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Fast in vitro methods to determine the speed of action and the stage-specificity of anti-malarials in Plasmodium falciparum.

Le Manach C, Scheurer C, Sax S, Schleiferböck S, Cabrera DG, Younis Y, Paquet T, Street L, Smith P, Ding XC, Waterson D, Witty MJ, Leroy D, Chibale K, Wittlin S - Malar. J. (2013)

Bottom Line: This has the advantage that initial results can be achieved within 4-7 working days, which helps to distinguish between fast and slow-acting compounds relatively quickly.The results obtained for the anti-malarials chloroquine, artesunate, atovaquone, and pyrimethamine are consistent with previous observations, suggesting the methodology is a valid way to rapidly identify fast-acting anti-malarial compounds.Another advantage of the approach is its ability to discriminate between static or cidal compound effects.

View Article: PubMed Central - HTML - PubMed

Affiliation: Parasite Chemotherapy Unit, Swiss Tropical and Public Health Institute, Basel, Switzerland. sergio.wittlin@unibas.ch.

ABSTRACT

Background: Recent whole cell in vitro screening campaigns identified thousands of compounds that are active against asexual blood stages of Plasmodium falciparum at submicromolar concentrations. These hits have been made available to the public, providing many novel chemical starting points for anti-malarial drug discovery programmes. Knowing which of these hits are fast-acting compounds is of great interest. Firstly, a fast action will ensure rapid relief of symptoms for the patient. Secondly, by rapidly reducing the parasitaemia, this could minimize the occurrence of mutations leading to new drug resistance mechanisms.An in vitro assay that provides information about the speed of action of test compounds has been developed by researchers at GlaxoSmithKline (GSK) in Spain. This assay also provides an in vitro measure for the ratio between parasitaemia at the onset of drug treatment and after one intra-erythrocytic cycle (parasite reduction ratio, PRR). Both parameters are needed to determine in vitro killing rates of anti-malarial compounds. A drawback of the killing rate assay is that it takes a month to obtain first results.

Methods: The approach described in the present study is focused only on the speed of action of anti-malarials. This has the advantage that initial results can be achieved within 4-7 working days, which helps to distinguish between fast and slow-acting compounds relatively quickly. It is expected that this new assay can be used as a filter in the early drug discovery phase, which will reduce the number of compounds progressing to secondary, more time-consuming assays like the killing rate assay.

Results: The speed of action of a selection of seven anti-malarial compounds was measured with two independent experimental procedures using modifications of the standard [3H]hypoxanthine incorporation assay. Depending on the outcome of both assays, the tested compounds were classified as either fast or non-fast-acting.

Conclusion: The results obtained for the anti-malarials chloroquine, artesunate, atovaquone, and pyrimethamine are consistent with previous observations, suggesting the methodology is a valid way to rapidly identify fast-acting anti-malarial compounds. Another advantage of the approach is its ability to discriminate between static or cidal compound effects.

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Related in: MedlinePlus

Schematic representation of the twoin vitroassays. The “IC50 speed assay” and “stage-specificity analysis” are performed with unsynchronized and synchronized parasite cultures, respectively. R, rings; S, schizonts.
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Figure 3: Schematic representation of the twoin vitroassays. The “IC50 speed assay” and “stage-specificity analysis” are performed with unsynchronized and synchronized parasite cultures, respectively. R, rings; S, schizonts.

Mentions: A schematic representation of the IC50 speed assay is shown in Figure 3. Briefly, parasite growth in the presence of anti-malarial compounds was assessed using the [3H]hypoxanthine incorporation assay and expressed as IC50 values [17]. For each compound, three incubation times were employed: 72 (standard assay time), 48 and 24 hours. In the case of the 72- and 48-hour assays, radioactive hypoxanthine was added for the last 24 hours. In the case of the 24-hour assay, [3H]hypoxanthine was added during the last eight hours. IC50 values in the standard 72-hour assay for chloroquine, artesunate, atovaquone, pyrimethamine, 1, 2 and 3 were previously found to be 5.1 ± 0.8 [14], 1.6 ± 0.1 [14], 0.38 ± 0.04 [18], 5.6 ± 0.5 [18], 18 ± 1 [13], 26 ± 4 [14] and 9.5 ± 2.6 [16] ng/mL.


