Small Molecule Probes for TAR DNA Binding Protein 43_TDP-43

描述（由申请人提供）：肌萎缩性侧索硬化症（ALS，也称为运动神经元或Lou Gehrig's病）和额颞叶痴呆（FTLD）是一种使人衰弱的神经退行性疾病，现在与TAR dna结合蛋白43 （TDP-43）有关。据估计，一半的FTLD患者有相关的TDP-43病理，使TDP-43相关的FTLD成为最大的单一亚型。对于ALS，特别是散发性ALS（约85-90%的患者）发病机制的一个关键进展是发现TDP-43是这些患者泛素化包涵体的关键组成部分，并且TDP-43的许多突变与家族性ALS和sALS有关。TDP-43是一种与核酸结合的主要核蛋白，据信在细胞中起多种作用，包括核运输和核酸加工。在将TDP-43与FTLD和ALS联系起来的最初报告以及多个证实性报告中，已经表明TDP-43和泛素化的TDP-43都存在于患者神经元的细胞内包裹体中，并且这种聚集伴随着细胞核中TDP-43的缺乏。有效的基于TDP-43的治疗方法将在这些人群中具有高水平的渗透；然而，目前尚不清楚TDP-43的确切作用以及它们的调节如何影响疾病进展。TDP-43探针分子的开发将极大地增强我们对其生物学的理解，并可能通过作为治疗有用的分子本身或使开发第二代TDP-43功能检测技术成为可能，导致治疗方法的快速发展。我们开发了第一个小分子探针与TDP-43结合的实验，适用于高通量化合物文库筛选，研究寡核苷酸结合的抑制作用，并确定了四种具有抑制TDP-43与寡核苷酸结合的新能力的化学型。我们将通过(1)继续购买和评估相关的小分子文库和(2)使用薛定谔软件套件进行包含计算药效团开发的探针文库药物化学，以及时和具有成本效益的方式探索这些热门药物的化学空间。在我们的初步试验中，具有足够效力的化合物将被测试其抑制TDP-43的CFTR外显子9剪接能力，改变TDP-43的细胞分布以及改变突变体TDP-43的毒性和分布的能力。我们将进行行业标准的选择性和代谢研究，以验证这些化合物在细胞和体内的使用。这些探针分子的未来发展可能会发现作为筛选化合物文库或检测TDP-43水平和模型系统运输的生化工具的效用。与TDP-43结合的成像工具将作为强有力的临床前研究工具，并可能成为人类疾病诊断和进展的潜在生物标志物。通过在该应用中的工作，探针分子的成功开发具有巨大的潜力，可以彻底改变我们对TDP-43的理解。

DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS, also motor neuron or Lou Gehrig's disease) and frontotemporal lobar dementia (FTLD) are debilitating neurodegenerative disorders that are now linked to TAR DNA-binding protein-43 (TDP-43). It is estimated that half of FTLD patients have associated TDP-43 pathology, making TDP-43-associated FTLD the largest single subtype. A key advance in the understanding of the pathogenesis of ALS, and especially sporadic ALS (sALS, ~85-90% of patients), was the discovery that TDP-43 is a key component of the ubiquitinated inclusions in these patients and that numerous mutations in TDP-43 are linked to both familial ALS and sALS. TDP-43 is a primarily nuclear protein that binds to nucleic acids and is believed to function in a variety of roles in the cell including nuclear transport and nucleic acid processing. In the initial report linking TDP-43 to FTLD and ALS and in multiple confirmatory reports, it has been shown that both TDP- 43 and ubiquitinated TDP-43 are present in intracellular inclusions in the neurons of patients and that this aggregation is accompanied by a lack of TDP-43 in the nucleus. Effective TDP-43 based therapeutics would have a high level of penetration into these populations; however, the precise roles of TDP-43 and how their modulation would effect disease progression are not currently understood. The development of TDP-43 probe molecules will dramatically enhance our understanding of its biology and may lead to the rapid development of therapeutics either by serving as therapeutically useful molecules themselves or enabling the development of second generation assays for TDP-43 function. We have developed the first ever assay for small molecule probe binding to TDP-43 suitable for high throughput compound library screening that looks at the inhibition of the binding of oligonucleotides and have identified four chemotypes that possess the novel ability to inhibit the binding of TDP-43 to oligonucleotides. We will explore the chemical space around these hits in a timely and cost-effective manner by (1) continuing to purchase and evaluate related small-molecule libraries and (2) conducting probe library medicinal chemistry incorporating computational pharmacophore development using the Schrodinger software suite. Compounds with sufficient potency in our primary assay will be tested for their ability to inhibit the CFTR exon 9 splicing ability of TDP-43, to alter the cellular distribution of TDP-43 and to alter the toxicity and distribution of mutant TDP-43s. We will perform industry standard selectivity and metabolic studies to validate these compounds for cellular and in vivo use. Future development of these probe molecules may find utility as biochemical tools for screening compound libraries or examining TDP-43 levels and trafficking in model systems. Imaging tools that binds to TDP-43 would serve as strong preclinical research tools and may lead to a potential biomarker for disease diagnosis and progression in humans. The successful development of probe molecules via the work in this application has enormous potential to revolutionize our understanding of TDP-43. PUBLIC HEALTH RELEVANCE: Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia (FTLD) are debilitating neurological disorders for which there are no effective, disease modifying therapies available, meaning that diagnosis carries with it the certainty of a decline in health leading to death. Not only are these diseases incurable, but their reach and devastation is much greater because of the impact upon caregivers and the health care system as ALS and FTLD both result in the need for constant around the clock care in their late stages. Both diseases are now linked a protein called TAR DNA binding protein 43 (TDP-43), which aggregates in the nerve cells of patients, and we propose to generate novel molecules that alter TDP 43's ability to bind to nucleic acids that will serve as the first small molecules probes of its function.