Carmaphycin B-Based Proteasome Inhibitors to Treat Human African Trypanosomiasis: Structure-Activity Relationship and In Vivo Efficacy

Abstract

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

Keywords: African trypanosomiasis; carmaphycin B; marine natural product; neglected tropical disease; proteasome inhibitor.

Figure 1

CPB analogs decrease T. brucei growth and engage the Tb20S β5 subunit. (A) Concentration response curves for three top-performing CPB analogs against T. brucei: the assay was performed once in triplicate and means ± SD values are shown. (B) Plot of log T. brucei EC₅₀ vs log HepG2 CC₅₀. Lines indicate SI cut-offs. The star represents the CPB progenitor compound, the triangles represent those compounds with a d-configuration at P3, and the blue, purple and green symbols represent J-71, J-77 and J-80, respectively. (C) Binding-competition assay in T. brucei lysates with CPB analogs using the fluorescent activity-based probe, Me₄BodipyFL-Ahx₃Leu₃VS.

Figure 2

Covalent and noncovalent docking simulations for P3 D- and L- inhibitor enantiomers in the β5 pocket of Tb20S and c20S. Predicted covalent docking poses of J-80 (A), J-79 (B), J-78 (C) and J-77 (D) using Glide XP in the modeled β5 pocket (gray) of Tb20S and c20S. All four inhibitors are predicted to engage Tb20S β5 with at least six interactions, i.e., eight for J-80, seven for J-77, and six for both J-79 and J-78. With c20S β5, J-78 and J-80 each have four interactions, whereas J-77 and J-79 have six interactions. Noncovalent docking simulations using two different scoring algorithms to predict binding affinity (kcal/mol) and GOLD score (CHEMPLP fitness) are also shown: higher values are colored in blue, with lower values in red. Hydrogen atoms are shown for clarity. Potential ligand interactions with residues are dashed in yellow. Images were generated with PyMOL (v2.5.7).

Figure 3

Effect of CPB analogs on blood parasitemia. Mice (n = 3) were infected with 100,000 T. brucei on day 0 and treated i.p. with the named compounds, at the doses indicated, 2 d.p.i. Parasitemia was measured as described in the main text: the limit of detection was 2.5 × 10⁵ parasites/mL blood, and data are represented as means ± SD. The Student’s paired t test was used for intragroup data, i.e., between 2 and 3 d.p.i., whereas the Student’s unpaired t test was used for intergroup data (vs vehicle control) 3 d.p.i. *p < 0.05, ***p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant.

Figure 4

J-80 extends the survival of T. brucei-infected mice. (A) Parasitemia was measured after i.p. administration of 50 mg/kg J-80 twice daily for 5 days (n = 5), or vehicle control once on day 2 d.p.i. (n = 3). Not shown for clarity is the parasitemia subsequent to the i.p. administration of 4 mg/kg pentamidine once on day 2 d.p.i (n = 5). Red triangles represent the first and last dosing days for J-80. (B) Kaplan–Meier survival curves for infected mice treated as indicated.