Modular Synthesis and Biological Evaluation of Structurally-Diverse Reactive Fragment Libraries

The development of small-molecule covalent inhibitors of protein targets has gained increasing interest in drug discovery within both academic and industrial settings. Many FDA-approved covalent pharmaceuticals are now available on the market. Covalent drug discovery has often focused on introducing mild electrophiles onto known and well-characterised inhibitors. However, in recent years, high-throughput reactive fragment screening has gained traction as a strategy to initiate the discovery of novel protein inhibitors. These workflows typically rely on commercially available fragments with pre-installed reactive warheads or the incorporation of electrophiles onto existing scaffolds via simple medicinal chemistry transformations. However, this approach limits the exploration of biologically relevant chemical space. Therefore, new synthetic strategies are required to develop reactive fragment libraries with more diverse three dimensional structures, which can serve as chemical tools to explore biological systems or as starting points for novel covalent pharmaceutical discovery.

The work presented herein begins with an introduction (Chapter 1) giving a brief overview of the covalent drug discovery process, followed by examples of reactive electrophilic warheads that have been used to target various amino acid residues in biological systems. This is followed by case studies employing covalent fragments to target proteins or pathogens of interest using high-throughput discovery platforms. Techniques used to validate the biological targets of covalent fragments will also be discussed, with a focus on modern chemical proteomics and mass spectrometry methods.

Subsequent results and discussion Chapters cover the successful implementation of highthroughput experimentation and Direct-to-Biology screening workflows to discover selective modifiers of protein targets (Chapter 2). To access compounds with more diverse structures, rhodium-catalysed chemistries are explored in Chapter 3 to enable the synthesis of reactive fragment sets. The products from successful reactions were then tested against a kinase and HeLa cell proteomes. Overall, this work establishes two complementary approaches, the high-throughput Direct-to-Biology discovery workflows and parallel rhodium-catalysed synthetic strategies. These have been used successfully to expand the chemical diversity of fragments to generate structurally diverse reactive fragment libraries, providing valuable tools and starting points for the discovery of novel covalent inhibitors.