Development of in vivo active small molecule selective inhibitors of ASK1

响应持续的细胞应激而改变的信号调节与许多复杂的 人类疾病，包括心血管疾病、炎症和神经退行性疾病。重要的是，基因 研究表明，沉默凋亡信号调节激酶(ASK1)可以改善许多 类风湿性关节炎、心肌肥大和神经退行性变小鼠模型的表型 精神错乱。因此，ask1已被强调为预期有拮抗剂的治疗靶点。 在多种疾病状态下提供显著的益处。在GM103957的支持下，我们的研究团队 最近完成了高通量的ASK1筛选活动，并鉴定了一系列小分子 来自六个不同结构类别的抑制剂。我们已经将我们的药物化学努力的重点缩小到 三种支架，本提案的目标是开发一种体内活性的ASK1小分子探针。在 研究目标1我们将使用迭代药物化学方法来优化效力、选择性和 针对外周靶点的ASK1抑制剂的体内特性。此外，要开发具有以下特性的化合物 合适的脑渗透特性，我们将应用以物理为重点的多参数优化策略 和化学性质是当前大多数食品和药物管理局(FDA)的标志- 批准的中枢神经系统渗透性小分子药物。我们将使用基于结构的设计来促进化学 策略，我们将通过一系列生化和基于细胞的分析对化合物进行分类，如 我们的研究操作计划(ROP)。随着改进的线索出现，我们将获得精选的酶选择性 很少，通过完整的动态组(反应生物学)和受体图谱(Riserca)。对于关键线索，我们还将 通过测量ASK1，通过生物化学和基于细胞的研究来评估目标参与寿命 下游底物磷酸化。集成到ROP中，我们还将通过一个 一系列药物代谢研究，包括评估微粒体的体外稳定性，抑制细胞色素P450， 溶解度、蛋白质结合和P-糖蛋白外流，并将铅标准纳入化合物的ROP 进步。在目标2中，外周和中枢神经系统暴露的高级导联将在小鼠中进行评估 急性剂量范围发现和28天毒性研究中的不良反应。我们将测量严重的毒性效应 VET扫描分析以及主要器官的TUNEL染色和组织病理学检查。最后，我们将使用 抑制MKK6、JNK和p38磷酸化作为体内抑制ASK1的报告 最大生物有效剂量和通过动力学研究建立了我们的铅在 活体探测器。我们在TSRI佛罗里达州斯克里普斯校区的多PI研究团队拥有专业知识和 基础设施来进行这些实验，我们提出，成功完成我们的研究将 确定一系列有效的、选择性的和体内活性的分子探针，这些探针将有助于确定 ASK1在正常的生理过程中发挥作用，也在疾病状态下发挥作用。

Altered regulation of signaling in response to persistent cellular stress is associated with a number of complex human diseases, including cardiovascular, inflammatory and neurodegenerative disorders. Importantly, genetic studies have shown that silencing of the apoptosis signal-regulating kinase (ASK1) ameliorates many of the phenotypes manifest in mouse models of rheumatoid arthritis, cardiac hypertrophy and neurodegenerative disorders. Consequently, ASK1 has been highlighted as a therapeutic target where antagonists are anticipated to provide significant benefit in multiple disease states. Under the auspices of GM103957 our research team has recently completed a high throughput ASK1 screening campaign and identified a series of small molecule inhibitors from six different structural classes. We have narrowed the focus of our medicinal chemistry efforts to three scaffolds and the goal of this proposal is to develop an in vivo active ASK1 small molecule probe. In the studies of Aim 1 we will use an iterative medicinal chemistry approach to optimize the potency, selectivity and in vivo properties of ASK1 inhibitors aimed at peripheral targets. In addition, to develop compounds with suitable brain penetrant properties we will apply a multi-parameter optimization strategy focusing on physical and chemical properties that are hallmarks of the majority of current Food and Drug Administration (FDA)- approved CNS penetrant small molecule drugs. We will use structure based design to facilitate chemistry strategies and we will triage compounds through a battery of biochemical and cell-based assays as outlined in our research operating plan (ROP). As improved leads emerge, we will obtain enzyme selectivity for a select few, through full scale kinome (Reaction Biology) and receptor profiling (Riserca). For key leads we will also assess target engagement lifetime through biochemical and cell-based studies through measurement of ASK1 downstream substrate phosphorylation. Integrated into the ROP, we will also triage new compounds through a series of drug metabolism studies including assessment of in vitro microsomal stability, CYP450 inhibition, solubility, protein binding and P-glycoprotein efflux, with lead criteria integrated into the ROP for compound progression. In Aim 2, advanced leads for both peripheral and CNS exposures will be assessed in mice for adverse effects in acute dose range finding and 28-day toxicity studies. We will measure gross toxicity effect and vet scan analyses as well as TUNEL staining and histopathology of major organs. Finally, we will use inhibition of MKK6, JNK and p38 phosphorylation as in vivo reporters of ASK1 inhibition to quantify the maximum biological effective dose and through kinetic studies establish the PK/PD relationships of our lead in vivo probes. Our Multi-PI research team at the Scripps Florida campus of TSRI has both the expertise and infrastructure to perform these experiments, and we submit that the successful completion of our studies will identify a series of potent, selective and in vivo active molecular probes that will help define the role(s) that ASK1 plays in normal physiological processes as well as in disease states.