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BioAssay: AID 2196

QFRET-based counterscreen for inhibitors of PFM18AAP: biochemical high throughput dose response assay for inhibitors of the Cathepsin L proteinase (CTSL1).

Grant Proposal PI: John Dalton and Donald Gardiner, Queensland Institute of Medical Research, Australia ..more
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 Tested Compounds
 Tested Compounds
All(125)
 
 
Inactive(125)
 
 
 Tested Substances
 Tested Substances
All(125)
 
 
Inactive(125)
 
 
AID: 2196
Data Source: The Scripps Research Institute Molecular Screening Center (CTSL1_INH_QFRET_1536_3XIC50)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-12-10
Hold-until Date: 2010-03-01
Modify Date: 2010-03-01

Data Table ( Complete ):           View All Data
Target
Tested Compounds:
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: Summary AID.
2178QFRET-based counterscreen for inhibitors of PFM18AAP: biochemical high throughput confirmation assay for inhibitors of the Cathepsin L proteinase (CTSL1).Screeningdepositor-specified cross reference: Confirmation screen (PFM18AAP inhibitors).
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 RBCsConfirmatorydepositor-specified cross reference
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 RBCsOtherdepositor-specified cross reference
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 rPfM18AAPScreeningdepositor-specified cross reference
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 lysateScreeningdepositor-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
2195QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (PFM18AAP).Confirmatorysame project related to Summary assay
588678QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (PFM18AAP)Confirmatorysame 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
588714Vero Cytoxicity Assay: A Cell Based Secondary Assay To Explore Cytotoxicity of Compounds that Inhibit Plasmodium falciparum M18 Aspartyl Aminopeptidase (PFM18AAP)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
602222QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (PFM18AAP) (1)Confirmatorysame project related to Summary assay
602225Vero Cytoxicity Assay: A Cell Based Secondary Assay To Explore Cytotoxicity of Compounds that Inhibit Plasmodium falciparum M18 Aspartyl Aminopeptidase (PFM18AAP) (2)Confirmatorysame project related to Summary assay
624174Counterscreen for inhibitors of PFM18AAP: QFRET-based biochemical high throughput dose response assay for inhibitors of the human M18 Aspartyl Aminopeptidase (hM18AAP) (3)Confirmatorysame project related to Summary assay
624175Counterscreen for inhibitors of PFM18AAP: QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M17 Leucine Aminopeptidase (PFM17LAP) (3)Confirmatorysame project related to Summary assay
624176Counterscreen for inhibitors of PFM18AAP: QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M1AAP (PFM1AAP) (3)Confirmatorysame project related to Summary assay
624177QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (PFM18AAP) (2)Confirmatorysame project related to Summary assay
624205Vero Cytoxicity Assay: A Cell Based Secondary Assay To Explore Cytotoxicity of Compounds that Inhibit Plasmodium falciparum M18 Aspartyl Aminopeptidase (PFM18AAP) (3)Confirmatorysame project related to Summary assay
Description:
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: John Dalton and Donald Gardiner, Queensland Institute of Medical Research, Australia
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R03 MH084103-01
Grant Proposal PI: John Dalton and Donald Gardiner, Queensland Institute of Medical Research, Australia
External Assay ID: CTSL1_INH_QFRET_1536_3XIC50

Name: QFRET-based counterscreen for inhibitors of PFM18AAP: biochemical high throughput dose response assay for inhibitors of the Cathepsin L proteinase (CTSL1).

