Development of Yeast-Based Assays for anti-influenza drug discovery

描述（由申请方提供）：基于酵母的生长试验将用于建立新型抗流感治疗药物的高通量筛选（HTS）程序。HTS的靶标将是甲型流感NS 1和M2蛋白以及细胞p58蛋白。当在S.啤酒。因此，阻断酵母中这些蛋白质功能的化合物有望恢复生长。以前我们已经证明腺病毒E1 A蛋白在酵母中是有毒的，并且有可能筛选出逆转这种效应的小分子。我们已经建立了携带PDR 1和PDR 3基因缺失的酵母，这些基因控制药物外排。PDR 1/PDR 3双突变体有效地保留了小分子，并允许筛选许多不同的化合物。NS 1和M2的HTS开发将分三个阶段进行。首先，将优化NS 1和M2在pdr 1/pdr 3遗传背景中的表达，以便可以观察到稳健的生长抑制表型。这将使用多种重组表达载体和培养基条件来实现。其次，将开发微量滴定板测定法，以明确区分生长抑制和生长恢复。第三，微量滴定板测定将使用弗吉尼亚大学医学院的功能高通量robitcs站自动化。还将建立涉及细胞p58蛋白的额外HTS程序。p58在流感感染期间释放并结合蛋白激酶R（PKR），从而防止PKR下调病毒蛋白质合成。在酵母中，PKR是高毒性的，并且p58的共表达可以完全阻断这种毒性，导致正常生长。因此，干扰p58与PKR结合的化合物将导致PKR诱导的毒性。预期此类化合物将在病毒感染的细胞中从p58释放PKR，从而抑制病毒复制。再次使用我们的pdr 1/pdr 3遗传背景，我们将建立PKR诱导的毒性和PKR的p58抑制的表达条件。该系统将用于建立微量滴定板测定和机器人筛选。

DESCRIPTION (provided by applicant): Yeast-based growth assays will be used to establish high-throughput screening (HTS) procedures for novel anti-influenza therapeutics. The targets for HTS will be the influenza A NS1 and M2 proteins, and the cellular p58 protein. NS1 and M2 have been shown to induce pronounced growth inhibition ("toxicity") when expressed in S. cerevisiae. Therefore chemical compounds that block the function of these proteins in yeast are expected to restore growth. Previously we have shown that the adenovirus E1A protein is toxic in yeast and that it is possible to screen for small molecules that reverse this effect. We have established yeast carrying deletions of the PDR1 and PDR3 genes, which control drug efflux. pdr1/pdr3 double mutants retain small molecules efficiently and allow screening of numerous diverse compounds. The development of the HTS for NS1 and M2 will involve three phases. First, expression of NS1 and M2 in the pdr1/pdr3 genetic background will be optimized such that a robust growth-inhibitory phenotype can be observed. This will be achieved using a variety of recombinant expression vectors and media conditions. Second, microtiter plate assays will be developed that allow clear differentiation between growth inhibition and growth restoration. Third, the microtiter plate assay will be automated using a functioning high-throughput robitcs station at the University of Virginia School of Medicine. An additional HTS procedure involving the cellular p58 protein will also be established. p58 is released during influenza infection and binds to protein kinase R (PKR), thereby preventing PKR from down regulating viral protein synthesis. In yeast, PKR is highly toxic and co-expression of p58 can completely block this toxicity, resulting in normal growth. Therefore compounds that interfere with p58 binding to PKR will result in PKR-induced toxicity. Such compounds would be expected to release PKR from p58 in virus-infected cells, thereby inhibiting virus replication. Again using our pdr1/pdr3 genetic background we will establish expression conditions for PKR-induced toxicity and p58 inhibition of PKR. This system will be used to establish microtiter plate assays and robotics screening.