Expanding the arsenal against leishmaniasis: Clemastine/Tamoxifen chimera as potential antileishmanials

Abstract

Leishmaniasis is a complex group of vector-borne zoonosis caused by parasites of the Leishmania genus. The prediction of annual cases of visceral leishmaniasis and cutaneous leishmaniasis combined is of more than 1 000 000 cases, with up to 20 000 deaths per year. The treatment of leishmaniasis relies on a few drugs presenting multiple shortcomings, which makes the discovery of new treatments urgent. In recent years, clemastine 27 and tamoxifen 28 have been identified in repurposing strategies and shown to display potent anti-leishmanial activity against both in vitro and in vivo models of the disease. Both molecules were proposed to target the same enzyme, inositol phosphorylceramide synthase (IPCS), which is found in the parasite but not in the host. These molecules also share similar chemical features, such as an aminoalkoxy side chain and a diaryl system. In this project, it was hypothesised that these chemical features are responsible for their antileishmanial activity. Therefore, the goal was to design and synthesise hybrid molecules between clemastine and tamoxifen and improve their antileishmanial activity. Firstly, a library of 40 compounds was synthesised varying the nature of the diaryl carbinol core and the aminoalkoxy side chain. Using a classic resazurin-based assay, all compounds were screened for antiparasitic activity against L. major and L. amazonensis promastigotes, and HepG2 cells for the evaluation of their cytotoxicity against human cells. Seven compounds with an EC50 < 2 µM against both species, and a selectivity index > 10 were the selected for testing against other two species of Leishmania promastigotes epidemiologically relevant in South America (L. braziliensis and L. infantum) as well as against two species of intracellular amastigotes (L. amazonensis and L. infantum). All compounds were highly active against L. braziliensis promastigotes, displaying EC50 values below 1 µM, and 6/7 compounds had EC50 approximate to 2 µM against L. infantum. In addition, they displayed promising activity against L. infantum amastigotes (EC50 ≤ 2 µM), but were slightly less active against L. amazonensis. Analogues 110 and 111 had the highest activity across the four species of promastigotes tested, and the most active compound against the intracellular amastigotes was 130. Collectively, these results contributed towards raising insights regarding the structure-activity relationship of this library, which led to the selection of clemastine/tamoxifen hybrids with high antileishmanial potency. Ongoing and future efforts are to explore the mechanism of action of this library. For this, two techniques were chosen to be explored; chemical proteomics, which makes use of chemical probes for the identification of binding proteins, and resistance selection followed by genomic sequencing, which allows for the identification of genes associated with the compound activity.