Development of CoraFluors: A Versatile, Target-agnostic Assay Technology Platform Optimized for Chemical Biology Research

Abstract

Biology uses strategic, coordinated proximity events to orchestrate complex cellular pathways essential to homeostasis, survival, and propagation. The interrogation of biological systems with chemogenomic approaches has led to a prolific era of research in the life sciences. Critical to these strategies are the availability of sensitive, quantitative, and flexible assay technologies that allow scientists to accurately measure binding events between biomolecules (or with their small molecule ligands), as well as their abundance – ideally in both biochemical and cellular contexts. Experimental designs based on time-resolved Förster resonance energy transfer (TR-FRET) are uniquely suited to study proximity at the biomolecular level, offering both high sensitivity and specificity. However, the paucity of synthetically accessible and biocompatible luminescent lanthanide complexes, which are essential reagents for TR-FRET-based approaches, and their poor cellular permeability have limited broader adaptation of TR-FRET beyond homogeneous and extracellular assay applications. In this Dissertation, I discuss the development of CoraFluors, a new class of synthetically tractable luminescent macrotricyclic terbium complexes optimized for TR-FRET-based chemogenomic applications. I apply CoraFluor technology in the development of novel, domain-selective assays for Keap1 (Kelch-like ECH-associated protein 1). Using cell-permeable CoraFluor derivatives, I also describe the adaptation of these reagents to enable isoform-selective, cell-free and live-cell ligand displacement assays for HDAC1 (histone deacetylase 1). In addition, I discuss a universal nanobody-based strategy that expands the toolbox of TR-FRET donor labeling approaches for a wide range of biochemical assays. Utilizing the novel CoraFluors, I develop a TR-FRET-based strategy for the quantification of target engagement and protein abundance of endogenous proteins of interest (POIs) in whole cell extracts. This methodology overcomes many inherent limitations of existing assay technologies such as Western blot, ELISA (enzyme-linked immunosorbent assay), and luminescence-based platforms. Importantly, this strategy is compatible with unmodified cell lines expressing native POIs. My approach enabled the quantitative characterization of the natural product celastrol, a p-quinone methide-containing pentacyclic triterpenoid, as a broad cysteine-targeting E3 ubiquitin ligase warhead for targeted protein degradation applications. Lastly, I develop a TR-FRET-based approach to resolve the often-deceptive isoform and complex selectivity of HDAC inhibitors. My findings show that widely used probes can display misleading activity in static biochemical assays, and I provide evidence that dynamic processes govern the isoform and complex selectivity of these ligands in cellular contexts. These data reconcile the previous apparent disconnect between biochemical inhibitory profiles and corresponding cellular activities of HDAC inhibitors and urge a comprehensive reinterpretation of studies that have used these chemical probes to interrogate epigenetic and other cellular processes. Overall, my research establishes CoraFluor TR-FRET technology as a target-agnostic, flexible, sensitive, and quantitative assay platform for a wide array of chemical biology approaches and has advanced our mechanistic understanding of epigenetic gene regulatory complexes.