Tuning aminoglycosides for treatment of genetic diseases

描述(申请人提供)：大量人类遗传病是由于突变导致突变基因编码的蛋白质合成提前终止所致。目前，已知数百个这样的无义突变，其中几个被证明与某些致命疾病有关，包括囊性纤维化(CF)、杜氏肌营养不良(DMD)、泰-萨克斯(Tay-Sachs)等。对于其中许多疾病，目前还没有有效的治疗方法。在过去的几年中，一些氨基糖苷类抗生素(包括庆大霉素)被证明能够使哺乳动物核糖体选择性地阅读通过假停止信号，而不是正常的终止信号，并产生全长功能蛋白。然而，这些药物对人体的高毒性限制了它们的治疗用途。本研究的主要目的是开发新的氨基糖苷类化合物，使其具有高效的终止抑制活性，同时降低对哺乳动物细胞的毒性。到目前为止，氨基糖苷类化合物的临床应用仅限于作为抗菌药物，还没有努力优化它们作为终止密码子通读诱导剂的活性。为此，几个不同的实验室正在进行一项合作努力，并结合一些互补学科的专业知识来合成和阐明所设计药物的结构-活性-毒性关系。该提议背后的假设是将导致毒性的氨基糖苷类结构的元素与诱导无意义抑制所需的元素分开：设计的结构对细胞质rRNA A位点具有广泛的特异性和选择性，可以缩小功能剂量范围，从而降低预期的毒性，包括对线粒体蛋白质合成机制的有害影响，使其成为治疗人类遗传疾病的潜在药物。我们的假设是基于最近的一系列观察，其中我们已经表明，通过降低对原核糖体的特异性，从而消除氨基糖苷类化合物的“天然”抗菌活性，我们可以减少它们对真核细胞线粒体蛋白质合成机制的作用，从而显著减少它们对人类的毒性影响。预计这项研究将带来显著的治疗和经济效益。这里给出的设计结构制备简单，初步测试已经发现了一些毒性更低、通读效率更高的变体，可以在体外和体内从突变基因中恢复功能性CFTR蛋白，包括与庆大霉素相比，在人类CF受试者中的结果更接近预测结果的模型。该项目目标的实现将为发现可能作为新药的新结构提供必要的知识和工具。

DESCRIPTION (provided by applicant): A large number of human genetic diseases result from mutations that cause premature termination of the synthesis of proteins encoded by mutant genes. Currently, hundreds of such nonsense mutations are known, and several where shown to account for certain cases of fatal diseases, including cystic fibrosis (CF), Duchenne muscular dystrophy (DMD), Tay-Sachs, and more. For many of those diseases there is presently no effective treatment. In the last several years, it was shown that some aminoglycoside antibiotics (including gentamicin) have the ability to allow the mammalian ribosome to selectively read past a false-stop signal, but not a normal termination signal, and generate full-length functional proteins. However, high toxicity of these drugs in humans limits their therapeutic use. The main objective of this research is to develop novel aminoglycosides that will have efficient termination suppression activity, and at the same time will have reduced toxicity against mammalian cells. To date, the clinical application of aminoglycosides is limited to their use as antibacterial drugs, and no efforts have been made to optimize their activity as stop codon read-through inducers. Toward these ends, a collaborative effort is under way between several different laboratories and combines expertise of number of complementary disciplines to synthesize and elucidate the structure-activity- toxicity relationships of the designed drugs. The hypothesis behind the proposal is to separate the elements of the aminoglycosides structures that cause toxicity from those that are required for inducing nonsense suppression: designed structures exhibiting extensive specificity and selectivity for the cytoplasmic rRNA A site can decrease the functional dosing ranges and subsequently decrease the anticipated toxicity, including deleterious effects on mitochondrial protein synthesis machinery, making them potential drugs for the treatments of human genetic disorders. Our hypothesis is based on a series of recent observations in which we have shown that by reducing the specificity to prokaryotic ribosome and as such wiping away from aminoglycosides their "natural" antibacterial activity we reduce their action on eukaryotic mitochondrial protein synthesis machinery and as such significantly reduce their toxic effects on humans. Substantial therapeutic and economic benefits are anticipated from this study. The designed structures presented here are simple for preparation and the preliminary tests already discovered some variants with lower toxicity and greater read-through efficacy to restore functional CFTR protein from the mutant gene both in vitro and in vivo, including models closely predictive of results with human CF subjects, than those of gentamicin. Fulfillment of the goals of this project will provide the necessary knowledge and tools for uncovering new structures, which may act as novel drugs.