Anti-Malarial Hsp90 Inhibitors Measured in Microorganism System Using Plate Reader - 2121-01_Inhibitor_Dose_CherryPick_Activity
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: 896
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 degrees C for less than 4 weeks) into 25 mL of SD-Trp synthetic yeast medium and shaken overnight at 23 degrees 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.
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 degrees 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 (CyBio)
7) After 48 hour incubation @ 30 degrees C luminescence reagent bactiterglo (Promega) is added, 20 uL per well.
8) Plates are read on an Envision plate reader (Perkin Elmer), standard luminescence, 0.1s/well.
PRESENCE OF CONTROLS: Neutral control wells (NC; n=36) and positive control wells (PC; n=36) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
The compounds were assayed in multiple independent instances using an identical protocol; each instance is called a 'test'. For each test, the following analysis was applied:
ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal (10)(Max_Concentration).
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: No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied.
MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): AC50
AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
AC values were calculated up to the active concentration limit described for each sample.
pAC was set to equal -1*log10(AC)
Activity_Outcome = 1 (inactive) when:
a) compound shows activity but in a direction opposite to the expected outcome
in these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to null
b) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
c) AC > AC_limit
Activity_Outcome = 2 (active) when:
AC <= AC_limit
Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= -70% but <= -30%, or
b) The fit was deemed not valid due to poor fit quality.
If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),
then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
When the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.
When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.
Once the data for each test was processed, the test number was appended to all column headers in that test's data set. The individual test results were then aggregated as follows:
1. The final PUBCHEM_ACTIVITY_OUTCOME was set to:
a. '2' (active) when all test outcomes were '2' (active), or
b. '1' (inactive) when all test outcomes were '1' (inactive), or
c. '3' (inconclusive) when the test outcomes were mixed.
2. The final ACTIVE_CONCENTRATION (AC) was set as follows:
a. If the final PUBCHEM_ACTIVITY_OUTCOME = 2, AC was set as the mean of the constituent test active concentrations;
b. If the final PUBCHEM_ACTIVITY_OUTCOME = 1 or 3, AC was left empty.
3. The final PUBCHEM_ACTIVITY_SCORE was calculated based on the aggregated ACTIVE_CONCENTRATION, using the same logic described above for individual test scores.
The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.
All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.
Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)