Division of Preclinical Innovation, National Center for Advancing Translational Sciences, NIH, Rockville, MD 20850.
Molecular Neurogenetics Section, Medical Genetics Branch, National Human Genome Research Institute, NIH, Bethesda, MD 20892.
Proc Natl Acad Sci U S A. 2024 Oct 15;121(42):e2406009121. doi: 10.1073/pnas.2406009121. Epub 2024 Oct 10.
Glucocerebrosidase (GCase) is implicated in both a rare, monogenic disorder (Gaucher disease, GD) and a common, multifactorial condition (Parkinson's disease, PD); hence, it is an urgent therapeutic target. To identify correctors of severe protein misfolding and trafficking obstruction manifested by the pathogenic L444P-variant of GCase, we developed a suite of quantitative, high-throughput, cell-based assays. First, we labeled GCase with a small proluminescent HiBiT peptide reporter tag, enabling quantitation of protein stabilization in cells while faithfully maintaining target biology. TALEN-based gene editing allowed for stable integration of a single HiBiT- transgene into an intragenic safe-harbor locus in -knockout H4 (neuroglioma) cells. This GD cell model was amenable to lead discovery via titration-based quantitative high-throughput screening and lead optimization via structure-activity relationships. A primary screen of 10,779 compounds from the NCATS bioactive collections identified 140 stabilizers of HiBiT-GCase-L444P, including both pharmacological chaperones (ambroxol and noninhibitory chaperone NCGC326) and proteostasis regulators (panobinostat, trans-ISRIB, and pladienolide B). Two complementary high-content imaging-based assays were deployed to triage hits: The fluorescence-quenched substrate LysoFix-GBA captured functional lysosomal GCase activity, while an immunofluorescence assay featuring antibody hGCase-1/23 directly visualized GCase lysosomal translocation. NCGC326 was active in both secondary assays and completely reversed pathological glucosylsphingosine accumulation. Finally, we tested the concept of combination therapy by demonstrating synergistic actions of NCGC326 with proteostasis regulators in enhancing GCase-L444P levels. Looking forward, these physiologically relevant assays can facilitate the identification, pharmacological validation, and medicinal chemistry optimization of small molecules targeting GCase, ultimately leading to a viable therapeutic for GD and PD.
葡萄糖脑苷脂酶 (GCase) 既与一种罕见的单基因疾病（戈谢病，GD）有关，也与一种常见的多因素疾病（帕金森病，PD）有关；因此，它是一个迫切需要治疗的靶点。为了鉴定由 GCase 的致病 L444P 变体引起的严重蛋白质错误折叠和转运障碍的校正因子，我们开发了一套定量、高通量、基于细胞的测定法。首先，我们用一个小的发光 HiBiT 肽报告标签标记 GCase，能够在细胞中定量稳定蛋白质，同时忠实地保持靶标生物学。基于 TALEN 的基因编辑允许将单个 HiBiT 转基因稳定整合到 - knockout H4（神经胶质瘤）细胞中的一个内含子安全港基因座。这种 GD 细胞模型适用于通过滴定定量高通量筛选进行先导化合物发现，并通过结构活性关系进行先导化合物优化。从 NCATS 生物活性化合物库中进行的 10779 种化合物的初步筛选鉴定出 140 种稳定 HiBiT-GCase-L444P 的化合物，包括药理学伴侣（氨溴索和非抑制性伴侣 NCGC326）和蛋白稳态调节剂（panobinostat、trans-ISRIB 和 pladienolide B）。两种互补的高内涵成像基于测定法被用来筛选命中物：荧光猝灭底物 LysoFix-GBA 捕获功能性溶酶体 GCase 活性，而具有抗体 hGCase-1/23 的免疫荧光测定法直接可视化 GCase 溶酶体易位。NCGC326 在两种二次测定法中均具有活性，并完全逆转了病理性葡萄糖神经鞘氨醇的积累。最后，我们通过证明 NCGC326 与蛋白稳态调节剂联合作用增强 GCase-L444P 水平的协同作用，测试了联合治疗的概念。展望未来，这些生理相关的测定法可以促进针对 GCase 的小分子的鉴定、药理学验证和药物化学优化，最终为 GD 和 PD 提供可行的治疗方法。
