Assay Development and Screening
Applications of CRISPR Technology in Drug Discovery
Cellular protein thermal stability provides a powerful method for assessing compound target engagement. Recently, there have been several high-throughput 384 well assay compatible detection formats published for cellular protein thermal stability using commercial luminescence complementation systems as well as homogenous antibody sandwich AlphaLISA assays. Similarly, we have utilized these detection formats and successfully developed and employed high-throughput format assays for a large number of diverse drug discovery projects ongoing at the SBP Prebys Center. In addition to serving the primary goal of assessing the target engagement, these efforts have provided in-depth knowledge of diverse detection systems, such as Promega HiBiT, DiscoveRx ePL, AlphaLISA and the classical approach relying on SDS-PAGE-Western Blot detection. In addition to monitoring thermal stability of intracellular proteins, we successfully employed these same detection approaches to assess small-molecule effects on steady-state protein level or localization in the cell. Not only do these approaches enable reporting on direct target binding, they also monitor the effects of small molecules on the protein target interactome governing homeostasis. This enables the identification of druggable partners from the entire target interaction network. Along with small molecule screening, all these methods provide unique and powerful tools to study targets of interest in their native state which exposes valuable information about the effects of cellular background and extracellular environment. From these studies, we have gleaned insight into the cellular and context specific target regulation and potential biological relevance of identified hits. We have successfully utilized both exogenous expression and CRISPR knock-in of detection tracers or AlphaLISA detection to assess protein stability of endogenous proteins learning advantages and disadvantages of each approach. Side-by-side assessment of these approaches helped to develop a decision tree for selecting of the most appropriate approach for each new project and target.