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BioAssay: AID 588678

QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (PFM18AAP)

Aminopeptidases (APs) are metalloproteases that cleave amino-terminal (N-terminal) amino acids during protein synthesis. These enzymes are characterized in part by their post-translational removal of leucine, aspartate, proline, methionine, etc from proteins and peptides, in order that proteins are properly regulated, targeted for degradation, and trafficked within both animal and plant cells. more ..
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 Tested Compounds
 Tested Compounds
All(63)
 
 
Active(23)
 
 
Inactive(40)
 
 
 Tested Substances
 Tested Substances
All(63)
 
 
Active(23)
 
 
Inactive(40)
 
 
AID: 588678
Data Source: Southern Research Specialized Biocontainment Screening Center (PlamM18_M18_1)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-10-25
Hold-until Date: 2012-09-30
Modify Date: 2012-10-01

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 23
Related Experiments
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AIDNameTypeComment
1855Summary of probe development efforts to identify inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (M18AAP)Summarydepositor-specified cross reference
588714Vero Cytoxicity Assay: A Cell Based Secondary Assay To Explore Cytotoxicity of Compounds that Inhibit Plasmodium falciparum M18 Aspartyl Aminopeptidase (PFM18AAP)Confirmatorydepositor-specified cross reference
602222QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (PFM18AAP) (1)Confirmatorydepositor-specified cross reference
602225Vero Cytoxicity Assay: A Cell Based Secondary Assay To Explore Cytotoxicity of Compounds that Inhibit Plasmodium falciparum M18 Aspartyl Aminopeptidase (PFM18AAP) (2)Confirmatorydepositor-specified cross reference
624174Counterscreen for inhibitors of PFM18AAP: QFRET-based biochemical high throughput dose response assay for inhibitors of the human M18 Aspartyl Aminopeptidase (hM18AAP) (3)Confirmatorydepositor-specified cross reference
624175Counterscreen for inhibitors of PFM18AAP: QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M17 Leucine Aminopeptidase (PFM17LAP) (3)Confirmatorydepositor-specified cross reference
624176Counterscreen for inhibitors of PFM18AAP: QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M1AAP (PFM1AAP) (3)Confirmatorydepositor-specified cross reference
624177QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (PFM18AAP) (2)Confirmatorydepositor-specified cross reference
624205Vero Cytoxicity Assay: A Cell Based Secondary Assay To Explore Cytotoxicity of Compounds that Inhibit Plasmodium falciparum M18 Aspartyl Aminopeptidase (PFM18AAP) (3)Confirmatorydepositor-specified cross reference
720736Late stage assay provider results from the probe development effort to identify inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (M18AAP): radiolabel-based cell-based assay to identify compounds that inhibit P. falciparum growth in RBCs, Set2Otherdepositor-specified cross reference
743024Late stage assay provider results from the probe development effort to identify inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (M18AAP): radiolabel-based cell-based dose response assay to identify compounds that inhibit P. falciparum growth in RBCs, Set 2.Confirmatorydepositor-specified cross reference
1822QFRET-based primary biochemical high throughput screening assay to identify inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (PFM18AAP).Screeningsame project related to Summary assay
1906QFRET-based counterscreen for PFM18AAP inhibitors: biochemical high throughput screening assay to identify inhibitors of the Cathepsin L proteinase (CTSL1).Screeningsame project related to Summary assay
2170QFRET-based biochemical high throughput confirmation assay for inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (PFM18AAP).Screeningsame project related to Summary assay
2178QFRET-based counterscreen for inhibitors of PFM18AAP: biochemical high throughput confirmation assay for inhibitors of the Cathepsin L proteinase (CTSL1).Screeningsame project related to Summary assay
2195QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (PFM18AAP).Confirmatorysame project related to Summary assay
2196QFRET-based counterscreen for inhibitors of PFM18AAP: biochemical high throughput dose response assay for inhibitors of the Cathepsin L proteinase (CTSL1).Confirmatorysame project related to Summary assay
489011Late stage assay provider results from the probe development effort to identify inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (M18AAP): radiolabel-based cell-based dose response assay to identify compounds that inhibit P. falciparum growth in RBCsConfirmatorysame project related to Summary assay
489015Late stage assay provider results from the probe development effort to identify inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (M18AAP): radiolabel-based cell-based assay to identify compounds that inhibit P. falciparum growth in RBCsOthersame project related to Summary assay
