Discovery of allosteric activators of phospholipase C-gamma2 to treat Alzheimer's disease

摘要 目前治疗阿尔茨海默病(AD)的方法并不能逆转甚至减缓疾病的进展。 这种情况是可怕的，针对两个更有希望的人的抗体的失败加剧了这种情况 靶点-磷酸化的tau和β-淀粉样蛋白-治疗这种疾病。显然，新的治疗方法迫在眉睫 需要的。 2017年，一项大规模的全基因组研究发现了PLCG2基因的一个自然发生的变体(P522R) 编码PLC-2，对迟发性AD具有保护作用。在后续研究中，这种遗传关联仍然存在 坚固耐用，重复性强。更令人鼓舞的是，在对轻度认知障碍患者的临床研究中 与非携带者相比，携带PLCG2(P522R)的人认知功能减退的速度较慢。 即使是ApoE4纯合子的患者也观察到了保护作用，ApoE4是一种与AD密切相关的生物标志物。在 大脑，PLC-2主要表达于小胶质细胞，在那里它控制吞噬和神经炎性反应 流程。在AD患者的病理区表达更高。在小胶质细胞中，PLC-2被激活 TREM2(以ApoE4为配体)和CSF1R下游的两个跨膜受体， 与阿尔茨海默病密切相关。同样，PLC-2激活蛋白激酶C，它也与AD相连。因此，基因和细胞 数据有力地支持PLC-2作为治疗AD的新的治疗靶点。 与野生型相比，PLC-2(P522R)的磷脂酶活性略有升高。 正是这种增加的小胶质细胞活性被普遍接受，以防止AD。我们建议确定 并优化选择性激活PLC-2的小分子，以重现PLC-DNA的神经保护作用。 -2(P522R)和治疗AD。该研究计划依赖于互补性的高通量分析，由两个 我们专门为这项研究发明的真核PLC的荧光底物。因此，我们将 追求三个目标。在目标1中，将对总计约300,000种化合物的内部收藏进行激活剂筛选 对PLC-2和一次命中的活性、选择性、成分和纯度进行验证；将使用化学信息学 从结构上对热门歌曲进行分类。在目标2中，一个高质量的全长PLC-2模型与分子动力学相耦合 模拟将用于对数千万种化合物的计算筛选。在《目标3》中，一套 生化、生物物理和细胞生物学研究将用于确定PLC-2的变构激活剂的优先顺序 良好的化学和药理特性。这些新的小分子将是无价的工具 进一步了解PLC-2(P522R)如何降低AD风险。小分子也将被用作引线。 开发治疗阿尔茨海默病的新疗法。

ABSTRACT Current therapies for Alzheimer’s disease (AD) do not reverse, or even slow, progression of the disease. This situation is dire and exacerbated by the failure of antibodies directed toward two of the more promising targets—phosphorylated tau and beta-amyloids—to treat the disease. Clearly, new treatments are urgently needed. In 2017, a large genome-wide study associated a naturally-occurring variant (P522R) of PLCG2, the gene encoding PLC-2, with protection from late onset AD. In follow-up studies, this genetic association has remained strong and highly reproducible. Even more encouraging, in clinical studies of patients with mild cognitive impairment, people that carried PLCG2 (P522R) had slower rates of cognitive decline compared to non-carriers. Protection was observed even for patients homozygous for ApoE4, a biomarker strongly linked to AD. In the brain, PLC-2 is primarily expressed in microglial cells where it controls phagocytic and neuroinflammatory processes. It is more highly expressed in pathological areas of patients with AD. In microglia, PLC-2 is activated downstream of both TREM2 (which uses ApoE4 as a ligand) and CSF1R, two transmembrane receptors that are strongly linked to AD. Similarly, PLC-2 activates PKC, which is also linked to AD. Thus, genetic and cellular data strongly support PLC-2 as a novel therapeutic target for treatment of AD. The phospholipase activity of PLC-2 (P522R) is modestly elevated relative to its wild-type counterpart and it is this increased activity in microglia that is generally accepted to protect against AD. We propose to identify and optimize small molecules that selectively activate PLC-2 to reproduce the neuroprotective effects of PLC- 2 (P522R) and treat AD. The research plan relies on complementary high-throughput assays enabled by two fluorogenic substrates for eukaryotic PLCs that we invented explicitly for this research. Consequently, we will pursue three Aims. In Aim 1, in-house collections totaling ~300,000 compounds will be screened for activators of PLC-2 and primary hits verified for activity, selectivity, composition, and purity; cheminformatics will be used to structurally classify hits. In Aim 2, a high-quality model of full-length PLC-2 coupled with molecular dynamics simulations will be used for computational screens of tens of millions of compounds. In Aim 3, a suite of biochemical, biophysical, and cell biological studies will be used to prioritize allosteric activators of PLC-2 with favorable chemical and pharmacological properties. These novel small molecules will be invaluable tools to further understand how PLC-2 (P522R) reduces the risk of AD. The small molecules will also be used as leads for the development of novel therapeutics to treat AD.