Shortcutting Lead Discovery via Combinatorial Methacrylamide-Based Covalent Probes

Interest in covalent probes is increasing due to their potency, selectivity, and duration of action. However, their discovery remains challenging. Furthermore, the ensemble of possible molecules relevant to drug discovery is enormous. A recent discovery in our lab of a new electrophile class, the α-substituted methacrylamides, has inspired us to explore the potential of combinatorial development of methacrylamide-based probes, which enables us to sample a space size of N2 methacrylamides with only 2N compounds. For that purpose, we performed amide coupling and substitution reactions with the same amine building-blocks library on a methacrylic acid and a 2-bromomethyl acrylamide (correspondingly). Performing the reactions in a nanomole-scale high-throughput synthesis manner and screening with a direct-to-biology approach have advantages such as saving time and reagents, but they come with their own unique set of challenges. Eventually, we successfully afforded a combinatorial binder for Keap1, however, it had reduced potency compared to its fragments. We assume that the binding of the fragments is mutually exclusive, which is unsuitable for screening combinatorically, making some proteins inappropriate for this method.