Development of NanoLuc-PEST expressing Leishmania mexicana as a new drug discovery tool for axenic- and intramacrophage-based assays

Berry, Sarah L.; Hameed, Hamza; Thomason, Anna G.; Maciej-Hulme, Marissa L.; Abou-Akkada, Somaia Saif; Horrocks, Paul; Price, Helen P.

doi:10.1371/journal.pntd.0006639

Cited by 25 publications

(32 citation statements)

References 58 publications

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“…Active compounds which demonstrated above 70% inhibition (red and orange colour) were selected for estimation of IC 50 (Table 1). Antileishmanial activity of MMV690102 and MMV690103 was previously reported [28,32], which strengthen the speculation of leishmanial DHFR to be target for these compounds.…”

Section: Primary Screening Resultssupporting

confidence: 73%

“…Antileishmanial activity of MMV690102 was reported earlier by Berry et al [28] and Duffy et al [32]. Both studies identified MMV690102 as promising starting point for further drug development.…”

Section: Activity Of Selected Compounds Against L Donovani Amastigotesmentioning

confidence: 54%

“…The infection index for each well in duplicate was determined by multiplying the percentage of infected macrophages (IR) with the average number of amastigotes in infected cells [28][29][30] as shown below.…”

Section: Thp-1 Cell Cytotoxicity Assay Cytotoxic Effect Of Selected mentioning

confidence: 99%

“…The lowest IC 50 (86.5 nm) was registered on L. donovani isolates obtained from Ethiopia, which substantiate our report. Berry et al[28] reported IC 50 value (1.780 μM) on L. mexicana. This variance might be caused by difference in the assay format or species variation.…”

mentioning

confidence: 99%

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Identification of Leishmania donovani inhibitors from pathogen box compounds of Medicine for Malaria Venture

Tadele

Abay

Makonnen

et al. 2019

Preprint

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Leishmaniasis is a collective term used to describe various pathological conditions caused by an obligate intracellular protozoan of the genus Leishmania. It is one of the neglected diseases and has been given low attention in drug discovery researches to narrow the existing gap in safety and efficacy of the currently used drugs to treat leishmaniasis. The challenge is further exacerbated by the emergence of drug resistance by the parasites. Aiming to look for potential anti-leishmanial hits and leads, we screened MMV Pathogen Box against clinically isolated L. donovani strain. Compounds were screened against promastigote, and then against amastigote stages; of which, 35 compounds showed >50% inhibition on promastigotes in the initial screen (1 μM). Out of these compounds, 9 compounds showed >70% inhibition with median inhibitory concentration (IC 50 ) ranges from 12 nM to 491 nM on anti-promastigote assay and 53 to 704 nM on intracellular amastigote assay. Identified compounds demonstrated good safety on THP-1 cell lines and sheep RBCs, and appropriate physico-chemical property suitable for further drug development. Two compounds (MMV690102 and MMV688262) were identified as lead compounds. Among these compounds, anti-tubercular agent MMV688262 (delamanid) showed synergistic effect with amphotericin B, indicating the prospect of this compound for combination therapy. The current study indicates the presence of additional hits which may hold promise as starting points for anti-leishmanial drug discovery and in-depth structure activity relationship studies. Future works also needs to investigate antiamastigotes activity of remaining 'hits', which were not covered in the present study. | P a g e Authors summaryVisceral leishmaniasis is a major public health problem in endemic regions. Different drugs have been used to treat visceral leishmaniasis. However, the available drugs are either toxic, noncompliance to the patient, painful upon administration, low in efficacy, or costly. New chemical entities that overcome the limitations of existing drugs are therefore desperately needed.Screening of 400 pathogen box compounds against of Leishmania donovani clinical isolate resulted in identification of 35 compounds with >50% inhibition against promastigotes at 1 μM.Out of these compounds, 9 showed >70% inhibition with median inhibitory concentration ranges from 12 nM to 491 nM on anti-promastigote assay, and 53 to 704 nM on intracellular amastigote assay. Our work identified new compounds which hold promise for further drug development.