Description:

Aminopeptidases (APs) are metalloproteases that cleave amino-terminal (N-terminal) amino acids during protein synthesis (1, 2) . 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 (3). As a result, these enzymes are involved in diverse processes, including meiosis (1), cellular senescence (1), blood pressure control (4, 5), angiogenesis (6), and inflammation (7). PFM18AAP is the sole aspartyl aminopeptidase (AAP) present in the genome of the malaria parasite Plasmodium falciparum (8). It exhibits exopeptidase activity exclusively against the N-terminal acidic amino acids glutamate and aspartate (9-11), is found in all intra-erythrocytic stages of the parasite (9), and functions to complete the hydrolysis of host hemoglobin into amino acids for use in de novo protein synthesis by the parasite (12, 13). Studies demonstrating that genetic knockdown of PFM18AAP results in a lethal parasite phenotype (9), and that inhibitors of methionine (14) and leucine (12, 15) 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.

References:

1. Walling, L.L., Recycling or regulation? The role of amino-terminal modifying enzymes. Curr Opin Plant Biol, 2006. 9(3): p. 227-33.
2. Meinnel, T., Serero, A., and Giglione, C., Impact of the N-terminal amino acid on targeted protein degradation. Biol Chem, 2006. 387(7): p. 839-51.
3. Jankiewicz, U. and Bielawski, W., The properties and functions of bacterial aminopeptidases. Acta Microbiol Pol, 2003. 52(3): p. 217-31.
4. Banegas, I., Prieto, I., Vives, F., Alba, F., de Gasparo, M., Segarra, A.B., Hermoso, F., Duran, R., and Ramirez, M., Brain aminopeptidases and hypertension. J Renin Angiotensin Aldosterone Syst, 2006. 7(3): p. 129-34.
5. Silveira, P.F., Gil, J., Casis, L., and Irazusta, J., Peptide metabolism and the control of body fluid homeostasis. Curr Med Chem Cardiovasc Hematol Agents, 2004. 2(3): p. 219-38.
6. Zhong, H. and Bowen, J.P., Antiangiogenesis drug design: multiple pathways targeting tumor vasculature. Curr Med Chem, 2006. 13(8): p. 849-62.
7. Proost, P., Struyf, S., and Van Damme, J., Natural post-translational modifications of chemokines. Biochem Soc Trans, 2006. 34(Pt 6): p. 997-1001.
8. Wilk, S., Wilk, E., and Magnusson, R.P., Purification, characterization, and cloning of a cytosolic aspartyl aminopeptidase. J Biol Chem, 1998. 273(26): p. 15961-70.
9. Teuscher, F., Lowther, J., Skinner-Adams, T.S., Spielmann, T., Dixon, M.W., Stack, C.M., Donnelly, S., Mucha, A., Kafarski, P., Vassiliou, S., Gardiner, D.L., Dalton, J.P., and Trenholme, K.R., The M18 aspartyl aminopeptidase of the human malaria parasite Plasmodium falciparum. J Biol Chem, 2007. 282(42): p. 30817-26.
10. Gyang, F.N., Poole, B., and Trager, W., Peptidases from Plasmodium falciparum cultured in vitro. Mol Biochem Parasitol, 1982. 5(4): p. 263-73.
11. Vander Jagt, D.L., Baack, B.R., and Hunsaker, L.A., Purification and characterization of an aminopeptidase from Plasmodium falciparum. Mol Biochem Parasitol, 1984. 10(1): p. 45-54.
12. Nankya-Kitaka, M.F., Curley, G.P., Gavigan, C.S., Bell, A., and Dalton, J.P., Plasmodium chabaudi chabaudi and P. falciparum: inhibition of aminopeptidase and parasite growth by bestatin and nitrobestatin. Parasitol Res, 1998. 84(6): p. 552-8.
13. Lauterbach, S.B. and Coetzer, T.L., The M18 aspartyl aminopeptidase of Plasmodium falciparum binds to human erythrocyte spectrin in vitro. Malar J, 2008. 7: p. 161.
14. Chen, X., Chong, C.R., Shi, L., Yoshimoto, T., Sullivan, D.J., Jr., and Liu, J.O., Inhibitors of Plasmodium falciparum methionine aminopeptidase 1b possess antimalarial activity. Proc Natl Acad Sci U S A, 2006. 103(39): p. 14548-53.
15. Stack, C.M., Lowther, J., Cunningham, E., Donnelly, S., Gardiner, D.L., Trenholme, K.R., Skinner-Adams, T.S., Teuscher, F., Grembecka, J., Mucha, A., Kafarski, P., Lua, L., Bell, A., and Dalton, J.P., Characterization of the Plasmodium falciparum M17 leucyl aminopeptidase. A protease involved in amino acid regulation with potential for antimalarial drug development. J Biol Chem, 2007. 282(3): p. 2069-80.

