The discovery of new drugs is a complex and long-term process that has made significant progress over time. It has evolved from serendipitous discoveries, often of natural products, to modern, more systematic approaches based on chemical library screening or rational design. In recent decades, fragment-based drug design has emerged as a successful approach and an alternative to high-throughput screening. It starts with screening small, structurally simple compounds as potential starting points for optimization into lead compounds.
In this dissertation, we investigated the potential of fragment-based drug design for the discovery of promising hit compounds for targets under investigation by our faculty. We assembled a library of over 1,000 fragments and evaluated their activity using various biochemical assays. We have developed a workflow for thorough hit evaluation to rule out false positives from screenings. This includes confirmation of purity and identity, as well as experimental evaluation of aqueous solubility, reactivity, and redox activity. For most promising hits, we confirmed activity or target binding using an orthogonal method. Previously known assays for the determination of redox activity did not have sufficient capacity or were not suitable for testing diverse chemical libraries. Using an expanded set of positive controls, we optimized them and established protocols to detect various mechanisms of redox activity. We demonstrated the broad applicability of reactivity and redox activity assays, particularly for the evaluation of hit compounds, known bioactive compounds and chemical probes, in the development of targeted covalent fragments and compounds with electrophilic warheads. The most promising results in fragment library screening were obtained with the bacterial enzyme D-alanine:D-alanine ligase and the β5i subunit of the immunoproteasome. In addition, we investigated alternative approaches to fragment screening, including virtual screening and the use of DNA-encoded chemical libraries. In all cases, hit evaluation was a bottleneck and a critical step for further success. A broader analysis of fragment screening results on different targets revealed that it is important to pay attention to compounds with a thiazole scaffold, which can often exhibit nonspecific activity due to their reactivity.
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