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

描述（由申请人提供）：肌萎缩侧索硬化症（ALS，也称为运动神经元或Lou Gehrig病）和额颞叶痴呆（FTLD）是目前与TAR DNA结合蛋白-43（TDP-43）相关的衰弱性神经退行性疾病。据估计，一半的FTLD患者具有相关的TDP-43病理学，使得TDP-43相关的FTLD成为最大的单一亚型。在了解ALS，特别是散发性ALS（sALS，约85-90%的患者）的发病机制方面的一个关键进展是发现TDP-43是这些患者中泛素化包涵体的关键组分，并且TDP-43中的许多突变与家族性ALS和sALS相关。TDP-43主要是一种与核酸结合的核蛋白，据信在细胞中发挥多种作用，包括核转运和核酸加工。在将TDP-43与FTLD和ALS联系起来的初始报告中以及在多个验证性报告中，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涵体中，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的治疗剂在这些人群中具有高水平的渗透性;然而，TDP-43的确切作用以及它们的调节如何影响疾病进展目前尚不清楚。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，并可能导致治疗的快速发展，无论是作为治疗有用的分子本身或使TDP-43功能的第二代测定法的发展成为可能。我们已经开发了第一个适合于高通量化合物文库筛选的小分子探针与TDP-43结合的测定法，该测定法着眼于寡核苷酸结合的抑制，并且已经鉴定了具有抑制TDP-43与寡核苷酸结合的新能力的四种化学型。我们将通过以下方式以及时和具有成本效益的方式探索这些命中周围的化学空间：（1）继续购买和评估相关的小分子文库;（2）使用薛定谔软件套件进行探针文库药物化学结合计算药效团开发。将测试在我们的初步测定中具有足够效力的化合物抑制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，以验证这些化合物的细胞和体内使用。这些探针分子的未来发展可能会发现作为筛选化合物库或检查TDP-43水平和模型系统中的贩运的生物化学工具的实用性。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。通过该应用中的工作成功开发探针分子具有巨大的潜力，可以彻底改变我们对TDP-43的理解。 公共卫生相关性：肌萎缩侧索硬化症（ALS）和额颞叶痴呆症（FTLD）是使人衰弱的神经系统疾病，没有有效的疾病改善疗法，这意味着诊断带有健康下降导致死亡的确定性。这些疾病不仅无法治愈，而且由于对护理人员和医疗保健系统的影响，它们的影响范围和破坏性更大，因为ALS和FTLD都需要在晚期进行全天候护理。这两种疾病现在都与一种称为TAR DNA结合蛋白43（TDP-43）的蛋白质有关，该蛋白质在患者的神经细胞中聚集，我们建议产生新的分子，改变TDP 43与核酸结合的能力，这些核酸将作为其功能的第一个小分子探针。

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.