Keywords: Cathepsin L, CTSL1, cathepsin L1, CATL, MEP, PFM18AAP, M18AAP, malaria, parasite, plasmodium falciparum, exopeptidase, counterscreen, dose response, HTS, high throughput screen, 1536, inhibitor, inhibition, fluorescence, MCA, QFRET, FLINT, peptide, cleavage, Scripps, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
Protocol
Assay Overview:

The purpose of this counterscreen assay is to determine dose response curves for compounds identified as active in a set of previous experiments entitled, "QFRET-based biochemical high throughput confirmation assay for inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (PFM18AAP)" (AID 2178). In this biochemical assay, a commercially available fluorogenic peptide substrate (Z-Leu-Arg-MCA) is incubated with purified recombinant cathepsin L1 protein in the presence of test compounds. Cleavage of the substrate by cathepsin L1 releases the fluorogenic MCA leaving group, leading to an increase in well fluorescence. As designed, compounds that inhibit cathepsin L1 will prevent substrate cleavage and liberation of the fluorescent leaving group, resulting in decreased well fluorescence. Test compounds were assayed in triplicate in a 10-point 1:3 dilution series starting at a nominal test concentration of 59.6 micromolar.

Protocol Summary:

Prior to the start of the assay, 2.5 microliters of assay buffer (25mM Tris HCl pH7.5, 1mM DTT, 0.1% BSA) containing 1.5micrograms/mL cathepsin L were dispensed into a 1536 microtiter plate. Next, 30 nL of test compound in DMSO, Z-Phe-Ala-diazomethylketone (1micromolar final concentration), or DMSO alone (0.59% final concentration) were added to the appropriate wells. The plates were then incubated for 30 minutes at 25 degrees Celsius.

The assay was started by dispensing 2.5 microliters of 100 micromolar Z-Leu-Arg-MCA substrate in buffer (25 mM Tris HCl, pH 7.5, 1mM DTT) into all wells. Well fluorescence was read immediately (T0) on the Viewlux (Perkin-Elmer) and again after 90 minutes (T90) of incubation at 25 degrees Celsius.

Prior to further calculations, T0 was subtracted from T90 for each individual well. The difference between RFU values read at T0 (RFU_T0) and T90 (RFU_T90), named delta RFU, was calculated as follows:

delta RFU = RFU_T90 - RFU_T0

The percent inhibition for each well was then calculated as follows:

Percent inhibition = ( test_compound_delta RFU - negative_control_delta RFU ) / ( positive_control_delta RFU - negative_control_delta RFU ) * 100

Where:

Test_Compound is defined as wells containing test compound.
Negative_Control is defined as the median of the wells containing Cathepsin L.
Positive_Control is defined as the median of the wells containing Z-Phe-Ala-diazomethylketone.

For each test compound, percent inhibition was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (Symyx Technologies Inc). The reported IC50 values were generated from fitted curves by solving for the X-intercept value at the 50% inhibition level of the Y-intercept value. In cases where the highest concentration tested (i.e. 59.6 micromolar) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 59.6 micromolar. Compounds with an IC50 greater than 10 micromolar were considered inactive. Compounds with an IC50 equal to or less than 10 micromolar were considered active.

Any compound with a percent activity value <50% at all test concentrations was assigned an activity score of zero. Any compound with a percent activity value >50% at any test concentration was assigned an activity score greater than zero. Activity score was then ranked by the potency, with the most potent compounds assigned the highest activity scores.

