Sterol endoperoxides: Influence on Leishmania and macrophages

Leishmaniasis is an infectious tropical disease caused by Leishmania parasites (protozoa), affecting at least two million people annually in 98 subtropical and tropical countries. It manifests in three forms: cutaneous, mucocutaneous, and visceral, each with varying severity. The disease is transmitted by infected female sand flies, injecting the motile flagellated promastigotes into hosts during blood meals. Inside host macrophages, the parasites transform into non-flagellated amastigotes and proliferate, evading the immune system. Increasing drug resistance and adverse effects of current treatments necessitate new antileishmanial drugs. Endoperoxides (EP) are promising candidates, having shown antileishmanial activity and being used as antimalarial agents. Their efficacy and mode of action depend on their structure beyond the EP group, which is of interest for drug development. Therefore, this thesis aimed to evaluate (i) the impact of sterol endoperoxides (ergosterol endoperoxide (ErgoEP), dehydrocholesterol endoperoxide (DHCholEP)) on the viability of Leishmania tarentolae promastigotes (LtP) and J774 murine macrophages, (ii) the effect of antioxidants (N-acetyl cysteine (NAC), butylated hydroxytoluene (BHT), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox)) on the viability of LtP treated with sterol EP, (iii) the influence of sterol EP on sterol synthesis in LtP and (iv) possible improvements of sterol analysis in LtP. To investigate these questions, viability assays of LtP and J774 were executed in 96-well plates and after 48 h incubation with substances, resazurin fluorescence was used to obtain corresponding IC50 values. To assess the impairment of antileishmanial viability of the substances by the antioxidants NAC, BHT and Trolox, alterations in IC50 values were recorded upon addition of said antioxidants. After incubation of LtP with ErgoEP and DHCholEP, sterol analysis was performed using high-performance liquid chromatography (HPLC) analysis. Furthermore, corticosterone (CCS) was evaluated as an internal standard for this type of analysis. The experiments revealed IC50 values in LtP vs. J774 for ErgoEP of 4.5 ± 1.2 μM vs. 22 ± 5 μM and for DHCholEP 2.0 ± 0.4 μM vs. 15 ± 5 μM. This indicates a selectivity factor of 5–8 for LtP over J774. For sterol EP actions on LtP, no major influence of antioxidants was observed. HPLC analysis demonstrated that both sterol EPs inhibited the biosynthesis of the major sterol 5-dehydroepisterol in LtP and CCS could so far not be used as a standard due to losses during extraction. In conclusion, sterol EPs could be suitable as antileishmanial agents but further effectivity and mechanistic studies in more complex and in vivo model systems are required.