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RNA helicases are proteins found in all kingdoms of life, and they are associated with all processes involving RNA from transcription to decay. They use NTP binding and hydrolysis to unwind duplexes, to remodel RNA structures and protein-RNA complexes, and to facilitate the unidirectional metabolism of biological processes. Viral, bacterial, and eukaryotic parasites have an intimate need for RNA helicases in their reproduction. Moreover, various disorders, like cancers, are often associated with a perturbation of the host's helicase activity. Thus, RNA helicases provide a rich source of targets for the development of therapeutic or prophylactic drugs. In this review, we provide an overview of the different targeting strategies against helicases, the different types of compounds explored, the proposed inhibitory mechanisms of the compounds on the proteins, and the therapeutic potential of these compounds in the treatment of various disorders.

International audience; DEAD-box RNA helicases are ubiquitous proteins found in all kingdoms of life and that are associated with all processes involving RNA. Their central roles in biology make these proteins potential targets for therapeutic or prophylactic drugs. The Ded1/DDX3 subfamily of DEAD-box proteins is of particular interest because of their important role(s) in translation. In this paper, we identified and aligned the protein sequences of 28 different DEAD-box proteins from the kinetoplast-protozoan parasite Leishmania infantum, which is the cause of the visceral form of leishmaniasis that is often lethal if left untreated, and compared them with the consensus sequence derived from DEAD-box proteins in general, and from the Ded1/DDX3 subfamily in particular, from a wide variety of other organisms. We identified three potential homologs of the Ded1/DDX3 subfamily and the equivalent proteins from the related protozoan parasite Trypanosoma brucei, which is the causative agent of sleeping sickness. We subsequently tested these proteins for their ability to complement a yeast strain deleted for the essential DED1 gene. We found that the DEAD-box proteins from Trypanosomatids are highly divergent from other eukaryotes, and consequently they are suitable targets for protein-specific drugs.

DEAD-box RNA helicases are ubiquitous proteins found in all kingdoms of life and that are associated with all processes involving RNA. Their central roles in biology make these proteins potential targets for therapeutic or prophylactic drugs. The Ded1/DDX3 subfamily of DEAD-box proteins is of particular interest because of their important role(s) in translation. In this paper, we identified and aligned the protein sequences of 28 different DEAD-box proteins from the kinetoplast-protozoan parasite Leishmania infantum, which is the cause of the visceral form of leishmaniasis that is often lethal if left untreated, and compared them with the consensus sequence derived from DEAD-box proteins in general, and from the Ded1/DDX3 subfamily in particular, from a wide variety of other organisms. We identified three potential homologs of the Ded1/DDX3 subfamily and the equivalent proteins from the related protozoan parasite Trypanosoma brucei, which is the causative agent of sleeping sickness. We subsequently tested these proteins for their ability to complement a yeast strain deleted for the essential DED1 gene. We found that the DEAD-box proteins from Trypanosomatids are highly divergent from other eukaryotes, and consequently they are suitable targets for protein-specific drugs.

RNA helicases are proteins found in all kingdoms of life, and they are associated with all processes involving RNA from transcription to decay. They use NTP binding and hydrolysis to unwind duplexes, to remodel RNA structures and protein-RNA complexes, and to facilitate the unidirectional metabolism of biological processes. Viral, bacterial, and eukaryotic parasites have an intimate need for RNA helicases in their reproduction. Moreover, various disorders, like cancers, are often associated with a perturbation of the host's helicase activity. Thus, RNA helicases provide a rich source of targets for the development of therapeutic or prophylactic drugs. In this review, we provide an overview of the different targeting strategies against helicases, the different types of compounds explored, the proposed inhibitory mechanisms of the compounds on the proteins, and the therapeutic potential of these compounds in the treatment of various disorders.

International audience; The antifungal agent 6-aminocholestanol targets the production of ergosterol, which is the principle sterol in many fungi and protozoans; ergosterol serves many of the same roles as cholesterol in animals. We found that it also is an effective inhibitor of the translation-initiation factor eIF4AI from mouse (eIF4AIMus) and the Trypanosomatid parasite Leishmania (LieIF4A). The eIF4A proteins belong to the DEAD-box family of RNA helicases, which are ATP-dependent RNA-binding proteins and RNA-dependent ATPases. DEAD-box proteins contain a commonly-shared core structure consisting of two linked domains with structural homology to that of recombinant protein A (RecA) and that contain conserved motifs that are involved in RNA and ATP binding, and in the enzymatic activity. The compound inhibits both the ATPase and helicase activities by perturbing ATP and RNA binding, and it is capable of binding other proteins containing nucleic acid-binding sites as well. We undertook kinetic analyses and found that the Leishmania LieIF4A protein binds 6-aminocholestanol with a higher apparent affinity than for ATP, although multiple binding sites were probably involved. Competition experiments with the individual RecA-like domains indicate that the primary binding sites are on RecA-like domain 1, and they include a cavity that we previously identified by molecular modeling of LieIF4A that involve conserved RNA-binding motifs. The compound affects the mammalian and Leishmania proteins differently, which indicates the binding sites and affinities are not the same. Thus, it is possible to develop drugs that target DEAD-box proteins from different organisms even when they are implicated in the same biological process.

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., Author summary Leishmaniases constitute a group of neglected parasitic diseases that inflict major burden on public health. Novel drugs and targets need to be identified since current therapies have adverse side effects. Herein, we focused on Leishmania infantum translation initiation factor 4A (LieIF), as a potential drug target. LieIF, a pivotal enzyme in the translation machinery, is also implicated in host-pathogen interactions. We modeled its 3D structure and identified two pockets, which were used in virtual screenings of a chemical compound library. Therefore, we selected and purchased 305 compounds. We established a reliable ATPase screening assay to test the molecules against the enzymatic activity of LieIF and its mammalian homologue. A promising hit was retained and further characterized. It inhibited both proteins but showed different kinetic properties. It was used as a basis to identify similar analogues and two additional inhibitors were identified. All three hits reduced the viability of the extracellular promastigote form of the parasite, but they had no significant cytotoxic effects on host cells. They also affected the viability of the intracellular amastigote form and reduced the macrophage infection. This selectivity is very promising and indicates that these inhibitors would constitute an avenue to develop strategies to fight leishmaniases.