SIK Activators to Treat PTH Pathway Bone Diseases

甲状旁腺激素受体1(PTHR1)及其下游信号通路(Gsα)是 涉及几种骨骼疾病，包括甲状旁腺功能亢进症，Jansen的干骺端软骨发育不良 (由于结构性活动性PTHR1)或纤维性发育不良-McCune-Albright综合征(由于结构性PTHR1 活性Gsα)，在遗传病的情况下，可能会导致重大的发育和终生问题 带着他们的骨架。在这三种情况下，唯一可用的治疗方法是手术或姑息治疗。蛋白激酶 A(PKA)是一种活性依赖于Gs、α活性和细胞内cAMP水平的酶。它是 在成骨细胞中受甲状旁腺素与甲状旁腺素受体1结合的调节。我们已经产生了一种可诱导的成骨细胞特异性 通过胶原蛋白1定向缺失调节亚基Prkar1a建立高活性PKA小鼠模型，并发现 在头骨、长骨、脊椎和尾骨中的一种高骨转换表型，模仿 PTHR1疾病。我们和其他人已经证明了PTH通过PKA控制基因对成骨细胞的作用 表达部分是通过盐诱导蛋白激酶(SIKs)的磷酸化抑制来实现的。这些激动酶 强磷酸化cAMP调节的转录辅助激活因子(CRTC1、2和3)和组蛋白 脱乙酰酶4/5(HDAC4/5)，将它们隔离在细胞质中。在甲状旁腺素作用时，PKA介导的SIK 抑制导致CRTC2和HDAC4/5的磷酸化水平随随后的核降低 介导破骨细胞基因RANKL转录的CRTC2/3和HDAC4/5易位， MMP13的调控和SOST转录的抑制。SIK抑制、缺失或敲除模仿 甲状旁腺素的作用。我们最近已经测试了几种可能激活SIKS及其结果的试剂 抑制PTH诱导的RANKL转录在小鼠颅骨成骨细胞分化中的作用 分子，9117014，作为“假定的”SIK激活剂，以满足这些目标。从这些培养中的细胞数据和 在体内的初步数据中，我们提出了SIKS的小分子激活剂将逆转的假说 甲状旁腺激素信号通路在骨骼中的不良影响。因此，这一行动的具体目标是 重新提交的R21申请是为了1)确定小分子激活SIKS是否会抑制PTH- 通过对甲状旁腺激素刺激RANKL进行剂量反应来调节成骨细胞的基因表达。 C.测试对其他甲状旁腺激素调节基因的影响，2)评估一个小分子 SIKS的激活剂将抑制小鼠的高骨转换表型，通过A.注射发育模型 高转换率，B.注射高转换率的成人模型，C.注射成年模型 甲状旁腺机能亢进症，并进行完整的骨骼分析。这样做，结果将是很高的。 影响，并可能导致进一步的临床前研究，可能形成第一个疾病修改口服的基础 甲状旁腺功能亢进症、Jansen‘s干骺端软骨发育不良或纤维性疾病的治疗 发育不良-麦昆-奥尔布赖特综合征。

An overactive Parathyroid Hormone Receptor 1 (PTHR1) and its downstream signaling pathway (Gsα) are involved in several bone diseases including hyperparathyroidism, Jansen’s metaphyseal chondrodysplasia (due to a constitutively active PTHR1) or Fibrous Dysplasia-McCune-Albright Syndrome (due to a constitutively active Gsα) which, in the case of the genetic diseases, can cause major developmental and lifelong problems with their skeletons. In all three situations, the only therapies available are surgical or palliative. Protein Kinase A (PKA) is an enzyme whose activity is dependent on the activity of Gsα and cellular levels of cAMP. It is regulated by PTH binding to PTHR1 in osteoblasts. We have generated an inducible, osteoblast-specific mouse model of hyperactive PKA by collagen 1-directed deletion of the regulatory subunit, Prkar1a, and found a high bone turnover phenotype in skulls, long bones, vertebrae and caudal bones of the tail that mimics the PTHR1 diseases. We and others have shown that PTH action on the osteoblast through PKA controls gene expression, in part, through inhibition by phosphorylation of salt-inducible kinases (SIKs). These kinases tonically phosphorylate cAMP-regulated transcriptional coactivators (CRTC1, 2 and 3) and histone deacetylases 4/5 (HDAC4/5), sequestering them in the cytoplasm. Upon PTH action, PKA-mediated SIK inhibition causes CRTC2 and HDAC4/5 phosphorylation levels to decrease with subsequent nuclear translocation of CRTC2/3 and HDAC4/5 which mediates transcription of the osteoclastogenic gene, Rankl, regulation of Mmp13 and suppression of Sost transcription. SIK inhibition, deletion or knockdown mimics the effects of PTH. We have recently tested several agents for possible activation of the SIKs and resultant inhibition of PTH-induced Rankl transcription in differentiating mouse calvarial osteoblasts and found a small molecule, 9117014, as a “putative” SIK activator, to fit these goals. From these data of cells in culture and preliminary data in vivo, we have developed the hypothesis that small molecule activators of SIKs will reverse the unwanted effects of the PTH signaling pathway in bone. Consequently, the specific aims of this resubmitted R21 application are to 1) determine if activation of SIKs by a small molecule will inhibit PTH- regulated gene expression in osteoblasts, by a. performing dose responses on PTH-stimulation of Rankl, b. knocking down SIK2/3, c. testing the effects on other PTH-regulated genes, 2) assess if a small molecule activator of SIKs will inhibit a high bone turnover phenotype in mice, by a. injecting a developmental model of high bone turnover, b. injecting an adult model of high bone turnover, c. injecting an adult model of hyperparathyroidism, and conducting complete bone analyses. In so doing, the results would be of high impact and may lead to further preclinical studies that could form the basis for the first disease-modifying oral treatments for patients with hyperparathyroidism, Jansen’s metaphyseal chondrodysplasia or Fibrous Dysplasia-McCune-Albright Syndrome.