Discovery of novel hit compounds with broad activity against visceral and cutaneous Leishmania species by comparative phenotypic screening

S Lamotte,G F Späth,E Prina,N Aulner

doi:10.1038/s41598-018-36944-6

Abstract

The limited success of recent phenotypic anti-leishmanial drug screening campaigns calls for new screening strategies for the discovery of clinically relevant hits. Here we present such a novel strategy based on physiologically relevant, ex vivo biology. We established high content phenotypic assays that combine primary murine macrophages and lesion-derived, virulent L. donovani and L. amazonensis amastigotes, which we applied to validate previously identified, anti-leishmanial hit compounds referred to as ‘GSK Leish-Box’. Together with secondary screens using cultured promastigotes, our pipeline distinguished stage- and/or species-specific compounds, including 20 hits with broad activity at 10 µM against intracellular amastigotes of both viscerotropic and dermotropic Leishmania. Even though the GSK Leish-Box hits were identified by phenotypic screening using THP-1 macrophage-like cells hosting culture-derived L. donovani LdBob parasites, our ex vivo assays only validated anti-leishmanial activity at 10 µM on intra-macrophagic L. donovani for 23 out of the 188 GSK Leish-Box hits. In conclusion, our comparative approach allowed the identification of hits with broad anti-leishmanial activity that represent interesting novel candidates to be tested in animal models. Physiologically more relevant screening approaches such as described here may reduce the very high attrition rate observed during pre-clinical and clinical phases of the drug development process.

Highlights

The limited success of recent phenotypic anti-leishmanial drug screening campaigns calls for new screening strategies for the discovery of clinically relevant hits
To increase the rate of hits targeting the clinically relevant, intracellular stage of L. donovani, we have developed a novel phenotypic High Content Assay (HCA) based on mouse primary macrophages that are infected with bona fide, tissue-derived L. donovani amastigotes
In contrast to a previous HCA we established for L. amazonensis[19] (Fig. S2) and for which the main readout of infected macrophages was the presence of large communal parasitophorous vacuoles (PVs) stained with a fluorescent acidotropic probe, the L. donovani HCA readout was strictly dependent upon the number of individual, immuno-stained parasites

Summary

Introduction

The limited success of recent phenotypic anti-leishmanial drug screening campaigns calls for new screening strategies for the discovery of clinically relevant hits. We present such a novel strategy based on physiologically relevant, ex vivo biology. Various screening campaigns tried to simulate this stage using axenic amastigote parasitesincubated with immortalized, macrophage-like cell lines[17,18], falling short in terms of biological relevance and infectivity. These screening conditions may explain the limited success of these efforts to yield novel, validated hit compounds. Current portfolios contain some new candidate leads, some of which have been identified using phenotypic screens, screening methodology still needs to be improved to correspond closer to the biology of clinical infection and to consider intra-species and inter-species variability in parasite tropism, drug susceptibility and intracellular infection

Methods

Results

Conclusion