Simple Colorimetric Trypanothione Reductase-Based Assay for High-Throughput Screening of Drugs against Leishmania Intracellular Amastigotes

Bogaart, Erika van den; Schoone, Gerard J.; England, Paul; Faber, Dorien; Orrling, Kristina M.; Dujardin, Jean‐Claude; Sundar, Shyam; Schallig, Henk D. F. H.; Adams, Emily R.

doi:10.1128/aac.00751-13

Cited by 30 publications

(15 citation statements)

References 42 publications

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“…IC 50 's calculated from dose-response curves in wild type strains corroborated with previous studies on AmB toxicity to both promastigotes (0.6 ± 0.07 μM) and amastigotes (0.2 ± 0.0.05 μM) (Fig. 1A–B and Supplementary Table 1) (Sereno and Lemesre, 1997, van den bogaart et al., 2014). In contrast the LdAI transfected parasites showed moderate sensitivity to AmB as reflected through nearly three- and eight-fold increase in IC 50 against promastigotes (2.2 ± 0.3 μM) and amastigotes (1.9 ± 0.6 μM) respectively, when compared to non-transfected strains (Supplementary Table 1).…”

Section: Resultssupporting

confidence: 88%

L-Asparaginase of Leishmania donovani: Metabolic target and its role in Amphotericin B resistance

Singh¹,

Khan²,

Yadav³

et al. 2017

International Journal for Parasitology: Drugs and Drug Resistan

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Emergence of Amphotericin B (AmB) resistant Leishmania donovani has posed major therapeutic challenge against the parasite. Consequently, combination therapy aimed at multiple molecular targets, based on proteome wise network analysis has been recommended. In this regard we had earlier identified and proposed L-asparaginase of Leishmania donovani (LdAI) as a crucial metabolic target. Here we report that both LdAI overexpressing axenic amastigote and promastigote forms of L. donovani survives better when challenged with AmB as compared to wild type strain. Conversely, qRT-PCR analysis showed an upregulation of LdAI in both forms upon AmB treatment. Our data demonstrates the importance of LdAI in imparting immediate protective response to the parasite upon AmB treatment. In the absence of structural and functional information, we modeled LdAI and validated its solution structure through small angle X-ray scattering (SAXS) analysis. We identified its specific inhibitors through ligand and structure-based approach and characterized their effects on enzymatic properties (Km, Vmax, Kcat) of LdAI. We show that in presence of two of the inhibitors L1 and L2, the survival of L. donovani is compromised whereas overexpression of LdAI in these cells restores viability. Taken together, our results conclusively prove that LdAI is a crucial metabolic enzyme conferring early counter measure against AmB treatment by Leishmania.

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Section: Resultssupporting

confidence: 88%

L-Asparaginase of Leishmania donovani: Metabolic target and its role in Amphotericin B resistance

Singh¹,

Khan²,

Yadav³

et al. 2017

International Journal for Parasitology: Drugs and Drug Resistan

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“…THP-1 cells were cultured in RPMI 1640 Medium (Sigma Aldrich, USA) supplemented with 10% HINBCS, 100 IU/ mL penicillin and 100 μg/mL streptomycin at 37°C in a 5% CO 2 humidified incubator. 22…”

Section: Thp-1 Cell Line Culturesmentioning

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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“…We have also identified new compounds that could potentially serve as a starting point for new drug discovery in leishmaniasis. in 5% CO 2 humidified incubator [22].…”

Section: Introductionmentioning

confidence: 99%

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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Simple Colorimetric Trypanothione Reductase-Based Assay for High-Throughput Screening of Drugs against Leishmania Intracellular Amastigotes

Cited by 30 publications

References 42 publications

L-Asparaginase of Leishmania donovani: Metabolic target and its role in Amphotericin B resistance

L-Asparaginase of Leishmania donovani: Metabolic target and its role in Amphotericin B resistance

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

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

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