Targeting Multiple Kinases to Treat Experimental Spinal Cord Injury.

靶向多种激酶治疗实验性脊髓损伤。

• 批准号: 10393353
• 负责人: John L Bixby
• 金额: $ 1.68万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-01 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10393353
• 关键词: Axon; Biological Assay; Catalytic Domain; Cell physiology; Cells; Contusions; Corticospinal Tracts; Development; Dorsal; Failure; Forelimb; Future; Growth; In Vitro; Individual; Information Theory; Injury; Machine Learning; Mediating; Modeling; Molecular Target; Motor; Natural regeneration; Nerve; Neurites; Neurons; Outcome; Pharmaceutical Preparations; Phosphotransferases; Process; Recovery of Function; Signal Transduction; Site; Small Interfering RNA; Source; Spinal cord injury; Spinal cord injury patients; Testing; Therapeutic; Transducers; Viral Vector; Walking; axon growth; axonal sprouting; behavioral outcome; central nervous system injury; clinically relevant; drug discovery; effective therapy; experience; experimental study; grasp; improved; in vivo; in vivo Model; inhibitor/antagonist; kinase inhibitor; knock-down; light microscopy; novel; small molecule; targeted agent

PROJECT SUMMARY Spinal cord injury (SCI) patients experience limited functional recovery, owing in part to the paucity of axon regrowth from injured CNS neurons. Effective treatments are lacking, likely because of multiple factors, intrinsic and extrinsic, that inhibit axon growth. Thus, we require agents that target more than one source of regeneration failure. Kinases are ubiquitous signal transducers that regulate most cellular processes, including axon growth. To begin to identify compounds that positively regulate axon growth, we screened 1600 small-molecule kinase inhibitors (KIs) in an in vitro CNS neurite outgrowth assay and identified “hit” KIs that reproducibly and strongly promote outgrowth. Due to homology of catalytic domains, KIs typically inhibit multiple kinases. This makes it difficult to identify the kinase(s) that mediate a KI’s effects on cells. We used information theory and machine learning to analyze the inhibition profiles of KIs in relation to their effects on neurite outgrowth. This enabled us to identify, and later validate via siRNA knockdown in primary neurons, multiple kinase targets (i.e. kinases that should be inhibited to promote neurite outgrowth). These included previously known targets that regulate intrinsic and extrinsic inhibitor factors, in addition to several novel candidates. Conversely, we identified kinases whose activity is critical for neurite outgrowth, and whose inhibition must be avoided (anti-targets). We discovered several KIs that inhibit multiple targets and no anti-targets. These KIs strongly promoted neurite outgrowth in vitro. We tested the KI, RO48, that had the largest effect in vitro in two in vivo models. Our preliminary experiments indicate that RO48 is remarkably effective in vivo. It promoted robust axonal growth of the corticospinal tract (CST) in three separate models of CST injury (pyramidotomy, funiculotomy, dorsal hemisection), and in the dorsal hemisection model, improved forelimb function. We propose to build on these remarkable results to test the working hypothesis that the simultaneous inhibition of RO48’s five target kinases (ROCK, PKC, PRKG1, PRKX, and RPS6K) promotes sprouting and regeneration of CST axons. This will be accomplished using viral vectors to knock down expression of the different target kinases individually and in combination. We will do knockdown in CST neurons in the cortex. We will assess CST axon growth at the injury site using light microscopy. We will also perform experiments to determine if RO48-induced CST axon growth promotes axon sprouting, regeneration, or both, and whether RO48 improves behavioral outcomes such as grasping and walking after a contusion injury.These experiments will 1) validate novel kinases as in vivo targets for future development of SCI therapeutics 2) determine whether these kinases regulate CST axon sprouting, regeneration, or both, and 3) confirm whether the substantial stimulation of axon growth induced by treatment with RO48 improves motor outcomes in a clinically relevant contusion model.

