High Throughput Screening of Peptide Pharmaceuticals

描述（由申请人提供）：尽管最近完成了人类基因组计划，但表面上更困难的后基因组挑战将是所有人类基因的功能注释，并将这些信息整合到合理的药物发现过程中。不幸的是，目前这是具有挑战性的，主要是由于合适的工具集的选择有限，无法快速描述潜在的药物靶点和大规模筛选新药。噬菌体展示是一种独特的工具，用于发现肽配体，设计肽模拟物，以及特定靶标的生物学验证。尽管取得了这些成功，但噬菌体展示技术在疾病相关细胞模型中用于生物活性肽的高通量筛选（HTS）的应用迄今尚未发展起来。为了解决这些问题，我们将开发一个新的实验平台，用于基于细胞的肽药物HTS，作为合成肽或化学小分子文库HTS的替代方法。当前资助申请的长期目标是开发和商业化下一代HTS技术，利用噬菌体展示库同时筛选数百万种肽化合物的生物活性，并与广泛的报告细胞系兼容这种HTS技术的肽导联。基于细胞的HTS技术简单，成本效益高，汇集了噬菌体文库，展示了数百万种肽化合物，远远超过了小分子文库的复杂性，将改变当前的药物发现策略并加速新疗法的发展。我们建议利用我们的新型生物活性肽HTS资源来描述癌细胞中不受控制的增殖过程，并应用该技术来鉴定癌细胞特异性的细胞毒性肽。在第一阶段的资助下，我们最初计划开发一个汇集的噬菌体文库，展示500种药物相关的细胞因子、生长因子、趋化因子和激素的肽，以及在基于细胞的实验中展示生物活性噬菌体肽的HTS技术。随后，我们将与克利夫兰临床基金会的生物学顾问合作，在转录报告细胞系模型中验证该文库，以鉴定NF-？B信号通路。噬菌体展示技术与基于细胞的HTS技术的结合，将使噬菌体展示技术成为鉴定和验证新型生物相关药物的有力工具。此外，我们设想对人类疾病机制的分子解剖产生重大影响。例如，这些试剂在确定治疗干预的新靶点和开发越来越相关的药物发现范例方面具有相当大的前景。因此，我们预计这些工具集将显著提高先导肽化合物HTS的效率、经济性和易用性，并将为学术和工业研究人员提供现有小分子文库的首选、成本效益高的替代方案。该项目的最终目标是开发并商业化新的、强大的药物发现工具：一种基于细胞的高通量筛选多肽药物的技术，该技术使用一套全基因组受体配体噬菌体展示文库和转录报告细胞系。我们建议验证并应用这些工具来发现NFkB信号通路的肽激动剂和拮抗剂。所开发的高通量筛选工具和技术将显著提高与多种人类疾病机制的分子解剖、新药开发相关的转化研究效率，因此对改进药物发现研究具有重要意义。

DESCRIPTION (provided by applicant): Despite the recent completion of the human genome project, an ostensibly more difficult post-genomic challenge will be the functional annotation of all human genes and integration of this information into a rational drug discovery process. Unfortunately, this is at present challenging, primarily due to the limited choice of suitable toolsets to rapidly delineate potential drug targets and screen new drugs en masse. Phage display is a unique tool for the discovery of peptide ligands, design of peptidomimetics, and biological validation of specific targets. Despite these successes, application of phage display technology for High Throughput Screening (HTS) of biologically active peptides in a disease-relevant cell model has not been developed so far. To address these issues, we will develop a novel experimental platform for cell-based HTS of peptide drugs as an alternative to HTS of synthetic peptide or chemical small molecule libraries. The long-term goal of the current grant application is the development and commercialization of next- generation HTS technology for the simultaneous screening of millions of peptide compounds for biological activity with pooled phage display libraries, concomitantly with a wide range of reporter cell lines compatible with this HTS technology of peptide leads. The technologically simple and cost-effective approach of cell- based HTS with pooled phage libraries displaying millions of peptide compounds, which far exceeds the complexity of small molecule libraries, will change current drug discovery strategies and accelerate the development of novel therapeutics. We propose to use our novel bioactive peptide HTS resource to delineate the processes which underlie deregulated proliferation in cancer cells, and to apply this technology to identify cytotoxic peptides specific for cancer cells. Under Phase I funding, we initially propose to develop a pooled phage library displaying peptides for 500 pharmaceutically relevant cytokines, growth factors, chemokines, and hormones, as well as technology for HTS of biologically active phage displayed peptides in a cell-based assay. In collaboration with our biology consultants at the Cleveland Clinical Foundation, we will subsequently validate this library in transcriptional reporter cell line models, to identify effector peptides of NF-?B signaling pathway. The combination of phage display technology with cell-based HTS will transform phage display technology into a powerful tool for identification and validation of novel biologically relevant drugs. Moreover, we envisage a major impact on the molecular dissection of human disease mechanisms. For example, these reagents harbor considerable promise to identify new targets for therapeutic intervention, and the development of increasingly relevant paradigms for drug discovery. As a result, we foresee that these toolsets will significantly improve the efficiency, economy, and ease of performing HTS of lead peptide compounds, and will provide both academic and industrial researchers with preferred, cost-effective alternatives to existing small molecule libraries. The ultimate goal of the proposed project is to develop and make commercially available new, powerful drug discovery tools: a technology for high throughput screening of peptide pharmaceuticals in a cell-based assay with a set of genome-wide receptor ligand phage display libraries and transcriptional reporter cell lines. We propose to validate and apply these tools for discovery peptide agonists and antagonists of NFkB signaling pathway. The developed high throughput screening tools and technologies will significantly improve the efficiency of translational research related to molecular dissection of diverse human disease mechanisms, development of new pharmaceuticals, and therefore, have major implications for improving drug discovery research.