Live-Cell Fluorescence Lifetime FRET Assays for HTS

 描述（由申请人提供）：我们寻求开发和商业化一种强大的技术平台，以使用基于活细胞中FRET的荧光寿命（FLT）读数的高通量筛选（HTS）来显著提高药物发现的有效性。这一突破是通过两种互补和协同技术的结合实现的：荧光生物传感器工程（明尼苏达大学，UMN）和荧光仪器工程（Fluorescence Innovations，FI）。在第一阶段，我们实现了我们的目标，在活细胞中产生了第一个真正高通量和高精度的FLT应用，应用于特定的生物传感器（SERCA）。我们还通过开发用于高通量光谱记录的新仪器，对50，000个化合物库进行SERCA筛选，以及开发基于几个新目标的FRET生物传感器，超越了我们的目标。此外，几家制药公司表示对该技术平台感兴趣，因此UMN和FI成立了一家新的药物发现公司Photonic Pharma，以实现这些组合技术的最终商业化。在第二阶段，我们将进一步开发这一技术平台，并将其应用于各种目标，从而清楚地展示 商业化潜力大。在目标1中，我们将寻求硬件和软件的改进，以提高未来的商业化潜力，包括将FLT和光谱记录相结合的新仪器，提高数字化仪速度，更高密度的板，与机器人的连接以及软件改进。在目标2中，为了证明我们的技术平台具有广泛的适用性，我们将扩大我们的生物传感器列表，重点关注具有高商业潜力的目标，世界领先的专家在UMN非常接近。这些疾病的靶点包括心力衰竭、药物滥用和成瘾、炎症和癌症以及肌肉张力障碍。生物传感器将在活细胞中进行工程化和表达，并针对标准测试和验证文库进行筛选，以优化筛选试验。然后，我们将使用优化的HTS测定法进行几次大规模（50，000种化合物）筛选。命中化合物将作为化合物浓度的函数（剂量-反应）进行重新测试，然后由UMN的上述世界级专家进行功能测定。有希望的命中可能成为未来药物化学发展的线索，但主要目标是验证发现平台。我们相信，在这些目标下进行的研究将通过验证我们的商业计划，将UMN在生物传感器工程和细胞培养方面的联合收割机专业知识与由新兴的早期药物发现初创公司Photonic Pharma领导的仪器方面的FI专业知识相结合，为我们的商业化阶段做好准备。因此，我们的商业计划不是销售仪器，而是利用我们独特的生物传感器和仪器专业知识的组合，开发和应用一个技术平台，加速药物发现的早期阶段。

 DESCRIPTION (provided by applicant): We seek to develop and commercialize a powerful technology platform to increase dramatically the effectiveness of drug-discovery using high-throughput screening (HTS) based on fluorescence lifetime (FLT) readouts of FRET in live cells. This breakthrough is enabled by a combination of two complementary and synergistic technologies: fluorescent biosensor engineering (University of Minnesota, UMN) and fluorescence instrumentation engineering (Fluorescence Innovations, FI). In Phase I we achieved our aims, producing the first truly high-throughput and high-precision applications of FLT in living cells, applied to a specific biosensor (SERCA). We also surpassed our aims by developing new instrumentation for high-throughput spectral recording, carrying out a SERCA screen on a 50,000-compund library, and developing FRET biosensors based on several new targets. In addition, several pharmaceutical companies expressed interest in this technology platform, so UMN and FI started a new drug-discovery company, Photonic Pharma, for the ultimate commercialization of these combined technologies. In Phase II, we will further develop this technology platform and apply it to a diverse array of targets, thus clearly demonstrating the high potential for commercialization. In In Aim 1 we will seek improvements in hardware and software that will enhance future commercialization potential, including a new instrument that combines FLT and spectral recording, increased digitizer speed, higher density plates, connectivity with robotics, and software improvements. In Aim 2, in order to demonstrate that our technology platform is widely applicable, we will expand our list of biosensors, focusing on targets of high commercial potential, for which world-leading experts are in close proximity at UMN. These disease targets include heart failure, drug abuse and addiction, inflammation and cancer, and muscle dystonia. Biosensors will be engineered and expressed in live cells, and screened against standard test and validation libraries to optimize the screening assay. We will then carry out several large-scale (50,000-compound) screens, using optimized HTS assays. Hit compounds will be retested as a function of compound concentration (dose-response), and then subjected to functional assays performed by the above world-class experts at UMN. Promising hits may become leads for future medicinal chemistry development, but the primary goal is to validate the discovery platform. We are confident that the research conducted under these aims will set us up nicely for the commercialization phase by validating our business plan to combine UMN expertise in biosensor engineering and cell culture with FI expertise in instrumentation, led by Photonic Pharma, an emerging early-phase drug discovery start-up company. Thus our business plan is not to sell instruments, but to leverage our unique combination of biosensor and instrumentation expertise to develop and apply a technology platform that accelerates the early phase of drug discovery.