Ookluc: A Plasmodium berghei Line for Identifying Transmission-blocking Compounds

Our research aims to identify and characterize transmission-blocking compounds and essential genes in Plasmodium sexual stages using high-throughput system to support novel strategies for interrupting malaria transmission. This protocol addresses the lack of standardized, high-throughput methods for identifying transmission-blocking compounds by using a transgenic P.berghei line with luminescence phase automated detection of post-fertilization parasite development. This protocol enables the screening of Plasmodium sexual stages, detects early transmission-blocking compounds, and supports precise IC50 determination, offering advantages over conventional methods.

To begin, prepare the compound samples by diluting the compound stock to one millimolar using ookinete medium. Prepare the working samples by diluting this one-millimolar stock in ookinete medium at a ratio of one-to-100 to achieve a final concentration of 10 micromolar. For one assay, prepare a final volume of 200 microliters of working sample in duplicate.

If the compound is dissolved in a solvent other than water, prepare a control sample by diluting the solvent in the ookinete medium in the same proportion used for the test sample. Using two 96-well plates, place 80 microliters of each sample in designated wells for positive control, negative control, solvent control, and test samples in duplicate following the given layout. Add four microliters of parasitized mouse blood to each well at a one-to-20 ratio, except the negative control wells.

Homogenize gently by pipetting up and down. For the negative control, add non-infected mouse blood to each well at a one-to-20 ratio and homogenize gently by pipetting. Place the plates in an incubator set to 21 degrees Celsius, and incubate for either six hours or 24 hours.

After incubation, homogenize each sample by pipetting up and down to ensure proper blood medium mixing. Prepare the lysis buffer and luciferase substrate mixture at a one-to-50 ratio immediately before use. Mix this lysis buffer substrate mixture with each sample at a one-to-one ratio, ensuring gentle mixing without introducing bubbles.

Incubate the plate at 37 degrees Celsius for three to five minutes. Now, transfer the samples to a 96-well white flat-bottom plate and measure the luminescence using a plate reader. After the luminescence assay, record the raw luminescence data displayed by the plate reader.

Using a spreadsheet, calculate the mean of each measurement using the given formula. Replace XX with the address of the first measurement cell and YY with the address of the second measurement cell. Repeat the formula for all samples.

Convert the data into percentage inhibition using the given formula, where A is the sample relative light unit value and B is the control relative light unit value. Select compounds that show more than 95%inhibition for the inhibitory concentration 50%determination. After infecting mice with Ookluc and collecting parasitized blood, prepare compound samples using ookinete medium as a diluent at the initial concentration that showed 100%inhibition of conversion.

Pipette the samples in triplicate into a 96-well plate following the specified layout. Using a pipette, perform twofold serial dilutions by transferring 80 microliters from well D1 into D2, mixing thoroughly, then transferring 80 microliters from D2 to D3 and continuing this process through to D12. Discard the final 80 microliters removed from D12 to maintain 80 microliters per well.

Now, add parasitized mouse blood to each well at a one-to-20 ratio and homogenize gently by pipetting. In the negative control wells, add non-infected mouse blood at a one-to-20 ratio and homogenize gently. Place the plate in an incubator set to 21 degrees Celsius, and incubate for either six or 24 hours before performing the luminescence assay.

After calculating percentage inhibition, open the analysis software and create an XY table for plotting. Set the y-axis to enter three replicate values in side-by-side subcolumns. Paste the data into the table with x representing compound concentration and y representing percentage inhibition.

Now, click on Analyze. Select Nonlinear regression, curve fit, then choose Dose-response curves, Inhibition, followed by Inhibitor vs. normalized response, variable slope, and click OK.View the dose-response curve on the left panel and apply customizations, such as color or point format changes.

Locate the nonlinear fit table to find the calculated IC50 and 95%confidence interval. Pyrimidine azepine 90 demonstrated a clear concentration-dependent inhibition of target activity with greater than 95%inhibition observed at concentrations from 0.125 to one micromolar, and complete loss of activity below 0.007 micromolar, suggesting a sharp threshold effect in its inhibitory response. The dose-response curve fitted to the pyrimidine azepine 90 data yielded an IC50 value of 0.013 micromolar, indicating high potency in inhibiting the targeted activity.