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

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.

Infectivity and Drug Susceptibility Profiling of Different Leishmania-Host Cell Combinations

“…In most of methods, the quantification is done by counting the stained or fluorescently engineered parasites, or by reading bioluminescent signal of Leishmania expressing luciferase [19,27,29,[49][50][51][52]. Many groups have reported differences of compound susceptibilities depending on the method mentioned, and a large portion of inhibitors found active in promastigotes or axenic amastigotes were found not active in intracellular amastigote assay [20][21][22]28,30]. This shows that evaluation of compound activity in physiologically relevant in the in vitro model-i.e., intracellular amastigote assay-represents a key step to successful drug development.…”

“…These axenically cultured amastigotes transformed from promastigotes have amastigote-like morphology but were shown to have different protein expression and drug susceptibility profiles compared to intracellular amastigotes [17,18]. Lastly, bona fide amastigotes developing inside Leishmania-infected host cells are widely used for susceptibility assays; favored cell lines for these assays include THP-1, U937, J774.1, and RAW263.7 or primary cells such as bone marrow-derived macrophages (BMDM) and peritoneal macrophages [19,20]. However, drug susceptibility is dependent on host cell type, and, given the slow or fast replication of intracellular Leishmania infections depending on their host cells in vitro, these assays may not reflect normal disease progression in an infected human patient.…”

Dynamic High-Content Imaging Reveals Surface Exposure of VirulentLeishmaniaAmastigotes in Infected Macrophages Undergoing Pyroptosis

“…Briefly, 200 μl of parasite culture was transferred into wells of a 96-well plate (Sigma, 655090) and incubated with 2.5 μg/mL of resazurin (Sigma, R7017) for 4 hours at 27°C. The fluorescence intensity of the resazurin-derived resorufin was determined using the Tecan Safire2 plate reader (558 nm excitation, 585 nm emission) (Lamotte et al, 2019).…”

Next-Generation Phenotypic Screening in Early Drug Discovery for Infectious Diseases

“…For example, a novel strategy, based on ex vivo biology, used phenotypic assays combining primary murine macrophages and lesion-derived, virulent L. donovani and Leishmania amazonensis amastigotes to validate antileishmanial hit compounds ('GSK Leish-Box'). Strikingly, the ex vivo approach validated antileishmanial activity on intramacrophagic L. donovani for only 23 out of the 188 GSK Leish-Box hits previously identified using immortalized THP1 macrophage cell lines [58]. Presumably the greater physiological relevance of ex vivo assays, compared with immortalized cells, provides for a more discriminating assay sensitivity.…”

“…During the last decade of drug screening, efforts in Leishmania have focused on extrapolating druggable targets in the parasite with some success (Box 1), reasoning that this could leverage rational development of chemotherapeutics suitable to address the public health challenges therein. However, while successful in identifying targets that allow large screening campaigns to be run, some groups have shifted their focus away from deconvolution of druggable targets in the parasite [56] and are turning their attention toward screening strategies targeting hostcell/pathogen interactions [57,58] (Figure 2) and phenotypic screening is well suited to this. For example, a novel strategy, based on ex vivo biology, used phenotypic assays combining primary murine macrophages and lesion-derived, virulent L. donovani and Leishmania amazonensis amastigotes to validate antileishmanial hit compounds ('GSK Leish-Box').…”