Fast in vitro methods to determine the speed of action and the stage-specificity of anti-malarials in Plasmodium falciparum.

Le Manach C, Scheurer C, Sax S, Schleiferböck S, Cabrera DG, Younis Y, Paquet T, Street L, Smith P, Ding XC, Waterson D, Witty MJ, Leroy D, Chibale K, Wittlin S - Malar. J. (2013)

Schematic representation of the twoin vitroassays. The “IC50 speed assay” and “stage-specificity analysis” are performed with unsynchronized and synchronized parasite cultures, respectively. R, rings; S, schizonts.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3842807&req=5

Figure 3: Schematic representation of the twoin vitroassays. The “IC50 speed assay” and “stage-specificity analysis” are performed with unsynchronized and synchronized parasite cultures, respectively. R, rings; S, schizonts.
Mentions: A schematic representation of the IC50 speed assay is shown in Figure 3. Briefly, parasite growth in the presence of anti-malarial compounds was assessed using the [3H]hypoxanthine incorporation assay and expressed as IC50 values [17]. For each compound, three incubation times were employed: 72 (standard assay time), 48 and 24 hours. In the case of the 72- and 48-hour assays, radioactive hypoxanthine was added for the last 24 hours. In the case of the 24-hour assay, [3H]hypoxanthine was added during the last eight hours. IC50 values in the standard 72-hour assay for chloroquine, artesunate, atovaquone, pyrimethamine, 1, 2 and 3 were previously found to be 5.1 ± 0.8 [14], 1.6 ± 0.1 [14], 0.38 ± 0.04 [18], 5.6 ± 0.5 [18], 18 ± 1 [13], 26 ± 4 [14] and 9.5 ± 2.6 [16] ng/mL.

Bottom Line: This has the advantage that initial results can be achieved within 4-7 working days, which helps to distinguish between fast and slow-acting compounds relatively quickly.The results obtained for the anti-malarials chloroquine, artesunate, atovaquone, and pyrimethamine are consistent with previous observations, suggesting the methodology is a valid way to rapidly identify fast-acting anti-malarial compounds.Another advantage of the approach is its ability to discriminate between static or cidal compound effects.

View Article: PubMed Central - HTML - PubMed

Affiliation: Parasite Chemotherapy Unit, Swiss Tropical and Public Health Institute, Basel, Switzerland. sergio.wittlin@unibas.ch.

ABSTRACT

Background: Recent whole cell in vitro screening campaigns identified thousands of compounds that are active against asexual blood stages of Plasmodium falciparum at submicromolar concentrations. These hits have been made available to the public, providing many novel chemical starting points for anti-malarial drug discovery programmes. Knowing which of these hits are fast-acting compounds is of great interest. Firstly, a fast action will ensure rapid relief of symptoms for the patient. Secondly, by rapidly reducing the parasitaemia, this could minimize the occurrence of mutations leading to new drug resistance mechanisms.An in vitro assay that provides information about the speed of action of test compounds has been developed by researchers at GlaxoSmithKline (GSK) in Spain. This assay also provides an in vitro measure for the ratio between parasitaemia at the onset of drug treatment and after one intra-erythrocytic cycle (parasite reduction ratio, PRR). Both parameters are needed to determine in vitro killing rates of anti-malarial compounds. A drawback of the killing rate assay is that it takes a month to obtain first results.

Methods: The approach described in the present study is focused only on the speed of action of anti-malarials. This has the advantage that initial results can be achieved within 4-7 working days, which helps to distinguish between fast and slow-acting compounds relatively quickly. It is expected that this new assay can be used as a filter in the early drug discovery phase, which will reduce the number of compounds progressing to secondary, more time-consuming assays like the killing rate assay.

Results: The speed of action of a selection of seven anti-malarial compounds was measured with two independent experimental procedures using modifications of the standard [3H]hypoxanthine incorporation assay. Depending on the outcome of both assays, the tested compounds were classified as either fast or non-fast-acting.

Conclusion: The results obtained for the anti-malarials chloroquine, artesunate, atovaquone, and pyrimethamine are consistent with previous observations, suggesting the methodology is a valid way to rapidly identify fast-acting anti-malarial compounds. Another advantage of the approach is its ability to discriminate between static or cidal compound effects.

Show MeSH
Related in: MedlinePlus