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

University Of Virginia, Charlottesville VA

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Abstract

[unreadable] 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. [unreadable] [unreadable] [unreadable]