A luciferase based viability assay for ATP detection in 384-well format for high throughput whole cell screening of Trypanosoma brucei brucei bloodstream form strain 427

Sykes, Melissa; Avery, Vicky M.

doi:10.1186/1756-3305-2-54

Cited by 32 publications

(38 citation statements)

References 33 publications

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“…24,25 Hydroxamic acid derivatives known to target human HDAC were found moderately potent against the parasites (EC 50 <11 lM), and as shown in Table 1, six of these HDACi are currently in Phase I, II, or III clinical trials. 5 Only one known sirtuin inhibitor was found active.…”

Section: Resultsmentioning

confidence: 98%

Evaluation of histone deacetylase inhibitors (HDACi) as therapeutic leads for human African trypanosomiasis (HAT)

Carrillo

Guiguemde

Guy

2015

Bioorganic & Medicinal Chemistry

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Section: Resultsmentioning

confidence: 98%

Evaluation of histone deacetylase inhibitors (HDACi) as therapeutic leads for human African trypanosomiasis (HAT)

Carrillo

Guiguemde

Guy

2015

Bioorganic & Medicinal Chemistry

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“…In the present study, we developed an automated, HTS-compatible assay of Naegleria viability that is suitable for identifying amebicidal compounds. This luciferase-based assay is modified from high-throughput screens previously used to measure proliferation, metabolic activ- ity, and cytotoxicity with a number of cell types, including trypanosomes (9,22,32,37). Because of the high virulence of N. fowleri, we first developed the assay for use with the nonpathogenic species N. gruberi and then rescreened active compounds against pathogenic N. fowleri.…”

Section: Discussionmentioning

confidence: 99%

Corifungin, a New Drug Lead against Naegleria, Identified from a High-Throughput Screen

Debnath

Tunac²,

Galindo-Gómez

et al. 2012

Antimicrob Agents Chemother

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c Primary amebic meningoencephalitis (PAM) is a rapidly fatal infection caused by the free-living ameba Naegleria fowleri. The drug of choice in treating PAM is the antifungal antibiotic amphotericin B, but its use is associated with severe adverse effects. Moreover, few patients treated with amphotericin B have survived PAM. Therefore, fast-acting and efficient drugs are urgently needed for the treatment of PAM. To facilitate drug screening for this pathogen, an automated, high-throughput screening methodology was developed and validated for the closely related species Naegleria gruberi. Five kinase inhibitors and an NFkappaB inhibitor were hits identified in primary screens of three compound libraries. Most importantly for a preclinical drug discovery pipeline, we identified corifungin, a water-soluble polyene macrolide with a higher activity against Naegleria than that of amphotericin B. Transmission electron microscopy of N. fowleri trophozoites incubated with different concentrations of corifungin showed disruption of cytoplasmic and plasma membranes and alterations in mitochondria, followed by complete lysis of amebae. In vivo efficacy of corifungin in a mouse model of PAM was confirmed by an absence of detectable amebae in the brain and 100% survival of mice for 17 days postinfection for a single daily intraperitoneal dose of 9 mg/kg of body weight given for 10 days. The same dose of amphotericin B did not reduce ameba growth, and mouse survival was compromised. Based on these results, the U.S. FDA has approved orphan drug status for corifungin for the treatment of PAM.

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“…Because of their easy, sensitive and fast readout, viability assays based on ATP detection (such as the luminescent CellTiter-Glo assay) have been proposed and used as an alternative viability assay for HTS in T.b. brucei strain 427 [5,7]. Currently, there is no reporter gene based in vitro assay employed for HTS compound screening, either for T.b.…”

Section: Introductionmentioning

confidence: 99%

Luminescent multiplex viability assay for Trypanosoma brucei gambiense

et al. 2013

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BackgroundNew compounds for the treatment of human African trypanosomiasis (HAT) are urgently required. Trypanosoma brucei (T.b.) gambiense is the leading cause of HAT, yet T.b. gambiense is often not the prime target organism in drug discovery. This may be attributed to the difficulties in handling this subspecies and the lack of an efficient viability assay to monitor drug efficacy.MethodsIn this study, a T.b. gambiense strain, recently isolated in the D.R. Congo, was made bioluminescent by transfection with Renilla luciferase (RLuc) without altering its in vitro and in vivo growth characteristics. A luminescent multiplex viability assay (LMVA), based on measurement of the Renilla luciferase activity and the ATP content of the cells within the same experiment, was investigated as an alternative to the standard fluorimetric resazurin viability assay for drug sensitivity testing of T.b. gambiense.ResultsIn a 96-well format, the RLuc transfected strain showed a detection limit of 2 × 104 cells ml-1 for the Renilla luciferase measurement and 5 × 103 cells ml-1 for the ATP measurement. Both assays of the LMVA showed linearity up to 106 cells ml-1 and correlated well with the cell density during exponential growth of the long slender bloodstream forms. The LMVA was compared to the fluorimetric resazurin viability assay for drug sensitivity testing of pentamidine, eflornithine, nifurtimox and melarsoprol with both the wild type and the RLuc transfected population. For each drug, the IC50 value of the RLuc population was similar to that of the wild type when determined with either the fluorimetric resazurin method or the LMVA. For eflornithine, nifurtimox and melarsoprol we found no difference between the IC50 values in both viability assays. In contrast, the IC50 value of pentamidine was higher when determined with the fluorimetric resazurin method than in both assays of the LMVA.ConclusionsLMVA has some advantages for viability measurement of T.b. gambiense: it requires less incubation time for viability detection than the fluorimetric resazurin assay and in LMVA, two sensitive and independent viability assays are performed in the same experiment.

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A luciferase based viability assay for ATP detection in 384-well format for high throughput whole cell screening of Trypanosoma brucei brucei bloodstream form strain 427

Cited by 32 publications

References 33 publications

Evaluation of histone deacetylase inhibitors (HDACi) as therapeutic leads for human African trypanosomiasis (HAT)

Evaluation of histone deacetylase inhibitors (HDACi) as therapeutic leads for human African trypanosomiasis (HAT)

Corifungin, a New Drug Lead against Naegleria, Identified from a High-Throughput Screen

Luminescent multiplex viability assay for Trypanosoma brucei gambiense

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