Small Molecules to Promote Regeneration and Recovery Following Spinal Cord Injury

A.摘要 脊髓损伤(SCI)后改善预后的一个重要障碍是轴突受限。 损伤部位轴突纤维再生不足所致的再生。RhoA介导的信号转导 GTP酶是生长的一个重要障碍。到目前为止，还没有小分子的RhoA拮抗剂 已经显示出体内疗效的药物。这归因于这样一个事实，即像RhoA这样的小GTP酶没有可药物 这使得直接与它们结合的小分子的开发变得极其困难。然而，结束了 多年来，已经发现了几种带有可下药口袋的蛋白质与RhoA相互作用。我们约定了 与这些可药物靶点结合的小分子将促进轴突再生和恢复到 与直接抑制RhoA活性的程度相似或更大。为了鉴定这种化合物，我们进行了一种独特的 包括基于结构的大型化学库与可药物靶标的计算对接的方法 与RhoA相互作用。我们测试了前50-100种化合物对轴突生长和轴突的影响。 再生。一种化合物，RLA-47，在体外抑制轴突生长和轴突再生。这个 在脊髓损伤的动物模型中，化合物也显示了大量的轴突交叉。我们的中央 假设RLA-47及其衍生物将促进脊髓后轴突的再生和恢复 受伤。我们的初步数据使我们处于有利地位，可以检验我们的假设。在我们的第一个目标，我们建议 设计和合成RLA-47的衍生物，将在我们的轴突生长和轴突再生中进行测试 体外试验。最有希望的候选者将在我们的活体脊髓半横切模型中进行探索 受伤。在我们的第二个目标中，我们建议开发具有合适的药代动力学和血液动力学的RLA-47衍生物。 促进体内轴突再生的大脑通透性。我们还测试了最有希望的化合物，使用 一种更具临床相关性的脊髓损伤挫伤模型。我们将使用标准的化学生物学策略 亲和纯化和质谱学以揭示RLA-47及其衍生物的靶标。与RNA一起 通过测序，这些研究将提供对化合物作用机制的更深层次的了解。我们预计 1-2个RLA-47的衍生物，显示出优越的药代动力学、脑渗透性和疗效。这些 化合物将导致研究性新药申请，我们预计最终将产生能够 可在临床试验中单独探索，或与其他疗法结合使用，以促进再生和 脊髓损伤患者的康复。

A. ABSTRACT A significant impediment towards improvement of outcome after spinal cord injury (SCI) is the limited axonal regeneration due to insufficient regrowth of the axonal fibers at the lesion site. Signaling mediated by RhoA GTPases presents a significant impediment to growth. To date, there exists no small-molecule RhoA antagonists that have shown in vivo efficacy. This is attributed to the fact that small GTPases like RhoA do not have druggable pockets, making the development of small molecules that directly bind to them extremely difficult. However, over the years, several proteins with druggable pockets have been discovered to interact with RhoA. We stipulated that small molecules that bind to these druggable targets will promote axonal regeneration and recovery to a similar or greater extent than direct inhibition of RhoA activity. To identify such compounds we pursued a unique approach that consisted of structure-based computational docking of large chemical libraries to druggable targets that interact with RhoA. We tested the top 50-100 compounds for their effect on neurite outgrowth and axonal regeneration. One compound, RLA-47, inhibited in vitro neurite outgrowth and axonal regeneration. The compound also showed substantial axon crossings in an animal model of spinal cord injury. Our central hypothesis is that RLA-47 and its derivatives will promote axonal regeneration and recovery following spinal cord injury. Our preliminary data puts us in a strong position to test our hypothesis. In our first aim, we propose to design and synthesize derivatives of RLA-47 that will be tested in our neurite outgrowth and axonal regeneration in vitro assays. The most promising candidates will be explored in our in vivo hemisection model of spinal cord injury. In our second aim, we propose to develop RLA-47 derivatives with suitable pharmacokinetics and blood- brain permeability that promote axonal regeneration in vivo. We also test the most promising compounds using a more clinically relevant contusion model of spinal cord injury. We will use a standard chemical biology strategy of affinity purification and mass spectrometry to uncover targets of RLA-47 and its derivatives. Along with RNA sequencing, these studies will provide deeper insight into the mechanism of action of compounds. We expect 1-2 derivatives of RLA-47 to exhibit superior pharmacokinetics, brain permeability and efficacy. These compounds will lead to investigational new drug filing that we expect will eventually lead to candidates that can be explored in clinical trials either alone, or in combination with other therapies to promote regeneration and recovery in spinal cord injury patients.