INtraCELLular delivery of PHOSphorylated tools to TAG proteins using next generation nucleoside triphosphate derivatives

使用下一代核苷三磷酸衍生物将磷酸化工具在细胞内递送至 TAG 蛋白

• 批准号: 426328168
• 负责人: Professor Dr. Chris Meier
• 金额: --
• 依托单位: Institut für Organische Chemie
• 依托单位国家: 德国
• 项目类别: Research Grants (Transfer Project)
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/426328168?language=en
• 关键词: INtraCELLular; delivery; PHOSphorylated; tools; TAG

Drug discovery heavily relies on assay technologies which analyze and monitor the physiological situation in human cell, in order to faithfully identify potent and selective modulators of disease processes. These modulators, whether they are small molecules, peptides or antibodies, all require pharmacologically relevant in-vitro assays to be used for quantification of their modulatory effects. Many approaches using in-vitro systems and overexpression of target proteins in heterologous cell types have often led to spectacular failures of drug candidates in later stages of clinical development. This is due to an intrinsic lack of biological relevance, in the test assay systems, to key disease efficacy and toxicological properties.We here aim to deliver a sensitive, consistent and accurate assay technology which enables the interrogation of key cellular drug targets in relevant live cell formats. The technology will be applied to major drug target classes including G-protein coupled receptors (GPCR) and protein kinases (PK) which are targeted by 30% and 6% of marketed drugs, respectively. Available assay technology is historically build on radiolabeled reagents, which is undesirable, and largely lacks applicability in physiological contexts, especially as reagents aren’t able to cross intact cell membranes.Our consortium combines unique expertise in generating cell-permeable, modified nucleoside triphosphate (NTP) substrates (TriPPPro technology), extensive experience in establishing and adapting cell-based assays for high throughput screening campaigns and in applying human induced pluripotent stem cell (hiPSC) technology to early stage (target-to-lead) drug discovery. In addition, we have access to state-of-the-art screening technology, access to several 100,000 "ready-to-screen" compounds and bring in the knowledge and experience of one of the leading companies world-wide providing assay technology to all major pharmaceutical companies.In the proposed project, we will generate novel NTP-derivatives that carry gamma-phosphate modifications for covalently labeling targets of interest in combination with a biodegradable masking moiety that enables cell membrane permeation. These compounds’ performance as live cell assay reagents will be interrogated focusing on specific G-protein or kinase functionalities in relevant physiological models, especially hiPS-cardiomyocytes, to finally yield a validated assay technology. This novel technology will be broadly applicable and has the potential to advance both basic research related to understanding disease mechanisms and industrial drug discovery once it has been successfully established on the market in “ready to use” commercial kit formats.

药物发现在很大程度上依赖于分析和监测人类细胞中的生理状况的测定技术，以便忠实地识别疾病过程的有效和选择性调节剂。这些调节剂，无论它们是小分子、肽还是抗体，都需要与体外试验相关的体外试验来定量它们的调节作用。使用体外系统和在异源细胞类型中过表达靶蛋白的许多方法经常导致候选药物在临床开发的后期阶段的惊人失败。这是由于在测试分析系统中，与关键疾病疗效和毒理学特性内在缺乏生物相关性。我们的目标是提供一种灵敏、一致和准确的分析技术，从而能够在相关活细胞形式中询问关键细胞药物靶标。该技术将应用于主要的药物靶点类别，包括G蛋白偶联受体（GPCR）和蛋白激酶（PK），分别有30%和6%的上市药物靶向这些靶点。现有的检测技术在历史上是建立在放射性标记试剂上的，这是不可取的，并且在生理环境中很大程度上缺乏适用性，特别是当试剂不能穿过完整的细胞膜时。我们的联盟结合了在产生细胞可渗透的修饰的三磷酸核苷（NTP）底物方面的独特专业知识（TriPPPro技术），在建立和调整基于细胞的高通量筛选试验以及应用人诱导多能干细胞（hiPSC）技术到早期（靶向先导）药物发现。此外，我们还拥有最先进的筛选技术，可获得数十万种“随时可筛选”的化合物，并将全球领先的公司之一的知识和经验引入其中，为所有主要制药公司提供分析技术。在拟议的项目中，我们将产生新的NTP衍生物，用于共价标记感兴趣的靶标的磷酸修饰与能够实现细胞膜渗透的可生物降解的掩蔽部分组合。这些化合物作为活细胞测定试剂的性能将被询问，重点关注相关生理模型中的特定G蛋白或激酶功能，特别是hiPS-心肌细胞，以最终产生经验证的测定技术。这项新技术将被广泛应用，并有可能推进与理解疾病机制和工业药物发现相关的基础研究，一旦它以“即用型”商业试剂盒形式在市场上成功建立。