Lead Optimization of Novel Inhibitors of Tissue Non-specific Alkaline Phosphatase

描述（由申请人提供）：本R 01申请题为“组织非特异性碱性磷酸酶新型抑制剂的先导优化”，是对PAR-12-060“体内化学探针发现验证命中请求”的回应。中膜血管钙化（MVC）是婴儿泛发性动脉钙化（GACI）的发病率和死亡率的主要原因，GACI是一种严重的儿童疾病，并且有助于川崎病（KD）、慢性肾病（CKD）以及糖尿病、肥胖和衰老中的心血管恶化。矿化抑制剂细胞外无机焦磷酸盐（ePPi）及其在这些条件下的体内平衡的相关性已经明确确立。ePPi水平的降低与组织非特异性碱性磷酸酶（TNAP）的表达水平升高有关，TNAP水解ePPi，从而消除其在被MVC攻击的组织中的抑制作用。例如，我们最近观察到TNAP在血管平滑肌细胞（VSMC）和尿毒症性动脉瘤中的上调，表明过度的TNAP活性是ePPi缺乏和中膜钙化的重要原因。我们假设有效的小分子TNAP抑制剂在全身给药后可能导致TNAP活性降低，导致局部ePPi量增加， 预防或改善血管钙化。因此，我们提出了通过用小分子TNAP抑制剂降低TNAP活性来增加ePPi水平的策略。我们最近报道了芳基磺酰胺衍生的TNAP抑制剂的表征和初步优化。这些化合物通过非竞争性机制发挥作用，可以进行全面的化学优化，为体内概念验证研究提供先导化合物。因此，我们的具体目标是：1）设计和合成在体内具有口服活性的优化的TNAP抑制剂; 2）在relevan体外测定中评估TNAP抑制剂的效力和选择性; 3）使用体外ADME/T和体内药代动力学（PK）测定评估新型小分子TNAP抑制剂;和4）在啮齿动物离体和体内血管钙化模型中表征前导TNAP抑制剂探针。所产生的TNAP抑制剂将为测试通过调节TNAP活性来增强ePPi水平以防止MVC的假设提供强有力的工具，同时为未来开发用于治疗由MVC引起的疾病家族的新型药物奠定基础。

DESCRIPTION (provided by applicant): This R01 application entitled "Lead Optimization of Novel Inhibitors of Tissue Non-specific Alkaline Phosphatase" is in response to PAR-12-060 "Solicitation of Validated Hits for the Discovery of in vivo Chemical Probes". Medial vascular calcification (MVC) is the major cause of morbidity and mortality in generalized arterial calcification of infancy (GACI), a severe childhood disease, and contributes to cardiovascular deterioration in Kawasaki disease (KD), chronic kidney disease (CKD), as well as in diabetes, obesity and aging. The relevance of the mineralization inhibitor extracellular inorganic pyrophosphate (ePPi) and its homeostasis in these conditions has been clearly established. Reduced levels of ePPi have been linked to elevated expression levels of tissue non-specific alkaline phosphatase (TNAP), which hydrolizes ePPi, thus eliminating its inhibitory effect in tissue stricken by MVC. For example, we have recently observed an upregulation of TNAP in vascular smooth muscle cells (VSMC) and also in uremic aortas, suggesting that excessive TNAP activity is an important cause of ePPi deficiency and medial calcification. We hypothesized that potent small molecule TNAP inhibitors are likely to cause a reduction in TNAP activity following systemic administration, resulting in an increase in the local amount of ePPi to prevent or ameliorate vascular calcification. Therefore, we propose a strategy for increasing ePPi levels by reducing TNAP activity with small molecule TNAP inhibitors. We recently reported the characterization and preliminary optimization of arylsulfonamide-derived inhibitors of TNAP. These compounds, which function via an uncompetitive mechanism, are ready for full-scale chemistry optimization to provide lead compounds ready for in vivo proof- of-concept studies. Therefore our Specific Aims are: 1) Design and synthesize optimized TNAP inhibitors that are orally active in vivo; 2) Assess the potency and selectivity of TNAP inhibitors in relevan in vitro assays; 3) Evaluate novel small molecule TNAP inhibitors using in vitro ADME/T and in vivo pharmacokinetic (PK) assays; and 4) Characterize lead TNAP inhibitor probes in rodent ex vivo and in vivo models of vascular calcification. The TNAP inhibitors generated will provide powerful tools for testing the hypothesis that enhancing ePPi levels by modulating TNAP activity protects against MVC, while laying a foundation for future development of a novel class of medications for the treatment of the family of diseases caused by MVC.