492974Late stage assay provider results from the probe development effort to identify inhibitors of the Plasmodium falciparum M18 Alanyl Aminopeptidase (PfM18AAP): fluorescence-based biochemical assay to identify inhibitors of rPfM18AAPScreeningsame project related to Summary assay
492975Late stage assay provider results from the probe development effort to identify inhibitors of the Plasmodium falciparum M18 Alanyl Aminopeptidase (PfM18AAP): fluorescence-based biochemical assay to identify inhibitors of malaria cell lysateScreeningsame project related to Summary assay
588679Counterscreen for inhibitors of PFM18AAP: QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M17 Leucine Aminopeptidase (PFM17LAP)Confirmatorysame project related to Summary assay
588680Counterscreen for inhibitors of PFM18AAP: QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M1AAP (PFM1AAP)Confirmatorysame project related to Summary assay
588696Counterscreen for inhibitors of PFM18AAP: QFRET-based biochemical high throughput dose response assay for inhibitors of the human M18 Aspartyl Aminopeptidase (hM18AAP)Confirmatorysame project related to Summary assay
602219Counterscreen for inhibitors of PFM18AAP: QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M1AAP (PFM1AAP) (2)Confirmatorysame project related to Summary assay
602220Counterscreen for inhibitors of PFM18AAP: QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M17 Leucine Aminopeptidase (PFM17LAP) (2)Confirmatorysame project related to Summary assay
602221Counterscreen for inhibitors of PFM18AAP: QFRET-based biochemical high throughput dose response assay for inhibitors of the human M18 Aspartyl Aminopeptidase (hM18AAP) (2)Confirmatorysame project related to Summary assay
Description:
Aminopeptidases (APs) are metalloproteases that cleave amino-terminal (N-terminal) amino acids during protein synthesis. These enzymes are characterized in part by their post-translational removal of leucine, aspartate, proline, methionine, etc from proteins and peptides, in order that proteins are properly regulated, targeted for degradation, and trafficked within both animal and plant cells. As a result, these enzymes are involved in diverse processes, including meiosis, cellular senescence, blood pressure control, angiogenesis, and inflammation. PFM18AAP is the sole aspartyl aminopeptidase (AAP) present in the genome of the malaria parasite Plasmodium falciparum. It exhibits exopeptidase activity exclusively against the N-terminal acidic amino acids glutamate and aspartate, is found in all intra-erythrocytic stages of the parasite, and functions to complete the hydrolysis of host hemoglobin into amino acids for use in de novo protein synthesis by the parasite. Studies demonstrating that genetic knockdown of PFM18AAP results in a lethal parasite phenotype, and that inhibitors of methionine and leucine aminopeptidases prevent malaria growth in culture and hemoglobin degradation, suggest that these enzymes are essential for parasite survival. As a result, the identification of selective inhibitors of PFM18AAP would elucidate this enzyme's role in the P. falciparum lifecycle, and serve as potential therapeutic agents to control malaria infection.
Protocol
The purpose of this assay is to determine dose response curves for compounds identified as active. In this biochemical assay, a commercially available fluorogenic peptide substrate (H-Glu-NHMec) is incubated with purified recombinant PFM18AAP enzyme (rPFM18AAP) in the presence of test compounds. Cleavage of the substrate by rPFM18AAP enzyme liberates the NHMec leaving group from the peptide, leading to increased well fluorescence. As designed, compounds that inhibit PFM18AAP will block rPFM18AAP-mediated cleavage of HGlu-NHMec and liberation of the NHMec leaving group from the substrate, resulting in decreased well fluorescence as measured at 340 nm excitation and 450 nm emission. Test compounds were assayed in triplicate in a 10-point 1:2 dilution series starting at a nominal test concentration of 100 micromolar.
Compound Dosing/Plating: For the dose response assays 10 concentrations of each compound ranging from 100-0.2 uM were dispensed into 1536-well black non-binding surface plates.
Assay Setup: 2.5 uL of PFM18AAP reagent mix, which included the fluorogenic peptide substrate (H-Glu-NHMec) in assay buffer, was added to each well of the previously compound dosed 1536-well plates. The reaction was initiated with the addition of 2.5 uL of thePFM18AAP diluted in assay buffer. The final concentrations in the reaction were 0.1 mM H-Glu-NHMec and 5 ug/ml PFM18AAP diluted in assay buffer (50 mM Tris-HCl (pH 7.5), 2 mM CoCl2, 0.1% BSA, 0.01% Triton X-100, and 2% DMSO). The test plate was incubated at room temperature for 90 minutes, then transferred to a Perkin Elmer Envision microplate reader and fluorescence (RFU)was measured at an excitation wavelength of 370nm and an emission wavelength of 460nm. Each plate had 256 control wells in the eight outside columns with 128 wells containing the complete reaction mixture with carrier control (Full Rxn) and 128 wells in which the PFM18AAP had been left out (Bkg).