项目摘要 脊髓损伤（SCI）患者的功能恢复有限，部分原因是轴突缺乏 从受损的CNS神经元再生。缺乏有效的治疗方法，可能是由于多种因素，内在的 和外源性的，抑制轴突生长。因此，我们需要针对多个再生源的代理 失败激酶是普遍存在的信号转导，调节大多数细胞过程，包括轴突生长。 为了开始鉴定积极调节轴突生长的化合物，我们筛选了1600种小分子激酶， 抑制剂（KI）在体外CNS神经突生长试验，并确定了"命中" KI，可重复和强烈 促进增长。由于催化结构域的同源性，KI通常抑制多种激酶。这使得 很难鉴定介导KI对细胞作用的激酶。我们用信息论和机器 学习分析KIs的抑制特征与其对神经突生长的影响。这使我们 为了在原代神经元中鉴定并随后通过siRNA敲低验证多个激酶靶（即 应被抑制以促进神经突生长）。这些包括以前已知的调节内源性 和外源性抑制因子，以及几种新的候选物。相反，我们鉴定了 活性对于神经突生长至关重要，必须避免其抑制（抗靶）。我们发现 几种抑制多个靶点的KI，没有抗靶点。这些KIs强烈促进神经突生长， 体外我们测试了KI，RO48，其在两种体内模型中具有最大的体外作用。我们的初步 实验表明，RO48在体内是显著有效的。它促进了轴突的生长， 皮质脊髓束（CST）损伤的三种不同模型（脊髓正中切开术，脊髓束切开术，背侧 半切），并且在背侧半切模型中，改善了前肢功能。我们建议在这些基础上， 显着的结果，以测试工作假设，同时抑制RO48的五个目标激酶 (ROCK、PKC、PRKG1、PRKX和RPS6K）促进CST轴突的发芽和再生。这将是 使用病毒载体单独敲低不同靶激酶的表达， 组合.我们将在皮层的CST神经元中进行敲除。我们将评估损伤处的CST轴突生长 使用光学显微镜检查部位。我们还将进行实验以确定RO 48诱导的CST轴突生长是否 促进轴突发芽，再生，或两者兼而有之，以及RO48是否改善行为结果， 这些实验将1）验证作为体内靶点的新型激酶 对于SCI治疗的未来发展，2）确定这些激酶是否调节CST轴突发芽， 再生，或两者兼而有之，以及3）确认治疗是否诱导轴突生长的实质性刺激 RO48改善了临床相关挫伤模型中的运动结果。

项目成果

AAV8 transduction capacity is reduced by prior exposure to endosome-like pH conditions.

通过事先暴露于内体样pH条件来降低AAV8转导能力。

• DOI: 10.4103/1673-5374.299272
• 发表时间: 2021-05
• 期刊: Neural regeneration research
• 影响因子: 6.1
• 作者: Lowell JA;Mah KM;Bixby JL;Lemmon VP
• 通讯作者: Lemmon VP

Computational Identification of Kinases That Control Axon Growth in Mouse.

• DOI: 10.1177/2472555220930697
• 发表时间: 2020-08
• 期刊: SLAS discovery : advancing life sciences R & D
• 作者: Devkota P;Danzi MC;Lemmon VP;Bixby JL;Wuchty S
• 通讯作者: Wuchty S

Compounds co-targeting kinases in axon regulatory pathways promote regeneration and behavioral recovery after spinal cord injury in mice.

• DOI: 10.1016/j.expneurol.2022.114117
• 发表时间: 2022-09
• 期刊: EXPERIMENTAL NEUROLOGY
• 影响因子: 5.3
• 作者: Mah, Kar Men;Wu, Wei;Al-Ali, Hassan;Sun, Yan;Han, Qi;Ding, Ying;Munoz, Melissa;Xu, Xiao-Ming;Lemmon, Vance P.;Bixby, John L.
• 通讯作者: Bixby, John L.

Phenotypic Screening Following Transcriptomic Deconvolution to Identify Transcription Factors Mediating Axon Growth Induced by a Kinase Inhibitor.

• DOI: 10.1177/24725552211026270
• 发表时间: 2021-12
• 期刊: SLAS DISCOVERY
• 影响因子: 3.1
• 作者: Lowell, Jeffrey A.;O'Neill, Nicholas;Danzi, Matt C.;Al-Ali, Hassan;Bixby, John L.;Lemmon, Vance P.
• 通讯作者: Lemmon, Vance P.

Identification of lead anti-human cytomegalovirus compounds targeting MAP4K4 via machine learning analysis of kinase inhibitor screening data.

• DOI: 10.1371/journal.pone.0201321
• 发表时间: 2018
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Strang BL;Asquith CRM;Moshrif HF;Ho CM;Zuercher WJ;Al-Ali H
• 通讯作者: Al-Ali H