Supramolecular Approaches to Protein Inhibitors Activated by MicroRNA-21

描述（由申请人提供）：由癌症生物标志物触发的前药具有吸引力，因为它们有可能在癌细胞中优先激活，从而减少与传统化疗相关的严重不良反应。虽然已经发现了许多癌症生物标志物，但酶触发的前药一直是主要焦点，因为酶可以通过共价转化激活前药。因此，使用非酶生物标志物进行前药激活存在一个障碍，因为我们对如何开发选择性识别并被不能促进共价转化的生物标志物激活的分子知之甚少。这种知识差距的存在阻碍了可以开发的前体药物的全部范围。因此，该项目的长期愿景是产生非共价触发的前药。这项基础科学应用的目标是建立microRNA-21 （miR-21）非共价激活蛋白抑制剂的基本超分子机制，这是实现我们长期目标的重要一步。miR-21是一种高度不受管制的致癌miR。我们将通过两种互补的设计策略（分别为目标1和目标2）对该应用程序的中心假设进行测试，即miR-21诱导蛋白拮抗剂可以通过开发dna -小分子嵌合体（DCs）来实现，该嵌合体在特异性结合miR-21序列时经历从失活构象到激活状态的结构转换。该项目的具体目标是开发（目标1）通过宿主/客体相互作用使发夹形成的DCs失活，（目标2）通过导链双链形成使双齿状DCs失活，作为临床相关碳酸酐酶ii （CA-II）的miR-21反应性抑制剂。在这两个目标中，我们将合成和表征各自的DC系统，并将通过一系列光谱实验（包括UV-vis和荧光猝灭）探测它们经历miR-21诱导的结构转换到激活状态的能力。随后，将评估失活和活性DC构象抑制CA-II的差异能力（通过比色酯酶测定）。在优化DC系统的结构（包括增强CA-II结合和核酸酶抗性）之后，我们将把DC转染到过表达（和控制表达）miR-21的细胞系中，并探测CA-II抑制引起的细胞生长停滞。这个提议是创新的，因为它开发了第一个激活蛋白抑制剂以响应miRs的例子。此外，该项目将推动一般研究转向检查前药激活的非共价机制。这项工作是重要的，因为(a)它是一个开创性的一步，在一个连续的研究目标，从长远来看，在开发miR-21触发的前药，和(b)它将发挥持续的影响超分子化学领域，通过加强我们的理解分子识别的关键原则（如宿主/客体相互作用和多价）结合DNA组装和结构转换现象，可用于开发动态蛋白质结合物。

DESCRIPTION (provided by applicant): Prodrugs that are triggered by cancer biomarkers are attractive because of their potential to be activated preferentially in cancer cells thereby reducing the serious adverse effects associated with conventional chemotherapy. While numerous cancer biomarkers have been discovered, prodrugs triggered by enzymes have been the main focus because enzymes can activate prodrugs via covalent transformations. Thus, a barrier exists in using non-enzyme biomarkers for prodrug-activation because of our poor understanding of how to develop molecules that selectively recognize, and are activated by, biomarkers that cannot facilitate covalent transformations. The existence of this knowledge gap hinders the full scope of prodrugs that can be developed. Thus the long-term vision of this project is to generate non-covalently triggered prodrugs. The objective of this basic-science application, which is a seminal step in achieving our long term goal, is to establish fundamental supramolecular mechanisms for the non-covalent activation of protein inhibitors by microRNA-21 (miR-21), a highly deregulated, oncogenic, miR. The central hypothesis of this application, which we will test via two complementary design strategies (Aim 1 and 2, respectively), is that miR-21 inducible protein antagonists can be achieved by developing DNA-small molecule chimeras (DCs), that undergo a structural switch from a de-activated conformation to an activated state upon binding sequence specifically to miR-21. The specific aims for this project are to develop, (Aim 1) hairpin-forming DCs de-activated by host/guest interactions and (Aim 2) bidentate DCs de-activated via duplex formation with a guide strand, as miR-21 responsive inhibitors of clinically relevant carbonic anhydrase-II (CA-II). In both aims, we will synthesize and characterize the respective DC systems and will probe their ability to undergo miR-21 induced structure-switching to an activated state via a number of spectroscopic experiments (including UV-vis and fluorescence quenching). Subsequently, the differential ability of the de-activated and active DC conformations to inhibit CA-II will be evaluated (via a colorimetric esterase assay). After optimization of the structure of the DC systems (including enhancing CA-II binding and nuclease resistance), we will transfect DCs into cell-lines over expressing (and control cell-lines under expressing) miR-21 and probe CA-II inhibition-derived cell growth arrest. This proposal is innovative because it develops the first examples of activating protein inhibitors in response to miRs. Further, this project will provide impetus to shift general research into examining non- covalent mechanisms for prodrug activation. This work is significant because (a) it is a seminal step in a continuum of research aimed, in the long-term, at developing miR-21 triggered prodrugs, and (b) it will exert a sustained influence on the field of supramolecular chemistry by enhancing our understanding of how key principles of molecular recognition (such as host/guest interactions and multivalency) in combination with DNA assembly and structure-switching phenomena can be used to develop dynamic protein-binders. )