Toward Improved Therapy for Classic Galactosemia

描述(申请人提供)：遗传性缺乏半乳糖-1-磷酸尿苷转移酶(GalT，E.C.2.7.7.12)活性可导致一种潜在的致命疾病，称为经典型半乳糖血症(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，但许多典型的半乳糖血症患者在晚年仍会出现生长发育、神经心理、语言发育迟缓以及卵巢功能不全(POI)等并发症。这种疾病的急性致死和长期并发症的发病机制在很大程度上仍不清楚，因此进一步阻碍了更有效的治疗方法的开发。然而，一些证据表明，半乳糖-1-磷酸半乳糖(GAL-1P)水平升高是典型的半乳糖血症的主要致病机制，如果不是唯一的话。因此，我们假设，通过抑制Galk来阻止Gal-1P的产生将缓解GALT缺乏的细胞的半乳糖毒性。为了验证这一假设，我们与NIH化学基因组中心(NCGC)的高通量筛选(HTS)设备合作，发起了一项定量高通量筛选(QHTS)活动，以确定人类Galk的小分子抑制剂。到目前为止，我们已经鉴定出149种小分子化合物，它们在体外微摩尔浓度下对对照Galk活性抑制80%或更多。通过对这些化合物的生化、结构和基于细胞的表征，我们已经确定了几种有希望的化学类型，它们是无毒的，在降低人GALT缺陷成纤维细胞中GAL-1P的积累方面有效。然而，要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，还需要进一步的化学修饰和在哺乳动物动物模型中的体内研究。在这一应用中，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流程，以(1)提高经过验证的Galk抑制剂在体外和基于细胞的检测中针对Galk的生物活性和类药物特性；(2)通过评估优化后的抑制剂在减少半乳糖挑战GalT基因敲除小鼠的GAL-1P产量方面的有效性，进行概念验证(POC)研究。拟议项目的完成将为新的靶点Galk确定体内化学探针，这具有重大意义，从了解半乳糖代谢在人类器官发育/功能中的作用，到开发改进的治疗方法，以满足令人衰弱的孤儿疾病经典半乳糖症的医疗需求。 公共卫生相关性：典型的半乳糖血症是一种罕见的代谢紊乱，包括在美国所有的新生儿筛查项目中。限制半乳糖饮食是目前治疗的主要方法，它可以防止受影响患者的新生儿死亡，但对卵巢早衰和言语延迟等长期并发症几乎没有效果。该项目的长期目标是将经过验证的人半乳糖激酶小分子抑制剂(Galk)转化为治疗这种衰弱疾病的新的、安全和更有效的疗法。

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. PUBLIC HEALTH RELEVANCE: Classic Galactosemia is a rare metabolic disorder included in all newborn screening programs in the USA. A galactose-restricted diet, which is currently the mainstay of treatment, can prevent neonatal lethality of the affected patients, but has little effet on long-term complications such as premature ovarian insufficiency and speech delay. The long-term goal of this project is to translate the validated small molecule inhibitors of human galactokinase (GALK) into novel, safe and more effective therapeutics for this debilitating disorder.