High throughput screening for small molecule therapy for Gaucher disease using patient tissue as the source of mutant glucocerebrosidase.
High throughput screening for small molecule therapy for Gaucher disease using patient tissue as the source of mutant glucocerebrosidase.
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DOI：
10.1371/journal.pone.0029861
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Sidransky E
中科院分区：
文献类型：
--
作者：
Goldin E;Zheng W;Motabar O;Southall N;Choi JH;Marugan J;Austin CP;Sidransky E
Gaucher病（GD）是最常见的溶酶体储存障碍，是由溶酶体酶葡萄糖辣椒酶（GCASE）的遗传缺乏引起的。以前，野生型GCASE用于大量化合物的高吞吐量筛选（HTS），以鉴定可以作为GD的新疗法开发的小分子伴侣。但是，从HTS鉴定出的化合物通常在基于验证细胞的测定中显示出降低性降低。另一种策略是在突变酶上执行HTS，以识别不同的铅化合物，包括增强突变酶活性的铅化合物。我们使用从具有基因型N370S/N370S的Gaucher疾病的患者的脾脏制备的酶提取物进行了新的筛查测定。在组织提取物中，GCASE处于更本地的生理环境中，并带有天然激活剂Saposin C和其他潜在辅助因子。使用此测定，我们筛选了一个250,000种化合物的库，并确定了突变GCASE的新型调节剂，其中包括14个新的铅抑制剂和30个铅激活剂。使用患者衍生的成纤维细胞在随后的基于细胞的测定中证实了某些主要热门的活性。这些结果表明，使用从组织中提取的酶进行的主要筛查是一种替代方法，可以鉴定出高质量，生理相关的铅化合物以进行药物发育。
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.
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影响因子：
12.8
作者：
Taddei, Tamar H.;Kacena, Katherine A.;Mistry, Pramod K.
通讯作者：
Mistry, Pramod K.
DOI：
10.1111/j.1742-4658.2010.07588.x
发表时间：
2010-04
期刊：
The FEBS journal
影响因子：
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作者：
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通讯作者：
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影响因子：
6.7
作者：
Marugan, Juan J.;Zheng, Wei;Motabar, Omid;Southall, Noel;Goldin, Ehud;Sidransky, Ellen;Aungst, Ronald A.;Liu, Ke;Sadhukhan, Subir Kumar;Austin, Christopher P.
通讯作者：
Austin, Christopher P.
影响因子：
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作者：
Charrow, J;Andersson, HC;Zimran, A
通讯作者：
Zimran, A
影响因子：
7.3
作者：
Marugan, Juan J.;Zheng, Wei;Austin, Christopher P.
通讯作者：
Austin, Christopher P.