Optimization of HDAC6 Inhibitors in the Treatment of CMT

描述（由申请人提供）：腓骨肌萎缩症（Charcot-Marie-Tooth disease， CMT）是最常见的遗传性神经系统疾病之一，在美国每2500人中就有1人患病。这种疾病是以1886年首次发现它的三位医生的名字命名的。CMT也被称为遗传性运动和感觉神经病变（HMSN）或腓肌萎缩症，是一组影响周围神经的疾病。CMT同时影响运动神经和感觉神经。一个典型的特征包括足部和小腿肌肉无力，这可能导致足下垂和高步伐步态，经常绊倒或跌倒。小热休克蛋白27 （HSPB1）的突变导致轴突CMT或远端遗传性运动神经病（远端HMN）。我们的团队已经开发并鉴定了仅在神经元中表达突变体（S135F） HSPB1的转基因小鼠。这些小鼠表现出CMT2的所有特征。突变体HSPB1的表达降低了乙酰化的微管蛋白水平，并诱导了严重的轴突运输缺陷。具有重要意义的是，我们现在已经证明，使用我们设计的HDAC6I [HDAC6抑制剂]Tubastatin A对组蛋白去乙酰化酶6 （HDAC6）诱导的-微管蛋白去乙酰化的药理学抑制纠正了HSPB1突变诱导的轴突运输缺陷，并挽救了有症状突变HSPB1小鼠的CMT表型。我们的研究结果证明了-微管蛋白去乙酰化在突变型hspb1诱导的神经病变中的致病作用，并为HDAC6抑制剂作为遗传性轴突病的治疗策略提供了有价值的观点。为了扩展这些高影响力的发现，并将最佳的HDAC6I疗法推向临床，用于治疗CMT和相关神经病变，我们打算更充分地探索Tubastatin a类似物家族的结构活性关系[SAR]， Tubastatin a类似物是我们研究中使用的关键HDAC抑制剂。本建议的目的如下：利用药物化学原理和计算机建模方法，设计合成改进的Tubastatin A类似物，并进一步优化其体外HDAC异构体的选择性和效价。为此目的，将进行计算机ADMET计算，以便仅制备最类似药物的化合物用于初步的体外研究。2. 对于那些对HDAC6表现出至少300倍选择性且IC50 < 50 nM的化合物，优化这些新的化学实体，使其能够增强有症状突变HSPB1小鼠培养神经元中的微管蛋白乙酰化和线粒体运动。3. 对于在Aim 2中表现出最佳效果的化合物，通过确定其实验ADMET参数进一步表征这些化合物，以确保适当的稳定性、细胞通透性、缺乏脱靶活性、安全性等。最后，我们在CMT转基因小鼠模型中测试了来自Aim 3的最佳化合物，正如我们之前所做的那样，表征了它们增强周围神经微管蛋白乙酰化和逆转突变HSPB1小鼠症状的能力。

DESCRIPTION (provided by applicant): Charcot-Marie-Tooth disease (CMT) is one of the most common inherited neurological disorders that affects ~ 1 in 2,500 people in the US. The disease is named for the three physicians who first identified it in 1886. CMT, also known as hereditary motor and sensory neuropathy (HMSN) or peroneal muscular atrophy, is made up of a group disorders that affect peripheral nerves. CMT affects both motor and sensory nerves. A typical characteristic includes weakness of the foot and lower leg muscles, which may result in foot drop and a high-stepped gait with frequent tripping or falls. Mutations in the small heat-shock protein 27 (HSPB1) cause axonal CMT or distal hereditary motor neuropathy (distal HMN). Our team has developed and characterized transgenic mice expressing mutant (S135F) HSPB1 in neurons only. These mice show all features of CMT2. Expression of mutant HSPB1 decreased acetylated �-tubulin levels and induced severe axonal transport deficits. Of great significance, we have now shown that pharmacological inhibition of histone deacetylase 6 (HDAC6)-induced �-tubulin deacetylation using our designed HDAC6I [HDAC6 Inhibitor] Tubastatin A corrected the axonal transport defects induced by HSPB1 mutations and rescued the CMT phenotype of symptomatic mutant HSPB1 mice. Our findings demonstrate the pathogenic role of �-tubulin deacetylation in mutant HSPB1-induced neuropathies and offer valuable perspectives for HDAC6 inhibitors as a therapeutic strategy for hereditary axonopathies. In order to extend these high impact findings and to advance the best HDAC6I therapies to the clinic for the treatment of CMT and related neuropathies, we intend to more fully explore the structure activity relationships [SAR] of a family of Tubastatin A analogs, the key HDAC inhibitor used in our studies. The aims of this proposal are as follow: 1. Using the principles of medicinal chemistry and computer modeling methods, design and synthesize improved analogs of Tubastatin A and further optimize these for their in vitro HDAC isoform selectivity and potency. In this aim, in silico ADMET calculations will be performed so that only the most drug-like compounds are prepared for the preliminary in vitro studies. 2. For those compounds showing at least 300-fold selectivity for HDAC6 over the other isoforms and having an IC50 of < 50 nM, optimize these new chemical entities for their ability to enhance tubulin acetylation and mitochondrial movement in cultured neurons from symptomatic mutant HSPB1 mice. 3. For compounds showing the best effects in Aim 2, further characterize these compounds by determining their experimental ADMET parameters in order to ensure appropriate stability, cell permeability, lack of off- target activity, safety, etc. 4. Lastly, tes the best compounds from Aim 3 in the CMT transgenic mouse models, characterizing these as we have done before on their capacity to enhance tubulin acetylation in peripheral nerves and to reverse the symptoms in the mutant HSPB1 mice.