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BioAssay: AID 588680

Counterscreen for inhibitors of PFM18AAP: QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M1AAP (PFM1AAP)

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 more ..
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 Tested Compounds
 Tested Compounds
All(63)
 
 
Active(26)
 
 
Inactive(37)
 
 
 Tested Substances
 Tested Substances
All(63)
 
 
Active(26)
 
 
Inactive(37)
 
 
AID: 588680
Data Source: Southern Research Specialized Biocontainment Screening Center (PlamM18_M1_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 ):           Active    All
Target
BioActive Compounds: 26
Depositor Specified Assays
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AIDNameTypeComment
1855Summary of probe development efforts to identify inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (M18AAP)summary
588714Vero Cytoxicity Assay: A Cell Based Secondary Assay To Explore Cytotoxicity of Compounds that Inhibit Plasmodium falciparum M18 Aspartyl Aminopeptidase (PFM18AAP)confirmatory
602219Counterscreen for inhibitors of PFM18AAP: QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M1AAP (PFM1AAP) (2)confirmatory
602225Vero Cytoxicity Assay: A Cell Based Secondary Assay To Explore Cytotoxicity of Compounds that Inhibit Plasmodium falciparum M18 Aspartyl Aminopeptidase (PFM18AAP) (2)confirmatory
624174Counterscreen for inhibitors of PFM18AAP: QFRET-based biochemical high throughput dose response assay for inhibitors of the human M18 Aspartyl Aminopeptidase (hM18AAP) (3)confirmatory
624175Counterscreen for inhibitors of PFM18AAP: QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M17 Leucine Aminopeptidase (PFM17LAP) (3)confirmatory
624176Counterscreen for inhibitors of PFM18AAP: QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M1AAP (PFM1AAP) (3)confirmatory
624177QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (PFM18AAP) (2)confirmatory
624205Vero Cytoxicity Assay: A Cell Based Secondary Assay To Explore Cytotoxicity of Compounds that Inhibit Plasmodium falciparum M18 Aspartyl Aminopeptidase (PFM18AAP) (3)confirmatory
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, Set2other
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.confirmatory
Description:
Southern Research Specialized Biocontainment Screening Center (SRSBSC)
Assay Provider: Donald Gardiner
Award: 1 R03 MH084103-01

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.
The intraerythrocytic stages of the human malaria parasite Plasmodium falciparum employs two cytosolic neutral aminopeptidases, an M1-family alanyl aminopeptidase (M1AAP) and an M17-family leucine aminopeptidase (M17LAP), in the terminal stages of host hemoglobin digestion. Thus, Plasmodium falciparum M1 alanyl-family leucine aminopeptidase (M1AAP) serves as a relevant counterscreen target for the M18AAP enzyme described above.
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-Leu-NHMec) is incubated with purified recombinant PFM1AAP enzyme (rPFM1AAP) in the presence of test compounds. Cleavage of the substrate by rPFM1AAP enzyme liberates the NHMec leaving group from the peptide, leading to increased well fluorescence. As designed, compounds that inhibit PFM1AAP will block rPFM1AAP-mediated cleavage of HLeu-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 PFM1AAP reagent mix, which included the fluorogenic peptide substrate (H-Leu-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 thePFM1AAP diluted in assay buffer. The final concentrations in the reaction were 0.1 mM H-Leu-NHMec and 5 ug/ml PFM1AAP 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 PFM1AAP 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 PFM1AAP 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.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Average PFM1AAP IC50*FloatμM
2PFM1AAP IC50 Rep 1FloatμM
3PFM1AAP IC50 Rep 2FloatμM
4PFM1AAP IC50 Rep 3FloatμM
5PFM1AAP IC50 Std Dev Rep 1Float
6PFM1AAP IC50 Hill Slope Rep 1Float
7PFM1AAP IC50 Normalized Chi2 Rep 1Float
8PFM1AAP IC50 Std Dev Rep 2Float
9PFM1AAP IC50 Hill Slope Rep 2Float
10PFM1AAP IC50 Normalized Chi2 Rep 2Float
11PFM1AAP IC50 Std Dev Rep 3Float
12PFM1AAP IC50 Hill Slope Rep 3Float
13PFM1AAP 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)
Classification
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