Structure-based optimization of a novel pharmacological chaperone therapy for MPSIIIC

项目摘要 溶酶体贮积病（LSD）是一种罕见的遗传性代谢紊乱， 分解代谢系统粘多糖样变性IIIC型（MPS IIIC或Sanfilippo病C型）就是这样一种LSD 这是由硫酸乙酰肝素乙酰辅酶A：α-氨基葡萄糖苷N-乙酰转移酶缺乏引起的， （HGSNAT）对于硫酸乙酰肝素的降解是必需的，硫酸乙酰肝素是一种重复的碳水化合物， 蛋白聚糖这种疾病引起硫酸乙酰肝素的积累，并导致进行性和严重的 早期神经系统退化大多数病人会变得痴呆，在成年前死亡，但有些人 活到40岁患有进行性痴呆目前没有针对MPS III的特异性治疗方法， 酶替代疗法可能是不可行的，因为重组酶可能难以穿过 血脑屏障然而，这种疾病可以被认为是所谓的伴侣蛋白的一个很好的候选者。 治疗（其中活性位点特异性抑制剂或其它小分子恢复突变体的某些活性 酶），因为正常水平的约10%的阈值活性应足以防止 基于体外数据的存储。因此，即使是由于酶的降解而导致的残留酶活性的这种微小增加， 伴侣治疗可能对疾病病理学有影响，并且对患者有益。最近我们 合作者Pshezhetsky博士已经确定了HGSNAT（AT 3784）的抑制剂和伴侣， 证明它可以部分恢复MPS IIIC患者细胞中缺乏的酶活性， 然而，需要鉴定更有效的伴侣蛋白用于治疗MPS IIIC。本STTR的目标是 解析HGSNAT的三级结构，并利用这些信息指导高效的合成。 HGSNAT的抑制剂/伴侣。合成的化合物将在体外测试其增加 MPS IIIC患者培养细胞中残留的HGSNAT活性。增加酶的活性化合物 将进一步测试它们减少硫酸乙酰肝素的储存和稳定硫酸乙酰肝素的能力。 突变酶在培养的患者细胞中的正确构象和靶向。作为一个延伸目标，我们将 研究最佳化合物穿过血脑屏障的能力。在本研究中鉴定的化合物 临床前研究将为未来的II期体内试验和优化提供线索， 使用CRISPR产生的MPS IIIC敲入小鼠模型扩展该项目（II期） Cas9技术 该建议利用了Geoffrey Chang教授（University of California）丰富的X射线晶体学经验， 加州圣地亚哥），Joel Freundlich博士丰富的药物化学经验 （罗格斯大学），Alexey Pshezhetsky博士（CHU Ste-Justine）的MPSIIIC生物学专业知识和药物 Sean Ekins（Phoenix Nest，Inc.）的发现知识。Pshezhetsky博士在这个项目上的工作将完全 由该资助之外的资金资助，包括Jonah's Just Begun或加拿大卫生研究院。 如果成功，第二阶段将导致临床候选人的研究，这将利用我们庞大的全球网络， 临床医生和其他科学家。

PROJECT SUMMARY Lysosomal storage diseases (LSD) are rare inherited metabolic disorders caused by defects in the cellular catabolic system. Mucopolysaccharidosis Type IIIC (MPS IIIC or Sanfilippo disease type C) is one such LSD that is caused by deficiency of the enzyme heparan sulfate acetyl CoA: α-glucosaminide N-acetyltransferase, (HGSNAT) essential for degradation of heparan sulfate, a repeating carbohydrate generally found attached to proteoglycans. This disease causes accumulation of heparan sulfate and results in progressive and severe neurological deterioration early in life. Most patients become demented and die before adulthood but some survive to the fourth decade with progressive dementia. Currently there is no specific treatment for MPS III and enzyme replacement therapy may not be viable as the recombinant enzyme may have difficulty crossing the blood brain barrier. The disease can however be considered as an excellent candidate for so-called chaperone therapy (where active site specific inhibitors or other small molecules restore some activity of a mutant enzyme) because a threshold activity of approximately 10% of the normal level should be sufficient to prevent storage based on in vitro data. Thus, even such a minor increase in residual enzyme activity as the result of chaperone therapy is likely to have an impact on disease pathology and be beneficial for patients. Recently our collaborator Dr. Pshezhetsky has identified an inhibitor and a chaperone for HGSNAT (AT3784) and demonstrated that it could partially restore the deficient enzyme activity in the cells from MPS IIIC patients, however more potent chaperones need to be identified for a therapy for MPS IIIC. The goal of this STTR is to resolve the tertiary structure of HGSNAT and use this information to direct the synthesis of potent inhibitors/chaperones of HGSNAT. Synthesized compounds will be tested in vitro for their ability to increase the residual HGSNAT activity in cultured cells from MPS IIIC patients. Active compounds that increase enzyme activity by > 10% will be further tested for their ability to reduce storage of heparan sulfate and to stabilize the proper conformation and targeting of the mutant enzyme in cultured patient cells. As a stretch goal we will investigate the ability of the best compound to cross the blood brain barrier. Compounds identified in this preclinical study will provide leads for future phase II in vivo testing and optimization that will be performed in the extension of this project (Phase II) using knock-in mouse models of MPS IIIC generated using the CRISPR Cas9 technology. This proposal leverages the vast X-ray crystallography experience of Professor Geoffrey Chang (University of California San Diego), extensive pharmaceutical medicinal chemistry experience of Dr. Joel Freundlich (Rutgers University), MPSIIIC biology expertise of Dr. Alexey Pshezhetsky (CHU Ste-Justine) and drug discovery knowledge of Sean Ekins (Phoenix Nest, Inc.). Dr. Pshezhetsky’s work on this project will be entirely funded by funds outside this grant including Jonah’s Just Begun or the Canadian Institutes of Health Research. If successful, Phase II will lead to a clinical candidate for studies which will leverage our large global network of clinicians and other scientists as needed.