Role of TBK1/IKK epsilon inhibition in pancreatic beta cell regeneration

糖尿病的特征是由于单独或与胰岛素抵抗相关的产生胰岛素的β−细胞不足或功能衰竭而导致的葡萄糖稳态受损。因此，恢复β细胞的质量和功能对于逆转糖尿病的发展至关重要。利用小分子诱导剂诱导β细胞增殖是一种特别有前途的策略，具有许多有价值的特点，如口服生物利用度和靶向性。然而，成人β细胞的缓慢增殖速度是使用这种方法需要克服的主要障碍。通过对斑马鱼β细胞再生小分子促进剂的化学遗传筛选，我们鉴定了几种ε抑制因子(Tbk1/IKKε-IS)。Tbk1/IKKε-1通过显著促进β-细胞的增殖而促进β-细胞的再生。哺乳动物靶标雷帕霉素(雷帕霉素)抑制剂雷帕霉素消除了tBK1/IKKε-IS对β细胞再生的影响，而tBK1/IKKε-IS增强了mTOR活性。有趣的是，经Tbk1/IKKε-1治疗后，cAMP水平显著升高。在包括人胰岛在内的原代哺乳动物胰岛中证实了Tbk1/IKKε-IS的增殖作用。本申请的目的是阐明如何通过抑制Tbk1/IKKε来增加功能性Tbk1/IKK细胞质量的机制和策略。首先，我们将设计并研究新型Tbk1/IKKε-IS对β细胞再生的效力。以显著促进β细胞再生的(E)-3-(3-苯基苯并[c]异恶唑-5-基)丙烯酸(简称PIAA)为先导化合物，对Tbk1/IKKε-PIAA相互作用进行构效关系分析。此外，我们将以PIAA为支架，设计新的分子结构，显示出有效和选择性的Tbk1/IKKε抑制活性，对β细胞再生的毒性最小。其次，我们将阐明Tbk1/IKKε介导的β细胞再生的潜在机制。鉴于雷帕霉素阻断了Tbk1/IKKε抑制对β细胞再生的影响，而Tbk1/IKKε-IS活性和cAMP水平增加，我们将测试Tbk1/IKKε抑制是否通过调节cAMP-mTOR信号级联来促进β细胞再生。我们将进行生化和功能分析，以确定Tbk1/IKKε-cAMP-mTor在体外和体内对β细胞再生的相互作用。第三，我们将研究Tbk1/IKKε-IS在哺乳动物系统中扩增功能性β细胞团的有效性。我们将使用原代胰岛来检测PIAA及其新合成的类似物对β细胞复制的效力。此外，我们将评估PIAA及其类似物在包括链脲佐菌素(STZ)诱导的糖尿病小鼠模型在内的小鼠中促进β细胞增殖和增强血糖控制的能力。

Diabetes mellitus is characterized by impaired glucose homeostasis resulting from insufficiency or functional failure of insulin-producing β−cells, alone or in association with insulin resistance. Therefore, restoring β-cell mass and function is essential to reverse the development of diabetes. Utilizing small molecule inducers of β-cell proliferation is a particularly promising strategy with numerous valuable features such as their oral bioavailability and target specificity. However, the slow rate of β-cell proliferation in adult humans is a major hurdle to overcome to use this approach. Through a chemical genetic screen for small molecule enhancers of β-cell regeneration in zebrafish, we identified several TBK1/IKKε inhibitors (TBK1/IKKε-Is). TBK1/IKKε-Is promoted β-cell regeneration by markedly increasing proliferation of β-cells. Mammalian target of rapamycin (mTOR) inhibitor rapamycin eliminated the effect of TBK1/IKKε-Is on regenerating β-cells, whereas TBK1/IKKε-Is augmented mTOR activity. Interestingly, treatment with TBK1/IKKε-Is led to pronounced increase in cAMP levels. The proliferation effect of TBK1/IKKε-Is was verified in primary mammalian islets including human islets. The goal of this application is to delineate the mechanisms and the strategies of how to increase functional β-cell mass with suppression of TBK1/IKKε. First, we will design and investigate the potency of novel TBK1/IKKε-Is on β-cell regeneration. Using (E)-3-(3-phenylbenzo[c]isoxazol-5-yl)acrylic acid (abbreviated as PIAA), which markedly increases β-cell regeneration, as a lead compound, we will perform structure-activity relationship (SAR) analyses of TBK1/IKKε-PIAA interaction. Furthermore, we will use the PIAA as a scaffold to design new molecular architectures that exhibit potent and selective TBK1/IKKε inhibition activities with minimum toxicity on β-cell regeneration. Second, we will elucidate underlying mechanisms of TBK1/IKKε-mediated β-cell regeneration. Given that rapamycin treatment abolished the effect of TBK1/IKKε suppression on β-cell regeneration and TBK1/IKKε-Is enhanced mTOR activity and cAMP levels, we will test whether TBK1/IKKε suppression promotes β-cell regeneration via modulating the cAMP-mTOR signaling cascade. We will perform biochemical and functional analyses to characterize the TBK1/IKKε-cAMP-mTOR interplay on β-cell regeneration in vitro and in vivo. Third, we will investigate the efficacy of TBK1/IKKε-Is on expanding functional β-cell mass in mammalian systems. We will use primary pancreatic islets to examine the potency of PIAA and its newly synthesized analogs on β-cell replication. Furthermore, we will evaluate the ability of PIAA and its analogs to promote β-cell expansion and enhance glycemic control in mice including a mouse model of streptozotocin (STZ)-induced diabetes.