Estrategias celulares y moleculares en el cribado de nuevos agentes anti-Leishmania

Abstract

Treatment of parasitic diseases caused by trypanosomatids requires the development of new agents endowed with both more selectivity and a pharmacological profile that is improved over that of currently used agents. We face this challenge by following two different strategies. On the one hand, as new drugs are often discovered serendipitously by testing large chemical libraries, we decided to implement an in‐house high‐throughput screening (HTS) that will enable us to test cytotoxicity both on Leishmania promastigotes/amastigotes and two different human cell lines (Jurkat and THP‐1). Using this HTS method we tested more than 160 compounds. The outcome in the treatment of amastigote‐infected THP‐1 cells was seven hits, four of them of comparable or even better activity than edelfosine, a reference standard antileishmanial drug. These derivatives thus represent new leads for further studies aimed at establishing their mechanism of action. On the other hand, we pursued an innovative approach in the treatment of leishmaniasis consisting of inhibiting the dimerization of the enzyme trypanothione reductase (TryR), an essential homodimeric flavoenzyme unique to kinetoplastid parasites including Trypanosoma, responsible for Chagas disease and sleeping sickness, and Leishmania, responsible for cutaneous and visceral leishmaniasis. TryR is thought to be the central enzyme in the redox metabolism of these protozoans because it alone couples the transfer of reducing equivalents from the NADP+/NADPH pair to thiol‐containing species. By means of structure‐based computational studies we found the hot‐spots in the dimeric interface presumably responsible for the largest part of the intermolecular affinity. Using molecular biology, biochemistry and oligomerization detection techniques, we have found that glutamic 436 (E436), one of the three initial candidates identified as putative hot‐spots, is a key residue for TryR dimerization. On the basis of these findings we then designed a peptide that inhibits both TryR dimerization and activity. Furthermore, we proved that this peptide has leishmanicidal activity on amastigote‐infected human macrophages. We also prensent some results suggestive of the existence of a biological sensor in the trypanosomatids’ TryR that allows them to detect an oxidative burst and react facing it. To sum up, herein we show two different approaches aimed at fulfilling the need to develop new, efficient and safe drugs for the treatment of leishmaniasis.