Controlled premature termination of translation to generate designated truncated

描述（由申请人提供）：许多疾病，包括几种形式的癌症，依赖于病理蛋白（如癌基因）的不适当表达。通常，这些蛋白质的结构-功能是模块化的，在蛋白质的c端部分有一个基本的功能域（例如转录因子的反活化结构域）。缺乏c端结构域的突变体可能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。这种变异有时是由选择性剪接/聚腺苷酸化自然产生的。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阴性变异的方法将是非常可取的，并且在包括癌症在内的广泛疾病中具有重要的治疗潜力。为了产生预先设计的，理想的截断变体而不是病理变体，我们提出了一种新的通用方法，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。这些自包膜反义翻译终止（SWATT）化合物通过引入一个不可逾越的空间块而起作用，该空间块不能被进入的翻译核糖体的解旋酶活性分解，而核糖体通常可以解开前mrna中的广泛二级结构。因此，这种障碍会导致核糖体下降，并表达更短的c端截断变体。我们将通过在专门设计的报告荧光素酶结构上评估SWATT化合物来验证这种新策略的可行性，并最终通过在癌细胞中使用Notch信号作为原理证明底物来显示它们的生物学相关性。缺乏细胞内信号域的Notch的c端显性阴性版本的诱导，应该通过参与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。SWATT化合物将提供一个额外的工具来剖析蛋白质在其生理背景下的结构-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.