Anti-Malarial Hsp90 Inhibitors Measured in Microorganism System Using Plate Reader - 2121-01_Inhibitor_SinglePoint_HTS_Activity_Set2
Assay Overview: Approximately 2 million deaths a year result from infections with the malarial parasite Plasmodium falciparum, the most deadly human parasitic disease. Drug-resistant strains of this parasite have emerged and threaten the utility of conventional anti-malarial compounds used to fight this disease. In a variety of fungal species, it has been shown that HSP90 inhibitors can reverse more ..
BioActive Compounds: 972
Keywords: Plasmodium falciparum, malaria, Hsp90
Assay Overview: Approximately 2 million deaths a year result from infections with the malarial parasite Plasmodium falciparum, the most deadly human parasitic disease. Drug-resistant strains of this parasite have emerged and threaten the utility of conventional anti-malarial compounds used to fight this disease. In a variety of fungal species, it has been shown that HSP90 inhibitors can reverse drug resistance, and this may be true for other eukaryotic pathogens such as P. falciparum. Furthermore, HSP90 inhibitors show potent anti-malarial activity against Plasmodium strains in culture. As global inhibition of HSP90 may be harmful in disease-compromised individuals, here we propose a strategy to discover compounds that specifically inhibit the malarial homolog of this protein.
The goal of this project is to identify inhibitors of the protein HSP90 specific to the malarial species P.falciparum. As strains of P. falciparum have developed resistance to conventional anti-malarial drugs and spread worldwide, creating drugs that operate through a novel mechanism is critically important. Previous work shows both that inhibition of HSP90 in fungi can reverse drug resistance and that inhibitors of HSP90 are very potent in killing P. falciparum in infected red blood cells, therefore new drugs that target HSP90 may provide an excellent method to combat this deadly disease.
Strains of yeast with PfHsp90 have shown some utility as a tool to understand biology of this important protein,
however a strains with wild-type PfHsp90 are limited in their utility for a small molecule screen by a slow growth phenotype. Previous work demonstrated that these yeast strains did not grow well enough to evaluate the effect of a drug on survival. We have created yeast strains with a single point mutation that allows them to grow at a much increased rate, and therefore makes them much more useful in a screening context. We have engineered yeast strains that survive and grow using either the P. falciparum (Pf) or human (Hs) ortholog to replace the fungal homolog of the essential protein Hsp90. These strains respond to known Hsp90 inhibitors, and show the expected selectivity of inhibition of HsHsp90 strains over PfHsp90 yeast.
Yeast strain W303-alpha-delta-pdr1-delta-pdr3 with a Trp-2micron plasmid with PfHsp90 under the GPD promoter are inoculated
from a plate culture (maintained at 4 deg C for less than 4 weeks) into 25 mL of SD-Trp synthetic yeast medium and shaken overnight at 23 deg C.Optical density is diluted down to a final OD600 of 0.00015 in assay media; radicicol is added to positive control wells. Test compounds are transferred using the 100 nL robotic pinner to the wells of 384-well plates that have been previously filled with 40 microL of synthetic yeast media with yeast inoculums. Negative control wells are pinned with an equal volume of DMSO. After 48 hour incubation @ 30 degrees C., luminescence reagent bactiterglo, which measures cellular ATP as a proxy for viability, is added and plates are read.
Expected Outcome: Inhibitors of the essential Pf Hsp90 will inhibit growth of the yeast, resulting in a loss of luminescent signal relative to the neutral controls. Later assays will determine whether this is due to specific inhibition of Hsp90 or due to general toxicity.l
SD-Trp synthetic medium: Per 1L:
1.7 g YNB without amino acids and ammomium sulfate (Sigma part #Y1250)
0.74g CSM-Tryp (Sunrise Science part # 1007-100)
1g glutamic acid monosodium salt (Sigma part # G1626)
20g D-glucose (Sigma part# G7021)
1) Inoculate S. cerevisiae (W303-alpha-delta-pdr1-delta-pdr3 with a Trp-2micron plasmid with PfHsp90 under the GPD promoter) into 25 mL of SD-Trp synthetic yeast medium
2) Shake overnight at 23 deg C.
3) Dilute down to a final OD600 of 0.00015 in assay media (SD-Trp medium)
4) Radicicol in assay medium is added to positive control wells to 20 uM final concentration.
5) Dispense 40 uL culture in 384-well plates using a Combi multidrop dispenser (Thermo)
6) Test compounds are transferred using the 100 nL robotic pin tool (HiRes Biosolutions)
7) After 48 hour incubation @ 30 degrees C., luminescence reagent bactiterglo (Promega) is added, 30 uL per well diluted 4x from manufacturers stock with PBS. Incubate 10 minutes at room temperature.
8) Plates are read on an Envision plate reader (Perkin Elmer), standard luminescence, 0.1s/well.
PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.
Experimental wells values were scaled to this range.
PATTERN CORRECTION: The plate pattern correction algorithm 'Runwise Pattern (Multiplicative)' in Genedata (v7.0.3) was applied to the normalized plate data.
This was set as equal to -1 * the mean of the normalized and corrected sample replicate activities, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at 75.
PERCENTAGE OF ACTIVE REPLICATES:
For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.
The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at 100.
Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_T
Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_T
Samples passing AT only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T
Samples passing PAR_T only were assigned an outcome of 2 (active) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T
** Test Concentration.
Data Table (Concise)