High throughput screen for discovery of N-acetyltransferase 8 Like (NAT8L) inhibitors

项目摘要 Canavan病（CD）是一种罕见的常染色体隐性脑白质营养不良， 乙酰化酶基因（ASPA），导致酶活性的丧失和增加的浓度， 底物N-乙酰天冬氨酸（NAA）在脑中。脑内NAA积累导致海绵状 白色物质变性、髓鞘形成异常、脑水肿、大头畸形、严重认知和运动障碍 赤字，最终死亡。CD没有治愈方法，也没有标准的治疗过程。治疗 目前仅限于支持性护理和症状管理。小鼠ASPA基因的遗传缺失 已经显示出再现了在患者中观察到的许多CD疾病症状。对这个提议很重要， 编码天冬氨酸N-乙酰转移酶8（NAT 8L）的基因的缺失，所述酶催化 从天冬氨酸和乙酰辅酶A生物合成NAA，预防CD小鼠模型中的脑白质营养不良。这些 小鼠显示NAA水平显著降低，没有证据表明星形胶质细胞空泡化、星形胶质细胞增生或 小脑或前脑的脱髓鞘。在脑池内给药中观察到类似的治疗获益， 施用针对NAT 8L的反义寡核苷酸。这些结果有力地表明，抑制NAT 8L 在CD的治疗中将是有用的。然而，目前还没有临床上有效的NAT 8L抑制剂。 已知的NAT 8L抑制剂具有低效力（IC 50值在μM - mM范围内）和/或具有 防止脑渗透的羧酸部分。本提案旨在进行高通量筛选 (HTS)一个大的和多样化的400，000人NAT 8L的小分子抑制剂的化合物库。 成功鉴定易处理的命中化合物，然后进行初步结构优化， 导致发现有前途的先导NAT 8L抑制剂，具有未来治疗药物开发的潜力 对于CD。我们准备抓住这个机会，执行以下三个具体目标：（目标1） 进行NAT 8L HTS和命中确认分析;（目标2）进行命中聚类、初步SAR和ADME （目标3）进行初步的潜在客户优化。

PROJECT SUMMARY Canavan disease (CD) is rare autosomal recessive leukodystrophy caused by mutations in the aspartoacylase gene (ASPA), leading to loss of enzyme activity and increased concentrations of the substrate N-acetylaspartate (NAA) in the brain. Accumulation of brain NAA results in spongiform degeneration of white matter, aberrant myelination, brain edema, macrocephaly, severe cognitive and motor deficits and ultimately death. There is no cure, nor is there a standard course of treatment for CD. Treatment is currently limited to supportive care and symptom management. Genetic deletion of the ASPA gene in mice has been shown to reproduce many of the CD disease symptoms seen in patients. Important to this proposal, deletion of the gene encoding for aspartate N-acetyltransferase 8 (NAT8L), the enzyme that catalyzes the biosynthesis of NAA from aspartate and acetyl CoA, prevented leukodystrophy in a CD mouse model. These mice showed substantial reduction in NAA levels and no evidence of astroglial vacuolation, astrogliosis, or demyelination in the cerebellum or forebrain. Similar therapeutic benefits were observed with intracisternal administration of antisense oligonucleotide to NAT8L. These results strongly suggest that inhibition of NAT8L would be useful in the treatment of CD. Currently, however, there are no NAT8L inhibitors that are clinically available; known NAT8L inhibitors have low potency (IC50 values in the μM - mM range) and/or possess carboxylate moieties that prevent brain penetration. This proposal aims to conduct high throughput screening (HTS) of a large and diverse 400,000 compound library for small molecule inhibitors of human NAT8L. Successful identification of tractable hit compounds followed by preliminary structural optimization should lead to the discovery of promising lead NAT8L inhibitors with potential for future development of therapeutics for CD. We are poised to seize this opportunity by executing the following three Specific Aims: (Aim 1) conduct NAT8L HTS and hit confirmation assays; (Aim 2) conduct hit clustering, preliminary SAR, and ADME profiling; (Aim 3) conduct preliminary lead optimization.