Rheumatoid arthritis (RA) is associated with chronic inflammation and overgrowth of synovial cells, and factors involved in these processes are potential targets for discovery of therapeutic drugs. Hrd1, also known as synoviolin, is an E3 ubiquitin ligase enzyme recently found to be present at high levels in rheumatoid synovial cells from arthritis patients, and further in vivo studies in mice identified it as a novel causative factor in arthropathy. Inhibitors of HrdVs ubiquitin ligase activity, which causes ubiquitination and ultimately proteasomal degradation of certain proteins, would thus be expected to be excellent candidates for activity against RA or other inflammatory disorders. With the goal of identifying such inhibitors, this proposal describes the development of a yeast-based assay in which human Hrd1 and a fluorescent GFP-fused version of its substrate CD3-d are expressed in Saccharomyces cerevisiae. When these components are functional, human Hrd1, in collaboration with endogenous yeast ubiquitin-proteasomal pathway elements (E1, E2, proteasomes), keeps the level of CD3-d-GFP at a minimum, resulting in no fluorescent signal. Inhibition of Hrd1 allows the CD3-d-GFP fusion to remain intact, producing a positive signal (fluorescence). The assay will be set up following cloning of the necessary components and expressing them in yeast. After adjusting assay conditions to produce an acceptable signal:noise ratio and other parameters of high throughput screening, collections of natural product extracts and compounds will be run through the screen to identify confirmed hits, to serve as a basis for identifying suitable leads. The ultimate commercial goal of the proposed work is to discover a drug with efficacy as a therapy against RA or other autoimmune diseases. ? ?
