High Throughput Screening Cascade To Identify Human Aspartate N-Acetyltransferase (ANAT) Inhibitors for Canavan Disease.
High Throughput Screening Cascade To Identify Human Aspartate N-Acetyltransferase (ANAT) Inhibitors for Canavan Disease.
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高通量筛选级联鉴定卡纳万病的人天冬氨酸 N-乙酰转移酶 (ANAT) 抑制剂。
DOI：
10.1021/acschemneuro.1c00455
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发表时间：
2021
影响因子：
5
通讯作者：
Slusher,BarbaraS
中科院分区：
文献类型：
--
作者：
Nešuta,Ondřej;Thomas,AjitG;Alt,Jesse;Hin,Niyada;Neužilová,Anna;Long,Shunyou;Tsukamoto,Takashi;Rojas,Camilo;Wei,Huijun;Slusher,BarbaraS
Canavan病（CD）是一种进行性、致死性的神经系统疾病，始于婴儿期，起因于N-乙酰基天冬氨酸（NAA）脱乙酰基转化为乙酸和天冬氨酸的酶--β-环化酶（ASPA）的突变。受影响儿童大脑中NAA水平的增加是CD的标志之一。有趣的是，编码天冬氨酸N-乙酰转移酶（ANAT）的N-乙酰转移酶-8-样（NAT 8L）的基因缺失导致了几种CD基因工程小鼠模型中NAA水平的正常化和症状的改善，ANAT是一种负责从l-天冬氨酸和乙酰辅酶A合成NAA的酶。因此，ANAT的药理学抑制为治疗CD提供了一种有前景的治疗策略。然而，目前还没有临床上可行的ANAT抑制剂。在这里，我们描述了基于荧光的高通量筛选（HTS）和基于放射性的正交试验使用重组人ANAT表达inE。杆菌在荧光法中，ANAT活性与孵育时间呈线性关系，最高孵育时间为30 min，蛋白质浓度为97.5 ng/μL，L-天冬氨酸和乙酰辅酶A的Km值分别为237 μM和11 μM。使用这种优化的分析，我们进行了试点筛选的10 000化合物库。使用L-[U-14 C]天冬氨酸盐作为底物，对基于荧光试验的命中物进行基于放射性的正交试验。两种化合物在两种试验中均证实具有剂量依赖性抑制作用。最有效的化合物的抑制动力学研究揭示了一个非竞争性的抑制机制，相对于门冬氨酸和乙酰辅酶A的非竞争性抑制机制。本文开发的筛选级联将使得能够进行大规模化合物文库筛选，以鉴定新型ANAT抑制剂作为进一步药物化学优化的先导。
Canavan disease (CD) is a progressive, fatal neurological disorder that begins in infancy resulting from a mutation in aspartoacyclase (ASPA), an enzyme that catalyzes the deacetylation ofN-acetyl aspartate (NAA) into acetate and aspartate. Increased NAA levels in the brains of affected children are one of the hallmarks of CD. Interestingly, genetic deletion ofN-acetyltransferase-8-like (NAT8L), which encodes aspartateN-aceyltransferase (ANAT), an enzyme responsible for the synthesis of NAA froml-aspartate and acetyl-CoA, leads to normalization of NAA levels and improvement of symptoms in several genetically engineered mouse models of CD. Therefore, pharmacological inhibition of ANAT presents a promising therapeutic strategy for treating CD. Currently, however, there are no clinically viable ANAT inhibitors. Herein we describe the development of fluorescence-based high throughput screening (HTS) and radioactive-based orthogonal assays using recombinant human ANAT expressed inE. coli. In the fluorescence-based assay, ANAT activity was linear with respect to time of incubation up to 30 min and protein concentration up to 97.5 ng/μL withKmvalues forl-aspartate and acetyl-CoA of 237 μM and 11 μM, respectively. Using this optimized assay, we conducted a pilot screening of a 10 000-compound library. Hits from the fluorescence-based assay were subjected to an orthogonal radioactive-based assay using L-[U-14C] aspartate as a substrate. Two compounds were confirmed to have dose-dependent inhibition in both assays. Inhibitory kinetics studies of the most potent compound revealed an uncompetitive inhibitory mechanism with respect tol-aspartate and a noncompetitive inhibitory mechanism against acetyl-CoA. The screening cascade developed herein will enable large-scale compound library screening to identify novel ANAT inhibitors as leads for further medicinal chemistry optimization.
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