transgenic AD mouse (APP/PS1) brain stained with ThS or NB-K. ThS emission was obtained at 488 nm (left panels) and NB-K fluorescence was obtained at 561 nm (middle panels). Merged images of ThS and NB-K are shown on the right panels. Hippocampus is shown in A-C, whereas cortex is shown in D-F. G-I are magnified images from dotted squares in D-F, respectively. Scale bar: 100 µ (A-F), 50 µ (G-I).
在Aβ聚集的过程中,核心缠结成不溶性的原纤维,周围是由可溶性寡聚物组成的环状结构,这些可溶性寡聚物正在慢慢向原纤维衍变。AD脑组织的ThS / NB-K双重染色清楚地表明了这种现象,如Figure 2所示,Aβ原纤维的ThS绿色荧光染色被Aβ寡聚物的NB-K红色荧光染色所包围。 另外,在正常对照小鼠的脑切片中,未观察到NB-K染色,进一步说明NB-K对Aβ寡聚物的特殊识别性和荧光信号响应性,这对AD早期诊断预防研究无疑是一个有价值的信息。
淮阴工学院
2021-05-11