An Optimized Route to the Syringolin Natural Products Enables Combinatorial Synthesis of Selective, Bioactive Inhibitors of the <i> <i>Plasmodium falciparum</i> </i> 20S Proteasome

The syringolin natural products are covalent inhibitors of the 20S proteasome that inspire therapeutic development. Here, we report a new route to the syringolins amenable to solution and solid-phase synthesis that overcomes a problematic macrocyclization. Exploiting our synthetic approach and substrate mimicry models for proteasome inhibition by the syringolins, we generated a collection of hypothetically selective inhibitors of the Plasmodium falciparum proteasome, which is an emerging target for antimalarial drugs. We identified compounds from the library having high second-order rate constants for Plasmodium proteasome inhibition and nanomolar antiparasitic activity. They exhibited selectivity for the Plasmodium proteasome over the human proteasome. We solved cryo-EM structures of an inhibitor bound to both 20S proteasomes, revealing key contacts favoring species-selective inhibition. Together, this work provides an improved route to syringolin analogs, sheds new light on substrate mimicry by the syringolins, and provides a structural basis for the pursuit of new antimalarial drugs.