Novel 2D and 3D Assays to Determine the Activity of Anti-Leishmanial Drugs
The discovery of novel anti-leishmanial compounds remains essential as current treatments have known limitations and there are insufficient novel compounds in development. We have investigated three complex and physiologically relevant in vitro assays, including: (i) a media perfusion based cell cul...
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| Format: | Article |
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MDPI AG
2020-06-01
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| Series: | Microorganisms |
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| Online Access: | https://www.mdpi.com/2076-2607/8/6/831 |
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| author | Alec O’Keeffe Christine Hale James A. Cotton Vanessa Yardley Kapish Gupta Abhishek Ananthanarayanan Sudaxshina Murdan Simon L. Croft |
| author_facet | Alec O’Keeffe Christine Hale James A. Cotton Vanessa Yardley Kapish Gupta Abhishek Ananthanarayanan Sudaxshina Murdan Simon L. Croft |
| author_sort | Alec O’Keeffe |
| collection | DOAJ |
| description | The discovery of novel anti-leishmanial compounds remains essential as current treatments have known limitations and there are insufficient novel compounds in development. We have investigated three complex and physiologically relevant in vitro assays, including: (i) a media perfusion based cell culture model, (ii) two 3D cell culture models, and (iii) iPSC derived macrophages in place of primary macrophages or cell lines, to determine whether they offer improved approaches to anti-leishmanial drug discovery and development. Using a <i>Leishmania major</i> amastigote-macrophage assay the activities of standard drugs were investigated to show the effect of changing parameters in these assays. We determined that drug activity was reduced by media perfusion (EC<sub>50</sub> values for amphotericin B shifted from 54 (51–57) nM in the static system to 70 (61–75) nM under media perfusion; EC<sub>50</sub> values for miltefosine shifted from 12 (11–15) µM in the static system to 30 (26–34) µM under media perfusion) (mean and 95% confidence intervals), with corresponding reduced drug accumulation by macrophages. In the 3D cell culture model there was a significant difference in the EC<sub>50</sub> values of amphotericin B but not miltefosine (EC<sub>50</sub> values for amphotericin B were 34.9 (31.4–38.6) nM in the 2D and 52.3 (46.6–58.7) nM in 3D; EC<sub>50</sub> values for miltefosine were 5.0 (4.9–5.2) µM in 2D and 5.9 (5.5–6.2) µM in 3D (mean and 95% confidence intervals). Finally, in experiments using iPSC derived macrophages infected with <i>Leishmania</i>, reported here for the first time, we observed a higher level of intracellular infection in iPSC derived macrophages compared to the other macrophage types for four different species of <i>Leishmania</i> studied. For <i>L. major</i> with an initial infection ratio of 0.5 parasites per host cell the percentage infection level of the macrophages after 72 h was 11.3% ± 1.5%, 46.0% ± 1.4%, 66.4% ± 3.5% and 75.1% ± 2.4% (average ± SD) for the four cells types, THP1 a human monocytic cell line, mouse bone marrow macrophages (MBMMs), human bone marrow macrophages (HBMMs) and iPSC derived macrophages respectively. Despite the higher infection levels, drug activity in iPSC derived macrophages was similar to that in other macrophage types, for example, amphotericin B EC<sub>50</sub> values were 35.9 (33.4–38.5), 33.5 (31.5–36.5), 33.6 (30.5—not calculated (NC)) and 46.4 (45.8–47.2) nM in iPSC, MBMMs, HBMMs and THP1 cells respectively (mean and 95% confidence intervals). We conclude that increasing the complexity of cellular assays does impact upon anti-leishmanial drug activities but not sufficiently to replace the current model used in HTS/HCS assays in drug discovery programmes. The impact of media perfusion on drug activities and the use of iPSC macrophages do, however, deserve further investigation. |
| first_indexed | 2024-03-10T19:28:08Z |
| format | Article |
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| institution | Directory Open Access Journal |
| issn | 2076-2607 |
| language | English |
| last_indexed | 2024-03-10T19:28:08Z |
| publishDate | 2020-06-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Microorganisms |
