Controlled premature termination of translation to generate designated truncated

描述(申请人提供)：许多疾病，包括几种形式的癌症，依赖于病理蛋白的不适当表达，如癌基因。通常，这些蛋白质的结构-功能掩盖了一种模块化的结构，在蛋白质的C末端部分有一个必要的功能域(例如转录因子的反式激活域)。缺乏C-末端结构域的突变体可以具有改变和拮抗功能，在某些情况下，它们可以表现为显性-负变异体。这种变体有时是通过选择性剪接/多聚腺苷基化自然产生的。然而，这些并不总是存在或不容易激活。一种专门诱导这些显性负变异体的方法将是非常可取的，并在包括癌症在内的广泛疾病中具有显著的治疗潜力。为了产生预先设计的、理想的截断变体而不是病理性的变体，我们建议开发一种新的通用方法学，基于一种全新的反义分子设计，能够将翻译终止从正常翻译的全长ORF重定向到上游指定位置。这些自包裹的反义翻译终止(SWATT)化合物的工作原理是引入一种无法克服的空间位阻，而这种空间位阻不能被进入的翻译核糖体的解旋酶活性所解决，后者通常可以解开Pre-mRNA中广泛的二级结构。这一障碍将导致核糖体丢失和表达一个更短的、C末端截短的变体。我们将通过评估特殊设计的报告荧光素酶结构上的SWATT化合物来验证这一新策略的可行性，并最终通过使用Notch信号作为癌细胞中的原理证明底物来显示它们的生物学相关性。诱导缺失细胞内信号转导结构域的C端显性负性的Notch，应该会通过与Notch配体的非生产性相互作用来抑制Notch信号，从而阻止其致癌功能。斯瓦特化合物将提供一个额外的工具来剖析蛋白质在其生理环境中的结构-功能特性，它们将构成治疗癌症和其他疾病的新治疗方法的基础。

DESCRIPTION (provided by applicant): Many diseases, including several forms of cancer, depend on the inappropriate expression of pathological proteins, such as oncogenes. Frequently, the structure-function of these proteins belies a modular construction, with an essential functional domain in the C-terminal portion of the protein (for example the transactivation domain of a transcription factor). Mutants lacking the C-terminal domain can have altered and antagonistic functions, and in some cases they can behave as dominant- negative variants. Such variants are sometimes naturally generated by alternative splicing/polyadenylation. However, these are not always present or not easily activatable. A method to specifically induce these dominant negative variants would be highly desirable and have a significant therapeutic potential in a broad range of diseases, including cancer. In order to generate a pre-designed, desirable truncated variant instead of the pathological one we propose development of a novel universal methodology, based on a brand new design of antisense molecules that are able to redirect termination of translation from the normally translated full-length ORF to an upstream specified location. These Self-Wrapping Antisense Translation Termination (SWATT) compounds work by introducing an insurmountable steric block that cannot be resolved by the helicase activity of incoming translating ribosomes, which can normally unwind extensive secondary structures within the pre-mRNA. This roadblock would thus result in ribosome drop-off and expression of a shorter, C-terminally truncated variant. We will validate the viability of this novel strategy by assessing SWATT compounds on specifically designed reporter luciferase constructs and, eventually, by showing their biological relevance using Notch signaling as a proof-of-principle substrate in cancer cells. Induction of a C-Terminal dominant negative version of Notch, lacking the intracellular signaling domain, should inhibit Notch signaling by engaging Notch ligands in non-productive interactions, and thus prevent its oncogenic functions. SWATT compounds will provide an additional tool to dissect the structure-function properties of proteins in their physiological contexts, and they wil constitute the basis for a novel therapeutic approach in the treatment of cancer and other diseases.