Genetic and Chemical Biological Studies of a Novel Wnt Inhibitor Tiki2

骨质疏松症和骨折是全球性的健康问题。确定新的治疗靶点和策略是至关重要的。Wnt家族分泌蛋白的信号传导已成为人类骨量调节的关键途径，因为Wnt共受体LRP5的功能丧失和“功能获得”突变分别与家族性骨质疏松症和高骨量疾病相关。Wnt/LRP5信号主要调控成骨细胞谱系和骨生成，为刺激骨生长的治疗提供了潜在的途径。显著分泌的Wnt拮抗剂在骨中表达并局部调节Wnt/LRP5信号，从而提供了特异性靶向骨的治疗机会。事实上，Sclerostin是一种结合并抑制LRP5的骨细胞特异性分泌因子，已成为治疗骨质疏松症的关键药物靶点。我们已经确定了一个新的Wnt拮抗剂家族，称为Tiki蛋白，它可能是翻译后修饰和灭活Wnt配体的新型酶。我们已经产生了Tiki2-/-突变小鼠，令人惊讶的是，这些小鼠能够存活，但骨量却很高，这表明Tiki2和Sclerostin一样，是骨稳态的重要负调节因子。由于其酶活性，人TIKI2可能是小分子抑制剂治疗骨质疏松症的理想靶点。我们提出了四个目标来研究Tiki2在骨生物学中的作用和潜在的治疗意义。在Aim 1中，我们将采用组织学、生化和生物力学方法表征Tiki2-/-突变小鼠的高骨量表型。我们还将详细研究Tiki2在骨细胞类型中的表达，以及它在骨中通过合成代谢和机械刺激的调节。在Aim 2中，我们将通过细胞类型特异性和他莫昔芬诱导的Cre系，在骨骼和成年期产生条件Tiki2缺失的突变小鼠。这些研究将确定Tiki2在骨骼和衰老过程中的作用部位和阶段。在Aim 3中，我们将在体外研究人类TIKI2（和TIKI1）骨相关功能和生化特性。我们将研究TIKI2和TIKI1在骨中的表达，研究TIKI2和TIKI1是否调节人成骨样细胞系的骨形成，并表征被TIKI蛋白修饰/失活的特定Wnt蛋白在骨量调节中的作用。在Aim 4中，我们将通过化合物筛选确定TIKI2的小分子抑制剂，并评估它们在体外和体内刺激骨生长的潜力。作为一类具有酶活性的新型Wnt拮抗剂，TIKI蛋白是小分子抑制剂用于实验操作和治疗干预的理想靶点。我们将进行高通量化合物筛选，以确定Tiki2抑制剂在刺激骨骼生长方面的潜在应用。这些研究共同代表了通过遗传、生化和化学生物学方法全面分析TIKI在骨生物学中的功能，并可能发现潜在的骨质疏松症和骨再生的新疗法。

DESCRIPTION (provided by applicant): Osteoporosis and bone fractures are global health problems. Identification of new therapeutic targets and strategies are of paramount importance. Signaling by the Wnt family of secreted proteins has emerged as a key pathway for human bone mass regulation, as loss-of-function and 'gain-of-function' mutations in Wnt coreceptor LRP5 are associated with familial osteoporosis and high bone mass diseases, respectively. Wnt/LRP5 signaling primarily regulates the osteoblast lineage and bone generation, providing a potential avenue for therapeutics that stimulates bone growth. Remarkably secreted Wnt antagonists are expressed in bone and modulate Wnt/LRP5 signaling locally, and thus offer treatment opportunities targeting bone specifically. Indeed Sclerostin, an osteocyte-specific secreted factor that binds to and inhibits LRP5, has become a key drug target for osteoporosis. We have identified a new family of Wnt antagonists, referred to as Tiki proteins, which likely are novel enzymes that post-translationally modify and inactivate Wnt ligands. We have generated Tiki2-/- mutant mice, which surprisingly are viable but exhibit high bone mass, suggesting that Tiki2, like Sclerostin, is an important negative regulator of bone homeostasis. Because of its enzymatic activity human TIKI2 may be an ideal target for small molecule inhibitors for potential therapeutic intervention for osteoporosis. We propose four aims to investigate the role of Tiki2 in bone biology and potential therapeutic implications. In Aim 1, we will characterize the high bone mass phenotype of Tiki2-/- mutant mice, employing histological, biochemical and biomechanical methods. We will also examine in details the expression of Tiki2 in bone cell types, its regulation in bone by anabolic and mechanical stimuli. In Aim 2, we will generate mutant mice with conditional Tiki2 deletion in the bone and in the adulthood via cell type- specific and a tamoxifen-inducible Cre lines. These studies will define the site and stage of Tiki2 action in bone and in the aging process. In Aim 3, we will investigate human TIKI2 (and TIKI1) bone-related functions and biochemical properties in vitro. We will investigate TIKI2 and TIKI1 expression in bone, and study whether TIKI2 and TIKI1 regulates bone formation in human osteoblast-like cell lines, and characterize the specific Wnt proteins that are modified/inactivated by TIKI proteins in bone mass regulation. In Aim 4, we will identify small molecule inhibitors of TIKI2 via chemical compound screening and to evaluate their potential in bone growth stimulation in vitro and in vivo. As a new class of Wnt antagonists with an enzymatic activity, TIKI proteins are ideal targets for small molecule inhibitors for use in experimental manipulation and therapeutic intervention. We will perform a high throughput chemical compound screen to identify Tiki2 inhibitors for their potential applications in stimulating bone growth. These studies together represent a comprehensive analysis of TIKI function in bone biology via genetic, biochemical and chemical biological methods, and may discover potential novel therapeutics for osteoporosis and bone regeneration.