| spelling | doaj.art-d2fc0f3ef4dc41758400f1ead02e723e2023-11-20T02:26:04ZengMDPI AGMicroorganisms2076-26072020-06-018683110.3390/microorganisms8060831Novel 2D and 3D Assays to Determine the Activity of Anti-Leishmanial DrugsAlec O’Keeffe0Christine Hale1James A. Cotton2Vanessa Yardley3Kapish Gupta4Abhishek Ananthanarayanan5Sudaxshina Murdan6Simon L. Croft7Department of Infection Biology, London School of Hygiene and Tropical Medicine, London WC1E 7HT, UKWellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge CB10 1SA, UKWellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge CB10 1SA, UKDepartment of Infection Biology, London School of Hygiene and Tropical Medicine, London WC1E 7HT, UKMechanobiology Institute, National University of Singapore, Singapore 117411, SingaporeInvitroCue Pte Ltd., Singapore 138667, SingaporeDepartment of Pharmaceutics, UCL School of Pharmacy, University College London, London WC1N 1AX, UKDepartment of Infection Biology, London School of Hygiene and Tropical Medicine, London WC1E 7HT, UKThe discovery of novel anti-leishmanial compounds remains essential as current treatments have known limitations and there are insufficient novel compounds in development. We have investigated three complex and physiologically relevant in vitro assays, including: (i) a media perfusion based cell culture model, (ii) two 3D cell culture models, and (iii) iPSC derived macrophages in place of primary macrophages or cell lines, to determine whether they offer improved approaches to anti-leishmanial drug discovery and development. Using a <i>Leishmania major</i> amastigote-macrophage assay the activities of standard drugs were investigated to show the effect of changing parameters in these assays. We determined that drug activity was reduced by media perfusion (EC<sub>50</sub> values for amphotericin B shifted from 54 (51–57) nM in the static system to 70 (61–75) nM under media perfusion; EC<sub>50</sub> values for miltefosine shifted from 12 (11–15) µM in the static system to 30 (26–34) µM under media perfusion) (mean and 95% confidence intervals), with corresponding reduced drug accumulation by macrophages. In the 3D cell culture model there was a significant difference in the EC<sub>50</sub> values of amphotericin B but not miltefosine (EC<sub>50</sub> values for amphotericin B were 34.9 (31.4–38.6) nM in the 2D and 52.3 (46.6–58.7) nM in 3D; EC<sub>50</sub> values for miltefosine were 5.0 (4.9–5.2) µM in 2D and 5.9 (5.5–6.2) µM in 3D (mean and 95% confidence intervals). Finally, in experiments using iPSC derived macrophages infected with <i>Leishmania</i>, reported here for the first time, we observed a higher level of intracellular infection in iPSC derived macrophages compared to the other macrophage types for four different species of <i>Leishmania</i> studied. For <i>L. major</i> with an initial infection ratio of 0.5 parasites per host cell the percentage infection level of the macrophages after 72 h was 11.3% ± 1.5%, 46.0% ± 1.4%, 66.4% ± 3.5% and 75.1% ± 2.4% (average ± SD) for the four cells types, THP1 a human monocytic cell line, mouse bone marrow macrophages (MBMMs), human bone marrow macrophages (HBMMs) and iPSC derived macrophages respectively. Despite the higher infection levels, drug activity in iPSC derived macrophages was similar to that in other macrophage types, for example, amphotericin B EC<sub>50</sub> values were 35.9 (33.4–38.5), 33.5 (31.5–36.5), 33.6 (30.5—not calculated (NC)) and 46.4 (45.8–47.2) nM in iPSC, MBMMs, HBMMs and THP1 cells respectively (mean and 95% confidence intervals). We conclude that increasing the complexity of cellular assays does impact upon anti-leishmanial drug activities but not sufficiently to replace the current model used in HTS/HCS assays in drug discovery programmes. The impact of media perfusion on drug activities and the use of iPSC macrophages do, however, deserve further investigation.https://www.mdpi.com/2076-2607/8/6/831<i>Leishmania</i>macrophagesdrug assaysmedia perfusion3D cellular modelsiPSC macrophages |
| spellingShingle | Alec O’Keeffe Christine Hale James A. Cotton Vanessa Yardley Kapish Gupta Abhishek Ananthanarayanan Sudaxshina Murdan Simon L. Croft Novel 2D and 3D Assays to Determine the Activity of Anti-Leishmanial Drugs Microorganisms <i>Leishmania</i> macrophages drug assays media perfusion 3D cellular models iPSC macrophages |
| title | Novel 2D and 3D Assays to Determine the Activity of Anti-Leishmanial Drugs |
| title_full | Novel 2D and 3D Assays to Determine the Activity of Anti-Leishmanial Drugs |
| title_fullStr | Novel 2D and 3D Assays to Determine the Activity of Anti-Leishmanial Drugs |
| title_full_unstemmed | Novel 2D and 3D Assays to Determine the Activity of Anti-Leishmanial Drugs |
| title_short | Novel 2D and 3D Assays to Determine the Activity of Anti-Leishmanial Drugs |
| title_sort | novel 2d and 3d assays to determine the activity of anti leishmanial drugs |
| topic | <i>Leishmania</i> macrophages drug assays media perfusion 3D cellular models iPSC macrophages |
| url | https://www.mdpi.com/2076-2607/8/6/831 |
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