Development of GSK-3 Beta Inhibitors for the Treatment of Parkinson's Disease

描述（由申请人提供）：帕金森病（PD）是一种慢性进行性神经系统疾病，也是一种衰老和年龄相关疾病，仅在美国就影响了大约150万人。PD不仅给患者带来了沉重的负担，也给患者的家庭和社会带来了沉重的负担。2003年，全世界用于治疗帕金森病的药物治疗费用约为23亿美元。虽然PD治疗药物的开发已经取得了很大的进步，但现有的PD药物如多巴胺（DA）激动剂、左旋多巴和儿茶酚- o -甲基转移酶抑制剂（COMT）仅能治疗疾病的症状，而且也充满了不良反应和长期并发症。因此，非常需要开发能够减缓或阻止疾病进展的疾病修饰和神经保护以及神经恢复药物。最近的研究表明，小分子抑制糖原合成酶激酶-3¿（GSK-3¿）可能为治疗包括阿尔茨海默病（AD）在内的许多神经退行性疾病提供了重要的策略，但其在PD中的作用尚未被描述。我们最近在帕金森氏症的细胞和动物MPTP模型中发现，诱导Tau蛋白p-Tau的过度磷酸化形式，并在许多位点看到过度磷酸化，包括在阿尔茨海默病的神经原纤维缠结中发现的那些。p-Tau水平的增加严格依赖于a-Syn的存在。这种对a-Syn的要求是强制性的，因为在a-Syn-/-小鼠中，以及在不表达任何a-Syn的转染细胞中，毒素无法诱导p-Tau。MPTP也引起a-Syn蛋白水平的增加。Tau的过度磷酸化导致其与细胞骨架分离，在萨科齐不溶性和Triton x -100不溶性组分中可见p-Tau的聚集体。a-Syn能够与p-Tau形成稳定的异质蛋白复合物，在a-Syn成熟包涵体中可见p-Tau聚集物。MPP+引起了几种p- tau特异性激酶的激活，如GSK-3¿和p-ERK。阻断GSK-3¿不仅可以阻止，而且可以降低MPP+诱导的p-Tau形成，a-Syn积累和细胞毒性。与Jeffrey Joyce博士合作，在人类死后PD大脑中获得了非常新的数据，显示了类似的病理模式：a- syn积累增加，与MPTP模型相似的Tau位点过度磷酸化[pSer262和pSer396/404]， MPTP模型中未见的位点缺乏磷酸化[pSer202]， GSK-3¿大量增加。有趣的是，这些病理变化在PD合并痴呆患者中进一步增强[PD + DEM]。总之，PD大脑的这些发现证实了我们的发现与MPTP模型的有效性。重要的是，在我们的发现之前，GSK-3¿在PD中的可能作用之前没有被描述过，尽管它在AD中的作用已经得到了很好的研究。除了我们的发现，另一项研究发现GSK-3¿基因的两个单核苷酸多态性与散发性帕金森病有很强的联系。因此，GSK-3¿在PD新疗法的开发中提供了一个新的靶点。到目前为止，我们已经确定了一些nM效价GSK-3¿抑制剂，这些抑制剂来自我们对斯陶孢素的SAR研究。许多这些设计的staurosporine类似物已经针对30个激酶家族进行了筛选。在测试的化合物中，我们发现一种吲哚-吲哚基马来酰亚胺，当浓度为10 μ M时，能够抑制GSK-3 - 98%的激酶活性。经过下面描述的进一步结构修饰，新的3-（吲哚-3-基）-4-（苯并呋喃-3-基）马来酰亚胺被鉴定出来，相对于30种其他激酶，其Ki值低至2 nM，选择性地对抗GSK-3 -¿。此外，我们已经能够证明这些配体中的一些能够在体外发挥神经保护和神经恢复作用。最终目标是确定一到两种GSK-3抑制剂，可以进一步开发以减缓或阻止PD的进展。通过这项str拨款，我们打算继续研究我们在寻找新的GSK-3抑制剂作为PD治疗的潜在疗法方面取得的令人兴奋的初步发现。为了实现这一目标，本研究计划的具体目标如下：1。化合物选择与合成：在现有化合物文库的基础上，重新合成10种有效的GSK- 3¿抑制剂进行进一步的体外药理研究。然后，根据体外轮廓，2或3种最有希望的配体将被放大（每种约5克）用于体内动物研究。2. 转染细胞和神经元的体外神经保护研究：研究化合物抑制GSK-3¿的时间过程和剂量反应，分析这些化合物调节的其他激酶和磷酸酶，并将我们的发现与使用锂的效果进行比较。3. 转染细胞和神经元的体外神经修复研究：在细胞毒性开始后，我们将检查神经修复所需的GSK-3¿抑制剂的时间和剂量。4. 体内动物研究：MPTP注射5天，然后同时或延迟注射不同时间段的GSK-3¿抑制剂，增加剂量。GSK-3¿，a-Syn, Tau，激酶和磷酸酶将被检查，具体目标2和3中规定。关键词：GSK-3抑制剂，激酶，选择性，帕金森病，治疗，Staurosporine类似物，神经保护剂，神经恢复剂，神经保护，a-Synuclein, p-Tau， tau病变，synucleopathy， MPTP模型，衰老，神经退行性，年龄相关疾病，阿尔茨海默病。帕金森病（PD）是仅次于阿尔茨海默病（AD）的第二大常见神经退行性疾病，是一种与衰老相关的进行性神经系统疾病。我们的建议需要化学合成新的staurosporine类似物作为有效的糖原合成酶激酶-3 （GSK-3）抑制剂，并在转染细胞和神经元的体外研究以及这些化合物的动物体内研究中，这些化合物具有巨大的潜力，可开发为神经保护和神经修复疗法，以减缓或阻止帕金森病的进展。公共卫生

DESCRIPTION (provided by applicant): Parkinson's disease (PD), a chronic and progressive neurological condition and one of aging and age- related diseases, affects approximately 1.5 million people in the US alone. PD not only places severe burden on the patients but also on their family and society. In 2003, approximately US $2.3 billion was spent on drug therapy worldwide to treat Parkinson's disease. Although great strides have been made in the development of agents to treat PD, the existing PD drugs such as dopamine (DA) agonists, levodopa and catechol-O-methyl transferase inhibitors (COMT) only treat the symptoms of the