Blocking Plasmodium falciparum Development via Dual Inhibition of Hemoglobin Degradation and the Ubiquitin Proteasome System by MG132

Abstract

Among key potential drug target proteolytic systems in the malaria parasite Plasmodium falciparum are falcipains, a family of hemoglobin-degrading cysteine proteases, and the ubiquitin proteasomal system (UPS), which has fundamental importance in cellular protein turnover. Inhibition of falcipains blocks parasite development, primarily due to inhibition of hemoglobin degradation that serves as a source of amino acids for parasite growth. Falcipains prefer P2 leucine in substrates and peptides, and their peptidyl inhibitors with leucine at the P2 position show potent antimalarial activity. The peptidyl inhibitor MG132 (Z-Leu-Leu-Leu-CHO) is a widely used proteasome inhibitor, which also has P2 leucine, and has also been shown to inhibit parasite development. However, the antimalarial targets of MG132 are unclear. We investigated whether MG132 blocks malaria parasite development by inhibiting hemoglobin degradation and/or by targeting the UPS. P. falciparum was cultured with inhibitors of the UPS (MG132, epoxomicin, and lactacystin) or falcipains (E64), and parasites were assessed for morphologies, extent of hemoglobin degradation, and accumulation of ubiquitinated proteins. MG132, like E64 and unlike epoxomicin or lactacystin, blocked parasite development, with enlargement of the food vacuole and accumulation of undegraded hemoglobin, indicating inhibition of hemoglobin degradation by MG132, most likely due to inhibition of hemoglobin-degrading falcipain cysteine proteases. Parasites cultured with epoxomicin or MG132 accumulated ubiquitinated proteins to a significantly greater extent than untreated or E64-treated parasites, indicating that MG132 inhibits the parasite UPS as well. Consistent with these findings, MG132 inhibited both cysteine protease and UPS activities present in soluble parasite extracts, and it strongly inhibited recombinant falcipains. MG132 was highly selective for inhibition of P. falciparum (IC50 0.0476 µM) compared to human peripheral blood mononuclear cells (IC50 10.8 µM). Thus, MG132 inhibits two distinct proteolytic systems in P. falciparum, and it may serve as a lead molecule for development of dual-target inhibitors of malaria parasites.

Targeting protein translation, RNA splicing, and degradation by morpholino-based conjugates in Plasmodium falciparum

“…PfCRT-VMO treatment resulted in deformed parasites with large digestive vacuoles ( Fig. 2A), a phenotype reminiscent of the effect of cysteine protease inhibitors (38). Using transgenic parasites expressing a luciferase reporter to monitor parasite development, PfPMT-VMO and PfCRT-VMO were effective in reducing luciferase activity by two-to three-fold compared with Ctrl-VMO after one or two cycles of treatment (Fig.…”

Protein Degradation Systems as Antimalarial Therapeutic Targets

“…Proteasome inhibitor-treated P. berghei sporozoites fail to establish a liver stage infection and cannot develop into exoerythrocytic forms in hepatocytes both in vitro and in vivo [9]. Moreover, asexual blood stage parasite growth is potently inhibited when exposed to proteasome inhibitors at the ring, trophozoite, or schizont stages [9–11, 16, 30]. Proteasome inhibition can also block parasite transmission by inhibiting stage V gametocyte development [13] and can prevent development in the mosquito by interfering with oocyst formation in the midgut [9].…”

Synthesis of novel benzimidazole acrylonitriles for inhibition of Plasmodium falciparum growth by dual target inhibition

“…Benzimidazole based compounds have been reported to exhibit FP‐2 inhibitory activity. On the basis of the core pharmacophoric features of these benzimidazole derivatives, novel benzimidazole acrylonitriles derivatives have been designed and synthesized. In this paper, we report synthesis of the designed compounds as inhibitors of falcipain‐2 enzyme and hemozoin formation which could be developed as potential antimalarial drugs.…”