Figure 5.

Covalent irreversible proteasome inhibitor 8304-vs is a potent, selective, and stable inhibitor of the P. falciparum proteasome. (A) Structures of pyrazole capped inhibitors with varying P3 or P1 groups and 8304-vs, a covalent version of a previously described noncovalent macrocyclic inhibitor of the proteasome. (B) Compound mean ± SEM EC₅₀ values for each inhibitor assayed against P. falciparum asexual blood stage W2 parasites. Data were generated using 72 h dose–response assays (N, n = 2,2). (C) EC₅₀ values against W2 parasites and cellular indexes comparing P. falciparum parasites to mammalian HFF cells (listed as the ratio of HFF EC₅₀ to parasite EC₅₀ values). (D) Potencies (IC₅₀) of compounds assayed either the β2 or the β5 subunits of the purified Plasmodium or human 20s proteasomes in 1 h inhibition assays. Biochemical selectivity was calculated as the ratio of potency for the two proteasomes (h20s/Pf20s). (E) Plot of rate of kill assay, in which compounds were dosed at their EC₅₀ and parasite viability was monitored over time and compared against known fast (chloroquine), medium (pyrimethamine), and slow (atovaquone) acting inhibitors of parasite growth ± SEM. N,n = 6,2. (F) Bar graphs depicting the half-life of compounds in either human (blue) or mouse (red) microsomes. Compound metabolism was measured by LC/MS/MS. *TDI-8304 data were previously reported.¹⁷