disease, and are also fraught with adverse effects and long-term complications. Consequently, there is a great need for developing disease-modifying and neuroprotective as well as neurorestorative drugs which can slow down or stop the disease from progressing. Recent research suggests that inhibition of the glycogen synthase kinase-3¿ (GSK-3¿) by small molecules may offer an important strategy in the treatment of a number of neurodegenerative diseases including Alzheimer's disease (AD), but its usefulness in PD has not been described. We have recently found in the cellular and animal MPTP model of parkinsonism, an induction in the hyperphosphorylated form of Tau, p-Tau, with hyperphosphorylation seen at many sites, including those found in neurofibrillary tangles of AD. The increase in p-Tau levels was strictly dependent upon the presence of a- Syn. This requirement for a-Syn was mandatory, since in a-Syn-/- mice, and in transfected cells not expressing any a-Syn, the toxin failed to induce p-Tau. MPTP also caused an increase in a-Syn protein levels. Hyperphosphorylation of Tau lead to its dissociation from the cytoskeleton, and aggregates of p-Tau were seen in sarkosyl-insoluble and Triton X-100-insoluble fractions. a-Syn was able to form stable heteromeric protein complexes with p-Tau, and p-Tau aggregates were seen in mature inclusion bodies of a-Syn. MPP+ caused the activation of several p-Tau-specific kinases, such as GSK-3¿ and p-ERK. Blockade of GSK-3¿ not only prevented, but also reduced, MPP+-induced p-Tau formation, a-Syn accumulation and cytotoxicity. Very new data obtained in human postmortem PD brains, in collaboration with Dr. Jeffrey Joyce, show a similar pattern of pathology: increased a-Syn accumulation, hyperphosphorylation of Tau at sites similar to the MPTP models [pSer262 and pSer396/404], lack of phosphorylation at sites not seen with MPTP [pSer202], and large increases in GSK-3¿. Interestingly, these pathological changes were further augmented in PD patients with dementia [PD + DEM]. Together, these findings in PD brains confirm the validity of our findings with the MPTP models. Importantly, prior to our findings, a possible role for GSK-3¿ in PD has not been previously described, although its role in AD is well studied. In addition to our findings, another study found strong linkage of two single nucleotide polymorphisms in the GSK-3¿ gene to sporadic PD. Thus, GSK-3¿ presents a novel target site in the development of novel therapies for PD. To date, we have identified some nM potency GSK-3¿ inhibitors that emerged from our SAR studies of staurosporine. A number of these designed staurosporine analogs have been screened against a family of 30 kinases. Among the compounds tested we found one, an indolyl-indazolylmaleimide, that was able to inhibit 98% of the kinase activity