Development of a Malarial Kinase-on-Phage Screening Platform

描述(由申请人提供)：新的恶性疟原虫激酶靶标的鉴定受到阻碍，因为已经纯化的激酶数量有限，并且缺乏可用的高通量分析来筛选潜在的候选药物。我们的长期目标是开发一个高通量的生化筛选平台，从伴随的平行合成文库中分析新型激酶抑制剂的选择性。这项应用的总体目标是开发一种展示恶性疟原虫T7噬菌体蛋白的模型平台和一种可用于高通量筛选的可修改的生物层干涉测定法(BLI)。这一应用的中心假设是，可以开发展示恶性疟原虫激酶的噬菌体来筛选可能的抗疟疾恶性疟原虫激酶抑制剂。我们计划验证我们的中心假设，并通过追求以下两个具体目标来实现这一应用的目标：(1)开发恶性疟原虫蛋白激酶的噬菌体展示平台；(2)开发一种可高通量修改的生化分析方法，能够直接测定激酶抑制剂的结合亲和力。开发可高通量修正的BLI筛选试验的基本原理是，与寄生虫增殖试验相结合，它将允许鉴定基本的疟疾激酶，并为平行合成药库提供筛选平台。拟议工作的预期结果是，将开发必要的使能技术，以建立使用噬菌体和BLI的疟疾激酶筛查平台。与抗疟疾分析相结合，将有可能确定关键的疟疾激酶作为新的药物靶点。此外，相伴的平行合成的假定的抗疟疾激酶抑制剂文库可以在以后筛选特定恶性疟原虫激酶的特异性。

DESCRIPTION (provided by applicant): The identification of new Plasmodium falciparum kinase targets have been hampered due to the limited numbers of kinases that have been purified and the lack of an available high-throughput assay to screen potential drug candidates. Our long-term goal is to develop a high-throughput biochemical screening platform to profile the selectivity of novel kinase inhibitors from companion parallel-synthetic libraries. The overall objective of this application is to develop both a model P. falciparum kinase-displaying T7 phage platform and a Bio-Layer Interferometry (BLI) assay amendable for high-throughput screening. The central hypothesis of this application is that P. falciparum kinase-displaying phage can be developed to screen putative anti-malarial P. falciparum kinase inhibitors. We plan to test our central hypothesis and accomplish the objective of this application by pursuing the following two specific aims: (1) Develop a phage display platform for P. falciparum kinases; and (2) Develop a biochemical assay amendable to high-throughput capable of direct determination of binding affinity of kinase inhibitors. The rationale for developing a BLI screening assay amendable for high- throughput is that in combination with parasitic proliferation assays it will allow for both the identification of essential malarial kinases and provide a screening platform for parallel-synthetic drug libraries. The expected outcome of the proposed work is that the necessary enabling technology will be developed for establishing a malarial kinase screening platform using phage and BLI. In combination with antimalarial assays it will then become possible to identify key malarial kinases as new drug targets. In addition, companion parallel-synthetic libraries of putative antimalarial kinase inhibitors can be later screened for specificity to particular P. falciparum kinases.