Proteasomal degradation typically requires post-translational modification of target proteins with K48-linked polyubiquitin chains. This process of protein proteolysis plays a key role in normal cellular function. The E3 ubiquitin ligase, Siah-1, facilitates the transfer of ubiquitin to its substrate proteins destined for degradation by way of its RING domain. Siah-1 is a member of a family of highly conserved RING domain proteins, which regulate a variety of cellular functions, including cell cycle arrest, tumor suppression, and apoptosis through the -catenin degradation pathway. Siah-1 has also been identified as a p53-inducible gene, functionally linking it to an important tumor suppressor. Chemical modulators of the Siah-1 pathway would provide powerful research tools for elucidating the roles of this signaling pathway in cancer development and progression. In this proposal, we describe the development of a High Throughput Screening (HTS) assay based upon fluorescence polarization, and utilizing a peptide ligand of Siah-family proteins with an attached flurochrome. This fluorescence polarization assay (FPA) forms the basis for a high-throughput competitive displacement assay that we have optimized for chemical library screening. We propose to screen the NIH compound library using this HTS assay. Additional downstream assays provided by the assay provider will be performed for deconvoluting hits. We expect to obtain candidate compounds for Structure Activity Relationship (SAR) studies to be performed for a prototypical RING- containing protein, Siah-1. Together, these efforts will result in validated chemical probes for studying the biology of Siah-family E3 ligases in a variety of biological settings.
Our goal is to identify chemicals capable of modulating the Siah-1 proteasomal degradation pathway. Through its activity as an ubiquitin ligase, this signaling pathway helps to regulate a variety of cellular events, including cell cycle arrests, tumor suppression, and apoptosis. Chemical modulators of the Siah-1 pathway will provide powerful research tools for studying pathways involved in human cancer development, progression, and resistance to therapy.