of GSK-3¿ when tested at a concentration of 10 ¿M. After further structural modifications described below, novel 3-(indol-3-yl)-4-(benzofuran-3-yl)maleimides having a Ki value as low as 2 nM selectively against GSK-3¿ relative to 30 additional kinases were identified. Moreover, we have been able to show that some of these ligands are able to exert a neuroprotective and neurorestorative action in vitro. The ultimate goal would be to identify one or two GSK-3¿ inhibitors that could be further developed for slowing or halting the progression of PD. Through funding from this STTR grant, we intend to follow-up on the exciting preliminary findings we have made in pursuit of novel GSK-3¿ inhibitors as potential therapeutics for the treatment of PD. To achieve this goal, the Specific Aims of this research proposal are as follows: 1. Compound selection and synthesis: Based upon the compound library in hand, 10 potent GSK- 3¿ inhibitors will be resynthesized for further in vitro pharmacological studies. Then 2 or 3 of the most promising ligands based upon the in vitro profile will be scaled up (about 5 grams each) for in vivo animal studies. 2. Neuroprotective In vitro studies in transfected cells and neurons: Investigation of time course and dose response of the compounds for inhibition of GSK-3¿, analyses of other kinases and phosphatases that are modulated by these compounds, and comparison of our findings with the effects using lithium. 3. Neurorestorative in vitro studies in transfected cells and neurons: After the initiation of cytotoxicity, we will examine the time and dose of GSK-3¿ inhibitors necessary for neurorestoration. 4. In vivo animal studies: Injection with MPTP for 5 days will be followed by simultaneous or delayed injections with increasing doses of the GSK-3¿ inhibitors for different time periods. GSK-3¿, a-Syn, Tau, kinases and phosphatases will be examined, as specified in Specific Aims 2 and 3. Key words: GSK-3¿ inhibitors, Kinases, Selectivity, Parkinson's disease, Therapeutics, Staurosporine analogs, Neuroprotective agents, Neurorestorative agents, Neuroprotection, a-Synuclein, p-Tau, Taupathies, Synucleopathies, MPTP model, Aging, Neurodegenerative, Age-related diseases, Alzheimer's disease. Project Des inson's disease (PD), the second most common neurodegenerative disease next to Alzheimer's disease (AD), is a progressive neurological condition associated with aging. Our proposal entails the chemical synthesis of novel staurosporine analogues as potent glycogen synthase kinase-3¿ (GSK-3¿) inhibitors, and in vitro studies in transfected cells and neurons as well as in vivo studies in animals of these compounds, which have the great potential to be developed as neuroprotective and neurorestorative therapies to slow down or stop Parkinson's disease from progressing. Public Health