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

药物的发现在很大程度上依赖于分析和监测人体细胞生理状态的检测技术，以忠实地识别有效的和选择性的疾病过程调节因子。这些调节剂，无论是小分子、多肽还是抗体，都需要药理学上相关的体外分析来量化它们的调节作用。许多使用体外系统和在异种细胞类型中过度表达靶蛋白的方法经常导致候选药物在临床开发的后期阶段出现惊人的失败。这是由于在测试分析系统中，关键疾病疗效和毒理学特性的内在生物学相关性。我们的目标是提供一种灵敏、一致和准确的测试技术，能够以相关的活细胞形式询问关键的细胞药物靶点。该技术将应用于主要的药物靶标类别，包括G蛋白偶联受体(GPCR)和蛋白激酶(PK)，这两类药物分别有30%和6%的上市药物针对这两类药物。现有的检测技术历来建立在放射性标记试剂的基础上，这是不可取的，在生理环境中很大程度上缺乏适用性，特别是在试剂无法穿过完整的细胞膜的情况下。我们的财团结合了在产生细胞渗透的、修饰的核苷三磷酸(NTP)底物(TriPPPro技术)方面的独特专业知识，在建立和调整基于细胞的分析以进行高通量筛选活动方面以及在将人类诱导多能干细胞(HiPSC)技术应用于早期(靶标对靶标)药物发现方面拥有丰富的经验。此外，我们还获得了最先进的筛选技术，获得了100,000种“随时可以筛选”的化合物，并引进了一家为所有主要制药公司提供检测技术的全球领先公司的知识和经验。在拟议的项目中，我们将生产新型的NTP-衍生物，它携带伽马-磷酸修饰，用于共价标记感兴趣的目标，并结合可生物降解的掩蔽部分，使细胞膜能够渗透。这些化合物作为活细胞分析试剂的性能将被询问，重点是相关生理模型中特定的G蛋白或激酶功能，特别是HIPS-心肌细胞，最终产生一种经过验证的测试技术。这项新技术将被广泛应用，并有可能推动与了解疾病机制有关的基础研究和工业药物发现，一旦它以“即用即用”的商业试剂盒形式成功上市。

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.