Identification of novel leishmanicidal molecules by virtual and biochemical screenings targeting Leishmania eukaryotic translation initiation factor 4A

Abstract

Leishmaniases are neglected parasitic diseases in spite of the major burden they inflict on public health. The identification of novel drugs and targets constitutes a research priority. For that purpose we used Leishmania infantum initiation factor 4A (LieIF), an essential translation initiation factor that belongs to the DEAD-box proteins family, as a potential drug target. We modeled its structure and identified two potential binding sites. A virtual screening of a diverse chemical library was performed for both sites. The results were analyzed with an in-house version of the Self-Organizing Maps algorithm combined with multiple filters, which led to the selection of 305 molecules. Effects of these molecules on the ATPase activity of LieIF permitted the identification of a promising hit (208) having a half maximal inhibitory concentration (IC50) of 150 ± 15 μM for 1 μM of protein. Ten chemical analogues of compound 208 were identified and two additional inhibitors were selected (20 and 48). These compounds inhibited the mammalian eIF4I with IC50 values within the same range. All three hits affected the viability of the extra-cellular form of L. infantum parasites with IC50 values at low micromolar concentrations. These molecules showed non-significant toxicity toward THP-1 macrophages. Furthermore, their anti-leishmanial activity was validated with experimental assays on L. infantum intramacrophage amastigotes showing IC50 values lower than 4.2 μM. Selected compounds exhibited selectivity indexes between 19 to 38, which reflects their potential as promising anti-Leishmania molecules.

Infection of Human Neutrophils With Leishmania infantum or Leishmania major Strains Triggers Activation and Differential Cytokines Release

“…The remaining cells were lysed by SDS (0.01 %), and the pellets were washed with PBS and resuspended in 100 μL Schneider medium supplemented with 10 % FBS and cultured at 22 °C in 96 well-plates. Parasites viability was assessed right after the lysis of the cells and 24 h later, by a Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay as described previously (Harigua-Souiai et al, 2018). Briefly, 20 μL of MTT (5 mg/mL) were added to each well and incubated at 22 °C.…”

The steroid derivative 6-aminocholestanol inhibits the DEAD-box helicase eIF4A (LieIF4A) from the Trypanosomatid parasite Leishmania by perturbing the RNA and ATP binding sites

“…Scientific, Waltham, MA, USA). The RNA products were separated by electrophoresis on a 15 8% PAGE gel containing 7 M urea, and the band corresponding to the desired product was 16 eluted, extracted with phenol, extracted with chloroform-isoamyl alcohol (24:1) and ethanol 17 precipitated as previously described [28]. The centrifuged and dried pellet was resuspended in 18 20 mM Tris-HCl, pH 7.5, 0.1 mM EDTA and stored at -20 °C until needed.…”

The In Silico Identification of Potential Members of the Ded1/DDX3 Subfamily of DEAD-Box RNA Helicases from the Protozoan Parasite Leishmania infantum and Their Analyses in Yeast

“…Moreover, the majority of the Leishmania and Trypanosoma genes are constitutively expressed as polycistronic RNAs that are subsequently processed into separate capped and polyadenylated mRNAs; thus, translation, and probably DDX3-like proteins, plays a particularly important role in the control of gene expression, especially for the differentiation into the different parasitic forms (reviewed by [ 49 , 50 , 51 ]). Indeed, we previously showed that the eIF4A-like translation protein, LieIF4A (LINF_010012800/LINF_010012900), is a viable target for therapeutic drugs [ 52 , 53 ].…”