We can provide a fee for service contract through UCSD to screen compounds against asexual blood stage of the parasite. If you are interested, please email ewinzeler@health.ucsd.edu for more details.
The services we are currently offering are as follows:
Plasmodium berghei liver stage screen
This assay is based on the murine Plasmodium berghei species transformed with Luciferase. Hepatic human transformed cells (HepG2), pretreated for 18 hours with the compound to investigate in 1536 well plates, are infected with freshly dissected P. berghei Luciferase sporozoites. After another 48 hours of incubation with the compound to investigate, the viability of P. berghei exoerythrocytic forms (EEF) is measured by bioluminescence.
This assay allows identification of compounds with an eventual activity against sporozoite infection of liver cell as well the viability of liver schizonts.
Number of compounds: 22 compounds in 1:3 12-point dose response in quadruplicates or 1300 compounds at concentration with one technical replicate.
Cyto-toxicity assay
The CellTiter-Glo® Luminescence Cell Viability Assay is a method to determine the number of viable cells in culture based on quantitation of the ATP present, which signals the presence of metabolically active cells. The assay allows us to identify compounds with cytoxic activity for the mammalian cells.
HepG2, Huh7.5.1 and HC04 hepatoma cell lines can be used. All cell lines are cultured at 37 °C and 5% CO2 in DMEM (Invitrogen, Carlsbad, USA) supplemented with 10% FBS , 0.29 mg/mL glutamine, 100 units of penicillin, and 100 μg/mL streptomycin.
Number of compounds: 22 in 1:3 12-point dose response in quadruplicates or 1300 compounds at single point concentration with one technical replicate.
Asexual blood stage assay
This assay is adapted from Plouffe et al 2008: The a-synchronized P. falciparum (DD2 strain) blood stage growth as indirectly measured through DNA amount with SYBR green. Blood stage parasite cultures are treated with compound in 1536 well plates and incubated for 72 hours. Whole well fluorescence is then measured after the addition of SYBR green and is used to evaluate parasite growth. This assay allows us to identify compounds with activity against the symptomatic parasite blood stages.
Additionally, the actives can be screened against parasite lines which have been genetically edited for some of the known anti-malarial resistance genes to identify compounds with novel mechanism of action.
Number of compounds: 22 in 1:3 12-point dose response in quadruplicates or 1300 compounds at single point concentration with one technical replicate.
MIR assay
The minimal inoculum for resistance (MIR) can be determined and is an indirect measure of the likelihood of a resistant genotype to occur and to be selected in vitro. In here, parasites at a starting inoculum ranging from 105 to 109 are exposed to a concentration of the compound nearing IC90 and monitored for 60 days for recrudescent parasites. MIR can be used to flag compounds at high risk of resistance. Compounds which show MIR> 8 can then be further assessed for genome-based target discovery.
Resistome selection
To find the target of the compound of interest, in vitro evolution and whole genome analysis (IVIEWGA) can be performed. In this method, 1 x 10^8 parasites will be exposed to a desired concentration of the compound (i.e.1x IC50, 3x IC50 etc.) Upon initiation, parasite health will be checked (i.e. count parasitemia and note morphological changes) every day for the first week (5-7 days). Once the parasites seem to be growing steadily, the dose may be increased by 20-50% of the previous concentration making sure that we have at least 1 x 10^8 parasites for each flask. Once again, the health of the parasites will be monitored. Once the parasites are growing steadily in drug, IC50 will be monitored for a stable shift. At this point the resistant parasite line can then be shipped to the requester for further analysis.
Note: Not all compounds will produce stable resistant mutants in selection experiments. Hence the experiment will be terminated at day 60 if no stable IC50 shift is observed.
We can also help in identifying the genetic basis of their resistance. For this the bulk parasite population will be cloned by limiting dilution. This typically takes 30 days. Once the clones are obtained, they will be evaluated for stable IC50 shift. The DNA will be isolated from the stable clones, followed by library prep and whole genome sequencing (WGS). The resistance mechanism will be identified by comparing the genomes of the resistant clones to the sensitive parent clone. The identified mutations will be provided in excel. The turn around time for this is approximately four months.