Aziridine-2,3-dicarboxylates, peptidomimetic cysteine protease inhibitors with antileishmanial activity

Abstract

Chemotherapy of leishmaniasis is mainly based on antimonials. However, they are extremely toxic and cause serious side effects, and there is a worldwide increasing frequency of chemoresistance to antimonials. These issues emphasize the urgent need for affordable alternative drugs against leishmaniasis. Leishmania cysteine proteases are essential for parasite growth, differentiation, pathogenicity, and virulence and are thus attractive targets for combating leishmaniasis. Herein we demonstrate that the cysteine protease inhibitors aziridine-2,3-dicarboxylates 13b and 13e impaired promastigote growth at mid-micromolar concentrations and decreased the infection rate of peritoneal macrophages at concentrations 8- to 13-fold lower than those needed to inhibit parasite replication. Simultaneous treatment of infected cells with compound 13b and gamma interferon resulted in an even further reduction of the concentration needed for a significant decrease in macrophage infection rate. Notably, treatment with the compounds alone modulated the cytokine secretion of infected macrophages, with increased levels of interleukin-12 and tumor necrosis factor alpha. Furthermore, the decreased infection rate in the presence of compound 13b correlated with increased nitric oxide production by macrophages. Importantly, at the concentrations used herein, compounds 13b and 13e were not toxic against fibroblasts, macrophages, or dendritic cells. Together, these results suggest that the aziridine-2,3-dicarboxylates 13b and 13e are potential antileishmanial lead compounds with low toxicity against host cells and selective antiparasitic effects.

FIG. 1.

(A) Schematic representation of the structures of the investigated aziridine-2,3-dicarboxylates: compounds 1 to 6 are aziridinyl dipeptides of the general structure X-Caa-Azi(OEt)₂ with Caa as cyclic amino acid, compound 7c is the aziridinyl dipeptide Cbz-Leu-Azi(OEt)₂, compounds 8 to 18 are aziridinyl tripeptides of the general structure Boc-Leu(or Gly)-Caa-Azi(OR)₂, and compound 19 is the aziridinyl tripeptide of the general structure Boc-Phe-Ala-Azi(OBn)₂. For detailed structures and absolute stereoconfiguration of the compounds see Table 1. Abbreviations used: Azet, azetidine-2-carboxylic acid; Azy, aziridine-2-carboxylic acid; Bn, benzyl; Boc, tert-butoxycarbonyl; Cbz, benzyloxy carbonyl; Et, ethyl; Gly, glycine; Ini, isonipecotic acid; Leu, leucine; Nip, nipecotic acid; Pip, pipecolic acid; Pro, proline; R, any residue. (B) Structures of compounds 13b [Boc-(S)-Leu-(R)-Pro-(S,S)-Azi(OBn)₂] and 13e [Boc-(R)-Leu-(S)-Pro-(S,S)-Azi(OBn)₂].

FIG. 2.

Dose-response curves showing the effects of compounds 13b (upper panel) and 13e (lower panel) on macrophage survival and macrophage infection rate in the absence or presence of IFN-γ. Macrophages were infected with stationary-phase L. major promastigotes for 24 h. After removal of extracellular parasites, the cells were incubated further for 48 h in the absence of drugs, or in the presence of increasing concentrations of compounds with or without IFN-γ (100 U ml⁻¹). The arrows point out normalized survival and infection rates of macrophages incubated without drugs, i.e., in the absence or presence of IFN-γ alone.

FIG. 3.

IL-6 secretion by macrophages. Macrophages were infected with stationary-phase L. major promastigotes for 24 h and treated with the compounds in the absence or presence of IFN-γ or LPS. (Upper panel) Percentages of IL-6 levels in the supernatants of noninfected macrophages cultured in the presence of compound 13b (40 μM) or compound 13e (40 μM), in the absence or presence of IFN-γ (100 U ml⁻¹) or LPS (15 μg ml⁻¹). (Lower panel) Change (n-fold) (infected/noninfected) of IL-6 levels in the supernatants of infected macrophages cultured in the presence of compound 13b or compound 13e, in the absence or presence of IFN-γ or LPS. The horizontal lines indicate changes in cytokine production induced by IFN-γ or LPS alone. A ratio of 1 means no change. Indicated comparisons 1, 5, and 6, P < 0.001; indicated comparisons 2, 3, and 4, P < 0.05.

FIG. 4.

IL-12 secretion by macrophages. Macrophages were infected with stationary-phase L. major promastigotes for 24 h and treated with the compounds in the absence or presence of IFN-γ or LPS. (Upper panel) Percentages of IL-12 levels in the supernatants of noninfected macrophages cultured in the presence of compound 13b (40 μM) or compound 13e (40 μM), in the absence or presence of IFN-γ (100 U ml⁻¹) or LPS (15 μg ml⁻¹). (Lower panel) Change (n-fold) (infected/noninfected) of IL-12 levels in the supernatants of infected macrophages cultured in the presence of compound 13b or compound 13e, in the absence or presence of IFN-γ or LPS. The horizontal lines indicate changes in cytokine production induced by IFN-γ or LPS alone. A ratio of 1 means no change. Indicated comparisons 1, 3, and 4, P < 0.001; indicated comparison 2, P < 0.005.

FIG. 5.

TNF-α secretion by macrophages. Macrophages were infected with stationary-phase L. major promastigotes for 24 h and treated with the compounds in the absence or presence of IFN-γ or LPS. (Upper panel) Percentages of TNF-α levels in the supernatants of noninfected macrophages cultured in the presence of compound 13b (40 μM) or compound 13e (40 μM), in the absence or presence of IFN-γ (100 U ml⁻¹) or LPS (15 μg ml⁻¹). (Lower panel) Change (n-fold) (infected/noninfected) of TNF-α levels in the supernatants of infected macrophages cultured in the presence of compound 13b or compound 13e, in the absence or presence of IFN-γ or LPS. The horizontal lines indicate changes in cytokine production induced by IFN-γ or LPS alone. A ratio of 1 means no change. Indicated comparisons 1 and 2, P < 0.005; indicated comparison 3, P < 0.001.

FIG. 6.

NO production by macrophages. Macrophages were infected with stationary-phase L. major promastigotes for 24 h. Cells were then washed and incubated for 48 h further with the compounds in the absence or presence of IFN-γ or LPS. Thereafter, the culture supernatants were collected for determination of the nitrite concentrations. (Upper panel) Micromolar concentrations of NO₂⁻ in the supernatants of noninfected macrophages cultured in the presence of compound 13b (40 μM) or compound 13e (40 μM), in the absence or presence of IFN-γ (100 U ml⁻¹) or LPS (15 μg ml⁻¹). (Lower panel) Change (n-fold) (infected/noninfected) of NO levels in supernatants of infected macrophages cultured in the presence of compound 13b or compound 13e, in the absence or presence of IFN-γ or LPS. The horizontal lines indicate changes in NO production induced by IFN-γ or LPS alone. A ratio of 1 means no change. Indicated comparisons 1 and 2, P < 0.005; indicated comparisons 3 and 4, P < 0.05.