Bookmark and Share
BioAssay: AID 624349

A screen for compounds that inhibit liver stage malaria

In humans, a malaria infection begins with a one-time, asymptomatic liver stage followed by a cyclic symptomatic blood stage. All high-throughput malaria drug discovery efforts have focused on the cyclic blood stage, and this focus has limited potential for the prophylaxis, transmission-blocking, and eradication efforts needed for the next stages of malaria treatment. A high-throughput more ..
_
   
 Tested Compounds
 Tested Compounds
All(3266)
 
 
Active(56)
 
 
Inactive(3243)
 
 
 Tested Substances
 Tested Substances
All(5370)
 
 
Active(59)
 
 
Inactive(5311)
 
 
 Related BioAssays
 Related BioAssays
AID: 624349
Data Source: ICCB-Longwood/NSRB Screening Facility, Harvard Medical School (HMS980)
Depositor Category: Other
Deposit Date: 2012-06-28

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 56
Related Experiments
AIDNameTypeComment
624392A screen for known kinase inhibitors that affect liver stage malariaOtherdepositor-specified cross reference
686982A confirmatory screen for known kinase inhibitors that affect liver stage malariaOtherdepositor-specified cross reference
Description:
In humans, a malaria infection begins with a one-time, asymptomatic liver stage followed by a cyclic symptomatic blood stage. All high-throughput malaria drug discovery efforts have focused on the cyclic blood stage, and this focus has limited potential for the prophylaxis, transmission-blocking, and eradication efforts needed for the next stages of malaria treatment. A high-throughput phenotypic liver-stage screen was developed to systematically identify molecules with liver stage efficacy that could address these unmet needs. The screen recapitulates liver-stage infection by isolating luciferase-expressing Plasmodium berghei parasites directly from the salivary glands of infected mosquitoes, adding them to confluent human liver cells in 384-well plates, and measuring luciferase activity after a suitable incubation period.
Protocol
The transgenic P. berghei line expresses firefly luciferase (LucIAV) and was generated in the reference clone of ANKA strain cl15cy1. Parasites contain the PbLuc gene fusion stably integrated as a single copy gene by double-crossover recombination.

One day before screening, HepG2 cells (biosource provider: ATCC, HB-8065) were seeded in 384-well plates at ~17,500 cell/well in DMEM (Invitrogen), 10% FBS (Sigma), and 1% penicillin-streptomycin-amphotericin B (Invitrogen), using a multichannel pipette.

On the day of screening, the cell medium was exchanged for new medium (25 microL), and 100 nL of each compound were pin-transferred to each plate. For every compound library plate, there were four daughter plates (2 plates containing HepG2 cells and another 2 plates containing HepG2 cells and Plasmodium berghei sporozoites). 1-4 hours after compound transfer, 4000 sporozoites (5 microL/well) were added to all wells in the 2 parasite plates, and these plates were centrifuged for 10 minutes at 1000 rpm. The sporozoites were isolated by dissection of the salivary glands of live malaria-infected mosquitoes, and they were added to the assay within 4 hours of dissection. Positive and negative controls were included on every assay plate (halofuginone at 1 microM final concentration and DMSO at 0.3% final concentration, respectively). Plates were then incubated at 37 C in a standard tissue culture incubator for 24 hrs. The cell medium was then exchanged again for new medium (30 microL), and 100 nL of each compound were pin-transferred to each plate. Plates were then incubated at 37 C in a standard tissue culture incubator for an additional 20-24 hours.

To obtain HepG2 liver cell viability data, CellTiter-Glo luciferase reagent (Promega) was added to all wells in the plates containing only HepG2 cells. To measure parasite load, Bright-Glo luciferase reagent (Promega) was added to all wells in the plates containing HepG2 cells infected with parasites. Plates were then read for luminescence signal in a PerkinElmer EnVision.

Library plates were screened in duplicate, with both assay plates in a given set prepared on the same day. For every compound library plate, there were four daughter plates (2 plates containing HepG2 cells and another 2 plates containing HepG2 cells and P. berghei sporozoites.
Comment
Experimental well luminescence values for P. berghei parasite load and for HepG2 liver cell viability were normalized to plate negative controls by dividing well luminescence by the plate average negative control luminescence and multiplying by 100. Parasite activity for each replicate was calculated by subtracting the parasite well luminescence value from plate average negative control luminescence, dividing by the difference between plate average negative and positive control parasite luminescence, subtracting the resulting value from 1, and multiplying by 100. Wells that had parasite activity < 5% (i.e., > 95% inhibition) for at least one parasite replicate and > 80% liver cell viability (percent of negative control > 80%) were considered active.

The activity score represents compound activity and was calculated by subtracting the average of replicate parasite activity from 100, rounded to the nearest integer. Activity values < 0 were scored as 0. An activity score of 100 indicates 100% inhibition of parasite load in HepG2 liver cells relative to the halofuginone positive control plate average.
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Luminescence Parasite_ALuminescence, P. berghei parasites expressing firefly luciferase (LucIAV)Integer
2Luminescence Parasite_BLuminescence, P. berghei parasites expressing firefly luciferase (LucIAV)Integer
3Luminescence Liver_ALuminescence, HepG2 liver cells without parasites, replicate AFloat
4Luminescence Liver_BLuminescence, HepG2 liver cells without parasites, replicate BFloat
5Parasite % Control_AP. berghei luminescence, normalized to plate average negative control for replicate AFloat%
6Parasite % Control_BP. berghei luminescence, normalized to plate average negative control for replicate BFloat%
7Liver % Control_AHepG2 luminescence, normalized to plate average negative control for replicate AFloat%
8Liver % Control_BHepG2 luminescence, normalized to plate average negative control for replicate BFloat%
9Activity Parasite_AP. berghei parasite luminescence, standardized to replicate A positive and negative control plate average luminescenceFloat%
10Activity Parasite_BP. berghei parasite luminescence, standardized to replicate B positive and negative control plate average luminescenceFloat%

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
PageFrom: