Time-Gated Luminescence Detection for High-Throughput Screening of Protein Interaction and Inhibitions

High throughput screening (HTS) assays are a key component of the drug discovery processes. They are designed to identify biologically active molecules that can ultimately be used as therapeutic agents. Such assays use the power of automation to test thousands of molecules per day, and they require high reproducibility and robustness to be reliable. Current HTS technologies often employ fluorescence-based detection methods as a means of identifying potentially useful compounds. However, often high levels of non-specific fluorescence background emanating from biological assay components and, especially, library compounds can reduce sensitivity and lead to unacceptable rates of false positive results. The use of time-gated detection in combination with organic complexes of lanthanides, particularly Tb3+ and Eu3+, that have millisecond-scale excited-state lifetimes as luminescent reporters alleviates this problem. By implementing a delay of 10 microseconds or more between sample excitation and detection, nanosecond-scale fluorescence background is eliminated, resulting in HTS assays with very large signal-to-background ratios. Interactions between proteins are essential for cellular function. Many diseases are caused by mutations that disrupt or otherwise give rise to aberrant protein-protein interactions (PPIs). While enzyme active sites and cell surface receptors have been the most common drug discovery targets, there is increasing interest in developing small molecule therapeutics that can disrupt PPIs. One of the key challenges in targeting PPIs with small molecules is that the interface between two interacting proteins is a relatively large, flat surface, as opposed to a distinct binding pocket. Moreover, there is often no structural information available about PPI interfaces, and this makes rational design of potential inhibitors difficult or impossible. For this reason, HTS has proven to be an important tool in the discovery process for PPI inhibitors that have been developed thus far, and it will continue to be play an important role in future efforts. The main objective of the studies described in this dissertation was to design HTS assays for PPI inhibition that can be readily adapted to almost any protein and that can be applied in purified biochemical preparations, cell lysates, or in living mammalian cells. The assay platform is based on the time-gated detection of Förster Resonance Energy Transfer between two overexpressed fusion proteins, one labeled with a luminescent Tb3+ complex and the other attached to a green fluorescent protein.