An Optimized Route to the Syringolin Natural Products Enables Combinatorial Synthesis of Selective, Bioactive Inhibitors of the Plasmodium falciparum 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.