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利用患者组织作为突变葡萄糖脑苷脂酶的来源,进行戈谢病小分子治疗的高通量筛选。

High throughput screening for small molecule therapy for Gaucher disease using patient tissue as the source of mutant glucocerebrosidase.

机构信息

Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, United States of America.

出版信息

PLoS One. 2012;7(1):e29861. doi: 10.1371/journal.pone.0029861. Epub 2012 Jan 17.

Abstract

Gaucher disease (GD), the most common lysosomal storage disorder, results from the inherited deficiency of the lysosomal enzyme glucocerebrosidase (GCase). Previously, wildtype GCase was used for high throughput screening (HTS) of large collections of compounds to identify small molecule chaperones that could be developed as new therapies for GD. However, the compounds identified from HTS usually showed reduced potency later in confirmatory cell-based assays. An alternate strategy is to perform HTS on mutant enzyme to identify different lead compounds, including those enhancing mutant enzyme activities. We developed a new screening assay using enzyme extract prepared from the spleen of a patient with Gaucher disease with genotype N370S/N370S. In tissue extracts, GCase is in a more native physiological environment, and is present with the native activator saposin C and other potential cofactors. Using this assay, we screened a library of 250,000 compounds and identified novel modulators of mutant GCase including 14 new lead inhibitors and 30 lead activators. The activities of some of the primary hits were confirmed in subsequent cell-based assays using patient-derived fibroblasts. These results suggest that primary screening assays using enzyme extracted from tissues is an alternative approach to identify high quality, physiologically relevant lead compounds for drug development.

摘要

戈谢病(GD)是最常见的溶酶体贮积症,是由于溶酶体酶葡萄糖脑苷脂酶(GCase)的遗传性缺乏所致。以前,野生型 GCase 曾被用于高通量筛选(HTS)大量化合物,以鉴定可开发为 GD 新疗法的小分子伴侣。然而,在确认基于细胞的测定中,HTS 鉴定出的化合物通常显示出降低的效力。另一种策略是对突变酶进行 HTS,以鉴定不同的先导化合物,包括那些增强突变酶活性的化合物。我们开发了一种新的筛选测定法,使用来自基因型 N370S/N370S 的戈谢病患者的脾脏酶提取物进行测定。在组织提取物中,GCase 处于更天然的生理环境中,并且存在天然激活剂神经鞘脂激活蛋白 C 和其他潜在的辅助因子。使用该测定法,我们筛选了 250,000 种化合物的文库,并鉴定出突变型 GCase 的新型调节剂,包括 14 种新型的先导抑制剂和 30 种先导激活剂。一些主要命中的活性在随后使用源自患者的成纤维细胞的基于细胞的测定中得到了证实。这些结果表明,使用从组织中提取的酶进行的初步筛选测定法是一种替代方法,可以鉴定用于药物开发的高质量、生理相关的先导化合物。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/063e/3260169/98fbe0c9f673/pone.0029861.g001.jpg

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