A Replicative In Vitro Assay for Drug Discovery against Leishmania donovani

Abstract

The protozoan parasite Leishmania donovani is the causative agent of visceral leishmaniasis, a disease potentially fatal if not treated. Current available treatments have major limitations, and new and safer drugs are urgently needed. In recent years, advances in high-throughput screening technologies have enabled the screening of millions of compounds to identify new antileishmanial agents. However, most of the compounds identified in vitro did not translate their activities when tested in in vivo models, highlighting the need to develop more predictive in vitro assays. In the present work, we describe the development of a robust replicative, high-content, in vitro intracellular L. donovani assay. Horse serum was included in the assay media to replace standard fetal bovine serum, to completely eliminate the extracellular parasites derived from the infection process. A novel phenotypic in vitro infection model has been developed, complemented with the identification of the proliferation of intracellular amastigotes measured by EdU incorporation. In vitro and in vivo results for miltefosine, amphotericin B, and the selected compound 1 have been included to validate the assay.T he leishmaniases are a complex of diseases, with visceral and cutaneous manifestations caused by protozoan parasites of the genus Leishmania. Visceral leishmaniasis (VL) has been the main focus for drug research and development over the past 2 decades, due to the large disease burden in East Africa and South Asia (1) and potential patient death if not treated. For VL, there has been progress in treatment over the past decade, with clinical evidence for efficacy of and registration for use of oral miltefosine, paromomycin, and the liposomal formulation of amphotericin B (AmBisome, Gilead, USA) in South Asia (2), as well as combinations of these standard drugs (3). The need for new drugs to treat VL remains, as (i) miltefosine is the only approved oral treatment but requires 28 days of treatment and potential teratogenicity limits its use (4), (ii) paromomycin requires 21 days of treatment and intramuscular administration (http://www.dndi.org/diseases-projects/diseases /vl/current-treatment/current-treatment-vl.html), and (iii) liposomal amphotericin B formulations, which have successful cure rates with a single dose (5), require intravenous (i.v.) infusion, have a high cost if not donated, and have a requirement for cold storage, limiting use in countries where the disease is endemic (6). As part of the drive to find new treatments, there has been a refocus on the assays and models used to identify and develop new molecules as antileishmanial drugs. For in vitro screens and assays, this has ranged from the need to develop methods that (i) are adaptable to and enable high-throughput screens against the replicative intracellular-macrophage amastigote stage of Leishmania donovani, one of the causative species of VL (7); and (ii) include high-throughput technologies that enable the collection of more information compared to the traditionally use...

Investigation of pyrimidine nucleoside analogues as chemical probes to assess compound effects on the proliferation of Trypanosoma cruzi intracellular parasites

“…For example, it has been demonstrated that following 72 hours exposure of HEK293 cells to EdU there was cytotoxicity displayed, with an IC 50 value of 0.060 ± 0.043 μM identified [32]. A recent report has described lack of toxicity of EdU toward L. donovani following 72 hours incubation [20]. It could be that these parasites have different methods of transport / variability in enzymes for nucleoside analogue incorporation into their DNA.…”

“…Incorporation of EdU into parasite DNA, detected with click chemistry, has recently been used as a chemical probe to identify replicating T. cruzi parasites, to assess variation in strain replication and responses to a selection of compounds at a single concentration [19]. Additionally, an image-based assay to assess parasite replication utilising EdU as a chemical probe was developed to determine compound activity against Leishmania donovani [20]. The incorporation of EdU and BrdU has been compared in T. cruzi epimastigotes and it was found that EdU was most effective for monitoring DNA replication because of its sensitivity and that DNA denaturation is not required [21].…”

Snapshot Profiling of the Antileishmanial Potency of Lead Compounds and Drug Candidates against Intracellular Leishmania donovani Amastigotes, with a Focus on Human-Derived Host Cells

“…Cell lines are often used over primary cells due to ease of culture and an argument of homogeneity. Phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 cells are widely used in antileishmanial drug research, but different stimulation conditions are reported (7,8,10,22). The host cell's ability to sustain infection with Leishmania parasites and comparison of the potencies of reference compounds to other host cells and assay formats have been the focus in developing protocols for antileishmanial drug evaluation using differentiated THP-1 cells.…”

Importance of secondary screening with clinical isolates for anti-leishmania drug discovery

“…The InMac assay developed by GSK was modified by the incorporation of horse serum which increased the robustness and the predictive potential of the assay by reducing the amount of extracellular promastigotes remaining after infection 17 . Moreover, it avoided the reinfection and increased the replication rate of intracellular amastigotes 17 . The assay was further optimized for the clinical isolates LdBPK_282 and LdBPK_275 (procedure described in Fig.…”