Targeting Human Ubiquitin Activating Enzyme UBE1 with Rationally Designed Copper-Based Inhibitors and the Application of a Fluorescent Chemosensor to the Development of an Assay for Adenylating Enzyme Activity.

摘要

Two projects were explored in this work. In the first, a previously developed, rationally-designed, potent, and selective cupric cyclen-based small molecule library of human ubiquitin activating enzyme 1 (UBE1) inhibitors was explored for its therapeutic potential in hematological malignancies. After significant difficulties reproducing inhibitor validation data, our further investigational findings suggest that the library's activity observed in western blot-based UBE1-UbcH6 E1-E2 transthiolation assays and a LifeSensors Fluorescence Resonance Energy Transfer (FRET)-based assay was simply the spurious result of excess unbound copper in the compound samples. This work also investigated the application of a dual-emission fluorescent chemosensor, ProxyPhos, to development of a robust high throughput assay for adenylating enzyme activity. With selective detection of pyrophosphate over ATP, ADP, and AMP in aqueous buffer, ProxyPhos was applied in buffer conditions suitable to adenylating enzymes to explore their effects on selectivity and signal.