Vitamin D Receptor-Cofactor Interactions as Targets for Novel Drug Discovery

描述(申请人提供)：维他命D在细胞分化、增殖和矿物质平衡中起关键作用。因此，这种荷尔蒙对健康骨骼的发育和维持是必不可少的。维生素D的活性代谢物1，25-二羟基维生素D3通过维生素D受体(VDR)介导其作用，VDR是核受体(NR)超家族配体调节转录因子的成员。维生素D对矿物质代谢的主要作用与其促进肾脏和小肠钙吸收的能力有关。最近的研究还揭示了维生素D在成骨细胞骨形成中的直接作用，表明VDR具有合成代谢作用。事实上，两种VDR配体，骨化三醇(1，25-(OH)2D3)及其合成类似物阿法骨化醇(1α-羟基维生素D3)已被批准用于治疗骨质疏松症。然而，VDR介导的肠道钙转运导致的高钙血症限制了这些药物的广泛使用。出于这个原因，在小肠中活性降低的促骨VDR调节剂是非常可取的，并且仍然是VDR药物发现计划的优先事项。传统上，VDR调节剂是以经验的方式确定的，化学主要通过体外配体结合分析来指导，其次是次要的转录报告基因分析来测量功能激动性/拮抗性。这些方法已经成功地确定了新的VDR配体，但没有一种方法显示出足够的组织选择性而被批准用于治疗骨质疏松症。下一代组织选择性VDR调节器的合理设计将需要更复杂的筛选方法。在这个第一阶段的计划中，我们将采用一种新的策略来解决这个重要的问题，使用一种方法，利用我们目前对VDR作用的分子决定因素的理解，以及观察到组织选择性NR药理在很大程度上是由这些转录因子以不同的方式与转录共激活因子或辅助抑制因子相互作用的能力介导的。这一过程的核心是一种优化的T7噬菌体展示技术，该技术用于快速鉴定组织特异性NR相互作用蛋白。这些相互作用的蛋白质然后被用来开发一种“受体构象分析工具”(RCPT)，该工具将允许识别能够实现不同辅因子相互作用的新化合物。因此，这项第一阶段提案的既定目标是利用优化的T7噬菌体展示技术从包括骨骼和小肠在内的多个目标组织中鉴定VDR相互作用蛋白的全谱。这些VDR-蛋白质相互作用随后将在细胞中得到证实，以确定它们在第二阶段资助期内未来开发基于辅因子的RCPT的有效性，该RCPT旨在确定VDR的新的促进骨骼的小分子调节剂，它降低了小肠中的钙吸收活动。与公共健康相关：维生素D通过维生素D受体(VDR)发挥作用，是维持和发育健康骨骼所必需的激素。事实上，维生素D的活性代谢物已被FDA批准用于治疗骨质疏松症，骨质疏松症是一种由骨骼虚弱引起的疾病。然而，维生素D激素治疗骨质疏松症的主要副作用是血钙水平升高。这是由于VDR在小肠中的活动，维生素D促进肠道钙的吸收。我们提出的目标是使用最先进的蛋白质-蛋白质相互作用筛选技术来开发和验证一种新的药物发现工具，该工具能够识别功能不同于维生素D的新的VDR调节剂。成功完成这一第一阶段项目将允许在第二阶段资助期内实施这种新的筛选工具，用于发现新类型的机械上不同的VDR调节剂，预计将以最小的副作用有效地治疗骨质疏松症。

DESCRIPTION (provided by applicant): The secosteroid vitamin D plays a critical role in cell differentiation, proliferation and mineral homeostasis. As such, this hormone is essential for the development and maintenance of healthy bone. The active metabolite of vitamin D, 1,25-dihydroxyvitamin D3, mediates its effects through the vitamin D receptor (VDR), a member of the nuclear receptor (NR) superfamily of ligand-regulated transcription factors. The primary effect of vitamin D on mineral metabolism relates to its ability to promote calcium absorption in the kidney and small intestine. Recent studies have also revealed a direct role of vitamin D on osteoblastic bone formation, suggesting an anabolic role for VDR. Indeed, two VDR ligands, Calcitriol (1,25-(OH)2D3) and its synthetic analog Alfacalcidol (1alpha-hydroxyvitamin D3), have been approved for the treatment of osteoporosis. However, hypercalcemia that develops as a result of VDR-mediated intestinal calcium transport limits the widespread use of these drugs. For this reason, bone-promoting VDR modulators with reduced activity in the small intestine are highly desirable and remain a priority in VDR drug discovery programs. Traditionally, VDR modulators have been identified in an empirical manner with chemistry being guided primarily by in vitro ligand binding assays, followed by secondary transcriptional reporter gene assays that measure functional agonism/antagonism. These approaches have been successful in identifying new VDR ligands but none have demonstrated sufficient tissue selectivity to be approved for osteoporosis. The rational design of next generation tissue- selective VDR modulators will require more sophisticated screening methodologies. During this Phase 1 program, we will employ a novel strategy to tackle this important problem by using an approach that takes advantage of our current understanding of the molecular determinants of VDR action and the observation that tissue-selective NR pharmacology is mediated in large part by the ability of these transcription factors to interact in a differential manner with either transcriptional co-activators or co-repressors. At the core of this process is an optimized T7 phage display technology that is used to rapidly identify tissue-specific NR interacting proteins. These interacting proteins are then used to develop a "receptor conformation profiling tool" (RCPT) that will allow for the identification of novel compounds that enable differential cofactor interactions. The stated objective of this Phase I proposal therefore is to utilize an optimized T7 phage display technology to identify the full spectrum of VDR interacting proteins from multiple target tissues including the bone and small intestine. These VDR-protein interactions will then be confirmed in cells to determine their utility in the future development, during a Phase 2 funding period, of a cofactor- based RCPT aimed at identifying novel bone-promoting small molecule regulators of VDR that have reduced calcium absorbing activities in the small intestine. PUBLIC HEALTH RELEVANCE: Vitamin D, acting through the vitamin D receptor (VDR), is a hormone essential for the maintenance and development of healthy bone. Indeed, the active metabolite of vitamin D is approved by the FDA for the treatment of osteoporosis, a disease that results from the weakening of bones. The treatment of osteoporosis with vitamin D hormones is however limited by their major side effect of elevated blood calcium levels. This is due to VDR activity in the small intestine, where vitamin D promotes intestinal calcium absorption. Our proposed goal is to use a state-of-art protein-protein interaction screening technology to develop and validate a novel drug discovery tool capable of identifying new VDR modulators that function differently than vitamin D. Successful completion of this Phase 1 project will allow, during a Phase 2 funding period, the implementation of this novel screening tool for use in the discovery of new classes of mechanistically distinct VDR modulators predicted to effectively treat osteoporosis with minimal side effects.