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

项目总结 溶酶体储存疾病(LSD)是一种罕见的遗传性代谢疾病，由细胞缺陷引起 分解代谢系统。粘多糖病IIIC(MPS IIIC或Sanfilippo病C型)就是这样一种LSD 这是由于缺乏乙酰辅酶A：α-氨基葡萄糖N-乙酰转移酶所致， (HGSNAT)对降解硫酸乙酰肝素至关重要，这是一种重复的碳水化合物，通常被发现附着在 蛋白多糖。这种疾病会导致硫酸乙酰肝素的蓄积，并导致进行性和严重的 生命早期的神经退化。大多数患者会变得精神错乱并在成年前死亡，但也有一些患者 患有进行性痴呆症的人可以活到第四个十年。目前还没有针对MPS III和MPS III的特效药 酶替代疗法可能不可行，因为重组酶可能难以跨越 血脑屏障。然而，这种疾病可以被认为是所谓的伴侣的极好候选者。 治疗(活性部位特定的抑制剂或其他小分子恢复突变的某些活性 酶)，因为阈值活性约为正常水平的10%应足以防止 基于体外数据的存储。因此，即使残留酶活性如此微小的增加，也是由于 伴侣疗法可能会对疾病病理产生影响，并对患者有利。最近我们的 合作者Pshezhetsky博士已经确定了HGSNAT(AT3784)的一种抑制剂和一种伴侣。 证明它能部分恢复MPS IIIC患者细胞中缺乏的酶活性， 然而，需要确定更有效的伴侣来治疗MPS IIIC。此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进行测试，以确定它们是否有能力增加 MPS IIIC患者培养细胞中残留HGSNAT活性的研究提高酶活力的活性化合物 将进一步测试&gt；10%的活性是否能够减少硫酸肝素的储存并稳定 突变酶在培养的患者细胞中的正确构象和靶向性。作为一个延伸目标，我们将 研究最佳化合物穿越血脑屏障的能力。本研究中鉴定的化合物 临床前研究将为未来的体内测试和优化阶段提供线索，这些测试和优化将在 该项目的扩展(第二阶段)使用使用CRISPR生成的MPS IIIC的敲入鼠标模型 Cas9技术。 这一建议利用了Geoffrey Chang教授丰富的X射线结晶学经验 加州圣地亚哥)，Joel Freundlich博士丰富的药物药物化学经验 (罗格斯大学)，阿列克谢·普谢日茨基博士(CHU Ste-Justine)的MPSIIIC生物学专业知识和药物 Sean Ekins(凤巢公司)的发现知识。普谢日茨基博士在这个项目上的工作将完全是 资金来自这笔赠款以外的资金，包括乔纳刚刚开始的基金会或加拿大卫生研究院。 如果成功，第二阶段将导致临床候选研究，这将利用我们庞大的全球网络 临床医生和其他需要的科学家。

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.