Inhibition of fumarate reductase in Leishmania major and L. donovani by chalcones

Abstract

Our previous studies have shown that chalcones exhibit potent antileishmanial and antimalarial activities in vitro and in vivo. Preliminary studies showed that these compounds destroyed the ultrastructure of Leishmania parasite mitochondria and inhibited the respiration and the activity of mitochondrial dehydrogenases of Leishmania parasites. The present study was designed to further investigate the mechanism of action of chalcones, focusing on the parasite respiratory chain. The data show that licochalcone A inhibited the activity of fumarate reductase (FRD) in the permeabilized Leishmania major promastigote and in the parasite mitochondria, and it also inhibited solubilized FRD and a purified FRD from L. donovani. Two other chalcones, 2,4-dimethoxy-4'-allyloxychalcone (24m4ac) and 2,4-dimethoxy-4'-butoxychalcone (24mbc), also exhibited inhibitory effects on the activity of solubilized FRD in L. major promastigotes. Although licochalcone A inhibited the activities of succinate dehydrogenase (SDH), NADH dehydrogenase (NDH), and succinate- and NADH-cytochrome c reductases in the parasite mitochondria, the 50% inhibitory concentrations (IC(50)) of licochalcone A for these enzymes were at least 20 times higher than that for FRD. The IC(50) of licochalcone A for SDH and NDH in human peripheral blood mononuclear cells were at least 70 times higher than that for FRD. These findings indicate that FRD, one of the enzymes of the parasite respiratory chain, might be the specific target for the chalcones tested. Since FRD exists in the Leishmania parasite and does not exist in mammalian cells, it could be an excellent target for antiprotozoal drugs.

FIG. 1

Effects of licochalcone A on the activities of FRD and SDH in permeabilized L. major promastigotes. Assays were carried out as described in Materials and Methods. Data are means ± standard deviations from five different experiments.

FIG. 2

Effect of licochalcone A on the activity of FRD in the crude mitochondria of the parasite. Assays were carried out as described in Materials and Methods. Data are means ± standard deviations from five different experiments.

FIG. 3

Effects of 3-MPA alone, licochalcone A alone, and licochalcone A plus 3-MPA on the activity of FRD in the crude mitochondria of L. major promastigotes. The crude mitochondria were incubated with various chemicals at 28°C for 5 min. Data are means ± standard deviations from five different experiments.

FIG. 4

Effects of licochalcone A on the activities of SDH (A), NDH (B), SCC (C), and NCC (D) in the crude mitochondria of L. major promastigotes. Assays were carried out as described in Materials and Methods. Data are means ± standard deviations from five different experiments.

FIG. 5

Effects of licochalcone A on the activities of SDH (A) and NDH (B) in the crude mitochondria of PBMC. Assays were carried out as described in Materials and Methods. Data are means ± standard deviations from five different experiments.

FIG. 6

Effects of licochalcone A (A), 24m4ac (B), and 24m4bc (C) on the activity of soluble FRD in L. major promastigotes. Assays were carried out as described in Materials and Methods. Data are means ± standard deviations from six different experiments.

FIG. 7

Effect of licochalcone A on the activity of purified FRD in L. donovani promastigotes. Assays were carried out as described in Materials and Methods. Data are means ± standard deviations from six different experiments.