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Section: Primary Screening Resultssupporting

confidence: 73%

Section: Activity Of Selected Compounds Against L Donovani Amastigotesmentioning

confidence: 54%

Section: Thp-1 Cell Cytotoxicity Assay Cytotoxic Effect Of Selected mentioning

confidence: 99%

mentioning

confidence: 99%

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Identification of Leishmania donovani inhibitors from pathogen box compounds of Medicine for Malaria Venture

Tadele

Abay

Makonnen

et al. 2019

Preprint

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“…The infection index for each well in duplicate was determined by multiplying the percentage of infected macrophages (IR) by the average number of amastigotes in infected cells, as shown below. [28][29][30] Infection index ¼…”

Section: Determination Of Ic 50 Against Intracellular Amastigotesmentioning

confidence: 99%

<p><em>Leishmania donovani</em> Growth Inhibitors from Pathogen Box Compounds of Medicine for Malaria Venture</p>

Tadele¹,

Abay²,

Makonnen³

et al. 2020

DDDT

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Introduction: Leishmaniasis is a collective term used to describe various pathological conditions caused by an obligate intracellular protozoan of the genus Leishmania. It is one of the neglected diseases and has been given minimal attention by drug discovery and development stakeholders to narrow the safety and efficacy gaps of the drugs currently used to treat leishmaniasis. The challenge is further exacerbated by the emergence of drug resistance by the parasites. Methods: Aiming to look for potential anti-leishmanial hits and leads, we screened Medicines for Malaria Venture (MMV) Pathogen Box compounds against clinically isolated Leishmania donovani strain. In this medium-throughput primary screening assay, the compounds were screened against promastigotes, and then against amastigote stages. Results: From the total 400 compounds screened, 35 compounds showed >50% inhibitory activity on promastigotes in the initial screen (1 μM). Out of these compounds, nine showed >70% inhibition, with median inhibitory concentration (IC 50 ) ranging from 12 to 491 nM using the anti-promastigote assay, and from 53 to 704 nM using the intracellular amastigote assay. Identified compounds demonstrated acceptable safety profiles on THP-1 cell lines and sheep red blood cells, and had appropriate physicochemical properties suitable for further drug development. Two compounds (MMV690102 and MMV688262) were identified as leads. The anti-tubercular agent MMV688262 (delamanid) showed a synergistic effect with amphotericin B, indicating the prospect of using this compound for combination therapy. Conclusion: The current study indicates the presence of additional hits which may hold promise as starting points for anti-leishmanial drug discovery and in-depth structure-activity relationship studies.

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Evaluation of NanoLuc, RedLuc and Luc2 as bioluminescent reporters in a cutaneous leishmaniasis model

et al. 2020

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New drugs for the treatment of human leishmaniasis are urgently needed, considering the limitations of current available options. However, pre-clinical evaluation of drug candidates for leishmaniasis is challenging. The use of luciferase-expressing parasites for parasite load detection is a potentially powerful tool to accelerate the drug discovery process. We have previously described the use of Leishmania amazonensis mutants expressing firefly luciferase (Luc2) for drug testing. Here, we describe three new mutant L. amazonensis lines that express different variants of luciferases: NanoLuc, NanoLuc-PEST and RedLuc. These mutants were evaluated in drug screening protocols. NanoLuc-parasites, in spite of high bioluminescence intensity in vitro, were shown to be inadequate in discriminating between live and dead parasites. Bioluminescence detection from intracellular amastigotes expressing NanoLuc-PEST, RedLuc or Luc2 proved more reliable than microscopy to determine parasite killing. Increased sensitivity was observed in vivo with RedLuc-expressing parasites as compared to NanoLuc-expressing L. amazonensis. Our data indicates that NanoLuc is not suitable for in vivo parasite burden determination. Additionally, RedLuc and the conventional luciferase Luc2 demonstrated equivalent sensitivity in an in vivo model of cutaneous leishmaniasis.

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Development of NanoLuc-PEST expressing Leishmania mexicana as a new drug discovery tool for axenic- and intramacrophage-based assays

Cited by 25 publications

References 58 publications

Identification of Leishmania donovani inhibitors from pathogen box compounds of Medicine for Malaria Venture

Identification of Leishmania donovani inhibitors from pathogen box compounds of Medicine for Malaria Venture

<p><em>Leishmania donovani</em> Growth Inhibitors from Pathogen Box Compounds of Medicine for Malaria Venture</p>

Evaluation of NanoLuc, RedLuc and Luc2 as bioluminescent reporters in a cutaneous leishmaniasis model

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