Data Analysis: 128 background control wells containing the peptide substrate only and 128 full reaction control wells containing peptide substrate and 5 ug/ml PFM18AAP were included on each assay plate and used to calculate a Z' value for each plate and to normalize the data on a per plate basis. Data were analyzed using the IDBS Activity Base software. Results for each concentration were expressed as percent inhibition (% Inhibition) and was calculated as: 100*((Med Full Rxn RFU- Med Bkg RFU) - (Cmpd RFU - Med Bkg RFU))/ ((Med Full Rxn RFU - Med Bkg RFU)). The dose response data was analyzed using a four parameter logistic fit to the data (Excel Fit equation 205) with the maximum and minimum locked at 100 and 0. From these curves IC50 values were calculated.
Comment
Possible artifacts in this assay include, but are not limited to, compounds that fluoresce at 370/460 nm, that absorb at either 370 or 460 nm, or that precipitate.
Outcome: Compounds showing 30% or greater inhibition at any concentration were considered "Active". IC50 values were calculated for these compounds and used to determine the relative score.
The following tiered system has been implemented at Southern Research Institute for use with the PubChem Score: Compounds in the primary screen are scored on a scale of 0-40 based on % activity; a score of 40 corresponds to 100% activity. In the confirmatory dose response screen of primary screen hits, active compounds are scored on a scale of 41-80 based on IC50 result while compounds where activity was not confirmed are given the score 0. Confirmatory dose response and secondary screens of purified and/or resynthesized compounds, indicating the highest degree of confidence) are scored on a scale of 81-100 based on IC50 result. Inactive compounds are given the score 0.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Biochemical
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Average IC50*FloatμM
2M18 IC50 Rep 1FloatμM
3M18 IC50 Rep 2FloatμM
4M18 IC50 Rep 3FloatμM
5M18 IC50 Std Dev Rep 1Float
6M18 IC50 Hill Slope Rep 1Float
7M18 IC50 Normalized Chi2 Rep 1Float
8M18 IC50 Std Dev Rep 2Float
9M18 IC50 Hill Slope Rep 2Float
10M18 IC50 Normalized Chi2 Rep 2Float
11M18 IC50 Std Dev Rep 3Float
12M18 IC50 Hill Slope Rep 3Float
13M18 IC50 Normalized Chi2 Rep 3Float
14% Inhibition @ 100 uM Rep 1 (100μM**)Float%
15% Inhibition @ 50 uM Rep 1 (50μM**)Float%
16% Inhibition @ 25 uM Rep 1 (25μM**)Float%
17% Inhibition @ 12.5 uM Rep 1 (12.5μM**)Float%
18% Inhibition @ 6.25 uM Rep 1 (6.25μM**)Float%
19% Inhibition @ 3.13 uM Rep 1 (3.13μM**)Float%
20% Inhibition @ 1.56 uM Rep 1 (1.56μM**)Float%
21% Inhibition @ 0.78 uM Rep 1 (0.78μM**)Float%
22% Inhibition @ 0.39 uM Rep 1 (0.39μM**)Float%
23% Inhibition @ 0.19 uM Rep 1 (0.19μM**)Float%
24% Inhibition @ 0.095 uM Rep 1 (0.095μM**)Float%
25% Inhibition @ 0.098 uM Rep 1 (0.098μM**)Float%
26% Inhibition @ 100 uM Rep 2 (100μM**)Float%
27% Inhibition @ 50 uM Rep 2 (50μM**)Float%
28% Inhibition @ 25 uM Rep 2 (25μM**)Float%
29% Inhibition @ 12.5 uM Rep 2 (12.5μM**)Float%
30% Inhibition @ 6.25 uM Rep 2 (6.25μM**)Float%
31% Inhibition @ 3.13 uM Rep 2 (3.13μM**)Float%
32% Inhibition @ 1.56 uM Rep 2 (1.56μM**)Float%
33% Inhibition @ 0.78 uM Rep 2 (0.78μM**)Float%
34% Inhibition @ 0.39 uM Rep 2 (0.39μM**)Float%
35% Inhibition @ 0.19 uM Rep 2 (0.19μM**)Float%
36% Inhibition @ 0.095 uM Rep 2 (0.095μM**)Float%
37% Inhibition @ 0.098 uM Rep 2 (0.098μM**)Float%
38% Inhibition @ 100 uM Rep 3 (100μM**)Float%
39% Inhibition @ 50 uM Rep 3 (50μM**)Float%
40% Inhibition @ 25 uM Rep 3 (25μM**)Float%
41% Inhibition @ 12.5 uM Rep 3 (12.5μM**)Float%
42% Inhibition @ 6.25 uM Rep 3 (6.25μM**)Float%
43% Inhibition @ 3.13 uM Rep 3 (3.13μM**)Float%
44% Inhibition @ 1.56 uM Rep 3 (1.56μM**)Float%
45% Inhibition @ 0.78 uM Rep 3 (0.78μM**)Float%
46% Inhibition @ 0.39 uM Rep 3 (0.39μM**)Float%
47% Inhibition @ 0.19 uM Rep 3 (0.19μM**)Float%
48% Inhibition @ 0.095 uM Rep 3 (0.095μM**)Float%
49% Inhibition @ 0.098 uM Rep 3 (0.098μM**)Float%
50VerificationString

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 MH084103-01

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