Structural stabilization of Parkinson's disease linked mutant DJ-1

描述（由申请人提供）：DJ-1基因中的A104T和M26I等功能丧失突变导致结构不稳定，并与早发性帕金森病有关。我们最近证明，通过引入二硫桥可以纠正DJ-1的结构缺陷，从而恢复突变蛋白的wt样特性。采用差示扫描荧光法对1080个FDA批准的化合物库进行了初步筛选，发现了许多提高突变体DJ-1稳定性的分子。此外，同样的分子也增加了WT DJ-1的稳定性。野生型DJ-1阻断a-synuclein聚集，这是家族突变体所不具有的特性。然而，从筛选中发现的分子似乎在突变的DJ-1中恢复了类似伴侣的功能，并增加了WT DJ-1长时间作为伴侣的能力。对其中一个命中进行了结构活性关系练习，鉴定出与DJ-1结合的类似物，解离常数为480nM。用等温滴定量热法测定了准确的解离常数。本提案的主要目标是将筛选扩展到更大的化学文库，以确定DJ-1的不同类别的分子，这些分子可以作为共同伴侣并阻断a-synuclein的聚集。高亲和力配体将代表PD治疗学发展的起点，以及更详细地了解DJ-1在基于细胞的模型中的作用的探针。

DESCRIPTION (provided by applicant): Loss-of-function mutations such as A104T and M26I in the DJ-1 gene lead to structural destabilization and have been linked to early onset Parkinson's disease. We have recently demonstrated that structural defects in DJ-1 can be corrected by introduction of disulfide bridges that restore WT-like properties in the mutant protein. A pilot screen carried out using differential scanning fluorimetry using a library of 1080 FDA approved compounds yielded a number of molecules that improve the stability of mutant DJ-1. In addition, the same molecules increased the stability of WT DJ-1 as well. Wild type DJ-1 blocks a-synuclein aggregation, a property not shared by the familial mutants. However, the molecules discovered from the screen seem to restore chaperone like function in mutant DJ-1, as well as increase the ability of WT DJ-1 to function as a chaperone for extended periods of time. A structure activity relationship exercise carried out on one of the hits led to the identification of an analog that bound to DJ-1 with a dissociation constant of 480nM. Accurate dissociation constants were determined using isothermal titration calorimetry. The major goal of this proposal is to extend the screening to a larger chemical library to identify diverse classes of molecules for DJ-1, which can work as co-chaperones and block aggregation of a-synuclein. High affinity ligands will represent a starting for development of PD therapeutics, as well as probes for understanding the role of DJ-1 in cell based models in greater details. PUBLIC HEALTH RELEVANCE: Parkinson's disease (PD) is the second most common neurodegenerative disorder. It affects over 1 million Americans and more than 60,000 patients are newly diagnosed each year. Mutation in a recently identified gene product of unknown function, DJ-1, has been implicated in autosomal- recessive early onset PD. The mutations appear to compromise the chaperone like function of DJ-1. The major objective of this proposal is to identify small molecules that would restore normal function in mutants of DJ-1. In addition, these molecules would enhance the function of wild-type DJ-1 and, thereby, have an indirect effect on a greater population of patients with PD and other synucleinopathies.