There are no active compounds in this assay.

List of Reagents:

Cathepsin L enzyme (supplied by Assay Provider)
Z-Leu-Arg-MCA substrate (Peptides International, part MCA-3210-v)
1536-well plates (Greiner, part 789176)
Tris (Amresco, part 0497)
DTT (Invitrogen, part 15508-013)
Z-Phe-Ala-diazomethylketone (Bachem, part N-1040)
BSA (Calbiochem, part 126609)
Comment
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. In this case the results of each separate campaign were assigned "Active/Inactive" status based upon that campaign's specific compound activity cutoff value. All data reported were normalized on a per-plate basis. In this assay, Z-Phe-Ala-diazomethylketone had an IC50 of approximately 5.2 nanomolar. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR. The MLSMR was not able to provide all compounds selected for testing in this AID.
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Binding
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1QualifierActivity Qualifier identifies if the resultant data IC50 came from a fitted curve or was determined manually to be less than or greater than its listed IC50 concentration.String
2IC50*The concentration at which 50 percent of the activity in the inhibitor assay is observed; (IC50) shown in micromolar.FloatμM
3LogIC50Log10 of the qualified IC50 (IC50) from the inhibitor assay in M concentration.Float
4Hill SlopeThe variable HillSlope describes the steepness of the curve. This variable is called the Hill slope, the slope factor, or the Hill coefficient. If it is positive, the curve increases as X increases. If it is negative, the curve decreases as X increases. A standard sigmoid dose-response curve (previous equation) has a Hill Slope of 1.0. When HillSlope is less than 1.0, the curve is more shallow. When HillSlope is greater than 1.0, the curve is steeper. The Hill slope has no units.Float
5Hill S0Y-min of the curve.Float
6Hill SinfY-max of the curve.Float
7Hill dSThe range of Y.Float
8Chi SquareA measure for the 'goodness' of a fit. The chi-square test (Snedecor and Cochran, 1989) is used to test if a sample of data came from a population with a specific distribution.Float
9RsquareThis statistic measures how successful the fit explains the variation of the data; R-square is the square of the correlation between the response values and the predicted response values.Float
10Number of DataPointsOverall number of data points of normalized percent inhibition that was used for calculations (includes all concentration points); in some cases a data point can be excluded as outlier.Integer
11Inhibition at 3.0 nM (0.003μM**)Value of %inhibition at 3.0 nanomolar inhibitor concentration; average of triplicate measurement.Float%
12Inhibition at 9.1 nM (0.009μM**)Value of %inhibition at 9.0 nanomolar inhibitor concentration; average of triplicate measurement.Float%
13Inhibition at 27.3 nM (0.0273μM**)Value of %inhibition at 27.3 nanomolar inhibitor concentration; average of triplicate measurement.Float%
14Inhibition at 81.8 nM (0.0818μM**)Value of %inhibition at 81.8 nanomolar inhibitor concentration; average of triplicate measurement.Float%
15Inhibition at 245.4 nM (0.245μM**)Value of %inhibition at 245 nanomolar inhibitor concentration; average of triplicate measurement.Float%
16Inhibition at 736.3 nM (0.736μM**)Value of %inhibition at 736 nanomolar inhibitor concentration; average of triplicate measurement.Float%
17Inhibition at 2.2 uM (2.2μM**)Value of %inhibition at 2.2 micromolar inhibitor concentration; average of triplicate measurement.Float%
18Inhibition at 6.6 uM (6.6μM**)Value of %inhibition at 6.6 micromolar inhibitor concentration; average of triplicate measurement.Float%
19Inhibition at 19.9 uM (19.9μM**)Value of %inhibition at 19.9 micromolar inhibitor concentration; average of triplicate measurement.Float%
20Inhibition at 59.6 uM (59.6μM**)Value of %inhibition at 59.6 micromolar inhibitor concentration; average of triplicate measurement.Float%

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

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