Toward Improved Therapy for Classic Galactosemia

描述（由申请人提供）：遗传性半乳糖-1-磷酸尿苷转移酶（GALT， E.C. 2.7.7.12）活性缺乏可导致一种潜在的致命性疾病，称为经典半乳糖血症（OMIM 230400）。尽管新生儿筛查、早期诊断和限制半乳糖饮食可以挽救生命，但许多经典半乳糖血症患者在生命后期会出现并发症，包括生长、神经心理、语言迟缓以及卵巢早衰（POI）。急性致死的致病机制和与这种疾病相关的长期并发症在很大程度上仍然未知，因此进一步阻碍了更有效治疗方法的发展。然而，一些证据表明，半乳糖激酶（GALK）的产物半乳糖-1-磷酸（gal-1P）水平升高是经典半乳糖血症的主要致病机制，如果不是唯一的致病机制。因此，我们假设通过抑制GALK来预防galt - 1p的产生将减轻galt缺陷细胞的半乳糖毒性。为了验证这一假设，我们与NIH化学基因组学中心（NCGC）的高通量筛选（HTS）设施合作，启动了定量高通量筛选（qHTS）活动，以确定人类GALK的小分子抑制剂。迄今为止，我们已经鉴定出149种小分子化合物，在微摩尔（¿M）浓度下，在体外抑制80%或更多的对照GALK活性。通过对这些化合物的生化、结构和基于细胞的表征，我们已经确定了一些有希望的化学型，它们无毒且有效地降低了人类galt缺陷成纤维细胞中gal-1P的积累。然而，将这些有希望的小分子GALK抑制剂转化为安全有效的治疗药物需要进一步的化学修饰和哺乳动物模型的体内研究。在这个应用中，我们建议建立一个创新的、具有成本效益的、迭代的和并行的优化过程，以：(1)提高经验证的GALK抑制剂在体外和基于细胞的GALK检测中的生物活性和药物样特性；(2)进行概念验证（POC）研究，评估优化的抑制剂在半乳糖挑战的galt敲除小鼠中减少gal-1P产生的功效。该项目的完成将确定新的靶点GALK的体内化学探针，这对了解人体器官发育/功能中的半乳糖代谢，以及开发一种改进的治疗方法，以解决使人衰弱的孤儿疾病经典半乳糖血症的未满足的医疗需求具有重要意义。

DESCRIPTION (provided by applicant): Hereditary deficiency of galactose-1-phosphate uridylyltransferase (GALT, E.C. 2.7.7.12) activity can lead to a potentially lethal disease called Classic Galactosemia (OMIM 230400). Despite the life-saving consequences of newborn screening, early diagnosis, and a galactose-restricted diet, many patients with Classic Galactosemia suffer later in life from complications including growth, neuropsychological, and speech delays as well as premature ovarian insufficiency (POI). The pathogenic mechanisms of the acute lethality and the long-term complications associated with this disorder remain largely unknown, thus further hamper the development of more effective therapies. Yet, several lines of evidence suggested that elevated level of galactose-1-phosphate (gal-1P), product of galactokinase (GALK), is a major, if not sole, pathogenic mechanism in Classic Galactosemia. We, therefore, hypothesize that prevention of gal-1P production by inhibiting GALK will relieve GALT-deficient cells from galactose toxicity. To test this hypothesis, we, in collaboration with th High-throughput Screening (HTS) Facility at the NIH Chemical Genomics Center (NCGC), had launched a quantitative high-throughput screening (qHTS) campaign to identify small molecule inhibitors of the human GALK. To date, we have identified 149 small molecule compounds that, at micromolar (¿M) concentration, inhibit 80% or more of control GALK activity in vitro. Through biochemical, structure- and cell-based characterization of these compounds, we have identified a few promising chemotypes that are non- toxic and effective in lowering gal-1P accumulation in human GALT-deficient fibroblasts. The translation of these promising small molecule GALK inhibitors into safe and useful therapeutics, however, requires further chemical modifications and in vivo studies in mammalian animal models. In this application, we propose to set up an innovative, cost-effective, iterative and parallel optimization process to (1) Improve the biologicl activities and drug-like properties of the validated GALK inhibitors against GALK in vitro and in cell-based assays; (2) perform proof-of-concept (POC) studies by evaluating the efficacy of the optimized inhibitors in reducing gal-1P production galactose-challenged GalT-knockout mice. The completion of the proposed project will identify in vivo chemical probes for a novel target, GALK, which has significant implications ranging from the understanding of galactose metabolism in human organ development/ functions to the development of an improved therapy to address the unmet medical needs of the debilitating orphan disease, Classic Galactosemia.