Cruzain inhibition by terpenoids

The cysteine protease cruzain has been identified as a potential target for treatment of American trypanosomiasis (Chagas disease). Because plants must deal with pathogenic fungi, bacteria, plant viruses, pathogenic mites, and herbivorous insects, which utilize cysteine proteases in order to infect the host plants, we hypothesize that plants have developed small-molecule cysteine protease inhibitors for protection from pathogenic pests and herbivory. In this study, we have identified a number of terpenoids from rainforest plants from Monteverde, Costa Rica, that show cruzain inhibitory activity (IC ₅₀<100µg/mL), including monoterpenoids (limonene and myrcene), sesquiterpenoids (β-caryophyllene, α-copaene, germacrene-D, α-humulene, and α-zingiberene), and triterpenoids (lupeol, canophyllol, tingenone, 29-hydroxyfriedelan-3-on-28-al, and 3-O-(3'-hydroxytetradecanoyl)-lupeol). We have carried out synergism studies of various essential oil components (in binary combinations). The cruzain-inhibitory sesquiterpenes (β-caryophyllene, α-copaene, germacrene-D, and α-humulene) showed enhanced activity in combination with other essential oil components. Thus, for example, β-caryophyllene, in combination with either endo-fenchol or p-cymene (neither of which is active) showed enhanced cruzain inhibitory activity. In addition, a mixture of caryophyllene oxide and eugenol (both inactive alone) showed cruzain inhibitory activity. A molecular docking analysis (ArgusDock 4.0.1) has been carried out using the monoterpenoid, sesquiterpenoid, and triterpenoid compounds that have shown cruzain inhibitory activity. The calculated binding energies of the docked ligands roughly correlate with their experimentally determined inhibitory activities. The orientations of the docked ligands are consistent with a mechanism whereby these hydrophobic compounds dock into a hydrophobic pocket near the active site, thereby blocking binding of the protein target to the protease.