Carmaphycin\nB‑Based Proteasome Inhibitors to\nTreat Human African Trypanosomiasis: Structure–Activity Relationship\nand <i>In Vivo</i> Efficacy

The proteasome is essential for eukaryotic cell proteostasis,\nand\ninhibitors of the 20S proteasome are progressing preclinically and\nclinically as antiparasitics. We screenedTrypanosoma\nbrucei, the causative agent of human and animal African\ntrypanosomiasis, <i>in vitro</i> with a set of 27 carmaphycin\nB analogs, irreversible epoxyketone inhibitors that were originally\ndeveloped to inhibit thePlasmodium falciparum20S (Pf20S). The structure–activity relationship was distinct\nfrom that of the human c20S antitarget by the acceptance of d-amino acids at the P3 position of the peptidyl backbone to yield\ncompounds with greatly decreased toxicity to human cells. For the\nthree most selective compounds, binding to the Tb20S β5 catalytic\nsubunit was confirmed by competition with a fluorescent activity-based\nprobe. For one compound, <b>J-80</b>, with its P3 d-configuration, the differential binding to the parasite’s\nβ5 subunit was supported by both covalent and noncovalent docking\nanalysis. Further, <b>J-80</b> was equipotent against both Trypanosoma brucei gambiense and Trypanosoma\nbrucei rhodesiense <i>in vitro</i>. In a\nmouse model of Stage 1 T. brucei infection,\na single intraperitoneal (i.p.) dose of 40 mg/kg <b>J-80</b> halted the growth of the parasite, and when given at 50 mg/kg i.p.\ntwice daily for 5 days, parasitemia was decreased to below the detectable\nlimit, with parasite recrudescence 48 h after the last dose. The <i>in vivo</i> proof of principle demonstrated by a potent, selective,\nand irreversible inhibitor of Tb20S reveals an alternative path to\nthe development of kinetoplastid proteasome inhibitors that differs\nfrom the current focus on allosteric reversible inhibitors.