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BioAssay: AID 463209

Late stage assay provider results from the probe development effort to identify inhibitors of the Plasmodium falciparum M1 Alanyl Aminopeptidase (M1AAP): radiolabel-based cell-based assay to identify compounds that inhibit P. falciparum growth in RBCs

Name: Late stage assay provider results from the probe development effort to identify inhibitors of the Plasmodium falciparum M1 Alanyl Aminopeptidase (M1AAP): radiolabel-based cell-based assay to identify compounds that inhibit P. falciparum growth in RBCs ..more
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 Tested Compounds
 Tested Compounds
All(14)
 
 
Inactive(14)
 
 
 Tested Substances
 Tested Substances
All(14)
 
 
Inactive(14)
 
 
 Related BioAssays
 Related BioAssays
AID: 463209
Data Source: The Scripps Research Institute Molecular Screening Center (P. FALCIPARUM GROWTH_INH_RAD_96_M1 ROUND 1 CSRUN)
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
Deposit Date: 2010-09-07
Hold-until Date: 2011-08-30
Modify Date: 2011-08-31

Data Table ( Complete ):           View All Data
Tested Compounds:
Related Experiments
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AIDNameTypeComment
1445Inhibitors of Plasmodium falciparum M1- Family Alanyl Aminopeptidase (M1AAP)Confirmatorydepositor-specified cross reference: Dose response (M1AAP inhibitors)
2214Counterscreen for inhibitors of M1 and M17 aminopeptidases: QFRET-based biochemical high throughput dose response assay for inhibitors of the Cathepsin L proteinase (CTSL1).Confirmatorydepositor-specified cross reference: Dose response counterscreen (CTSL1 inhibitors in triplicate)
2215Counterscreen for inhibitors of M1 and M17 aminopeptidases: QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (PFM18AAP).Confirmatorydepositor-specified cross reference: Dose response counterscreen (PFM18AAP inhibitors in triplicate)
434966Summary of the probe development effort to identify inhibitors of the Plasmodium falciparum M1- Family Alanyl Aminopeptidase (M1AAP)Summarydepositor-specified cross reference: Summer (M1AAP inhibitors)
492976Late stage assay provider results from the probe development effort to identify inhibitors of the Plasmodium falciparum M1 Alanyl Aminopeptidase (PfM1AAP): fluorescence-based biochemical assay to identify inhibitors of malaria cell lysateScreeningsame project related to Summary assay
492978Late stage assay provider results from the probe development effort to identify inhibitors of the Plasmodium falciparum M1 Alanyl Aminopeptidase (PfM1AAP): fluorescence-based biochemical assay to identify inhibitors of rPfM1AAPScreeningsame project related to Summary assay
588682Counterscreen for inhibitors of PFM1AAP: QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M17 Leucine Aminopeptidase (PFM17LAP)Confirmatorysame project related to Summary assay
588686QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M1 Alanyl Aminopeptidase (PFM1AAP)Confirmatorysame project related to Summary assay
588687Counterscreen for inhibitors of PFM1 aminopeptidases: QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (PFM18AAP)Confirmatorysame project related to Summary assay
588717Vero Cytoxicity Assay: A Cell Based Secondary Assay To Explore Cytotoxicity of Compounds that Inhibit Plasmodium falciparum M1 Aspartyl Aminopeptidase (PFM1AAP)Confirmatorysame project related to Summary assay
602209Counterscreen for inhibitors of PFM1AAP: 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
602212Counterscreen for inhibitors of PFM1 aminopeptidases: QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (PFM18AAP) (2)Confirmatorysame project related to Summary assay
602213QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M1 Alanyl Aminopeptidase (PFM1AAP) (2)Confirmatorysame project related to Summary assay
602228Vero Cytoxicity Assay: A Cell Based Secondary Assay To Explore Cytotoxicity of Compounds that Inhibit Plasmodium falciparum M1 Aspartyl Aminopeptidase (PFM1AAP) (2)Confirmatorysame project related to Summary assay
624163QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M1 Alanyl Aminopeptidase (PFM1AAP) (3)Confirmatorysame project related to Summary assay
624164Counterscreen for inhibitors of PFM1AAP: 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
624165Counterscreen for inhibitors of PFM1 aminopeptidases: QFRET-based biochemical high throughput dose response assay for inhibitors of the 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 (SRIMSC)
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: John Dalton and Donald Gardiner, Queensland Institute of Medical Research
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R03 MH082342-01A1
Grant Proposal PI: John Dalton, Queensland Institute of Medical Research
External Assay ID: P. FALCIPARUM GROWTH_INH_RAD_96_M1 ROUND 1 CSRUN

Name: Late stage assay provider results from the probe development effort to identify inhibitors of the Plasmodium falciparum M1 Alanyl Aminopeptidase (M1AAP): radiolabel-based cell-based assay to identify compounds that inhibit P. falciparum growth in RBCs

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). 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 (8). Their action results in the release of free amino acids that are used for the anabolism of parasite proteins and, hence, are critical to the development of the parasite in red blood cells (9). Inhibitors of the two exopeptidases prevent the growth of P. falciparum parasites in vitro, and protect mice from infection with rodent malaria P. chabaudi, providing strong evidence that these enzymes are targets which can be used to develop new anti-malarial drugs (10-12). Thus, P. falciparum M1-family alanyl aminopeptidase (M1AAP) is an attractive chemotherapeutic target and was used to screen a large (200K) chemical library to identify novel inhibitors as probes for this enzyme in P. falciparum.

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. Klemba M, Gluzman I, Goldberg DE. A Plasmodium falciparum dipeptidyl aminopeptidase I participates in vacuolar hemoglobin degradation. J Biol Chem. 2004 Oct 8;279(41):43000-7.
9. Dalal S, Klemba M. J Biol Chem. 2007 Dec 7;282(49):35978-87. Roles for two aminopeptidases in vacuolar hemoglobin catabolism in Plasmodium falciparum.
10. Skinner-Adams TS, Lowther J, Teuscher F, Stack CM, Grembecka J, Mucha A, Kafarski P, Trenholme KR, Dalton JP, Gardiner DL. Identification of phosphinate dipeptide analog inhibitors directed against the Plasmodium falciparum M17 leucine aminopeptidase as lead antimalarial compounds. J Med Chem. 2007 Nov 29;50(24):6024-31.
11. Gavigan CS, Machado SG, Dalton JP, Bell A. Analysis of antimalarial synergy between bestatin and endoprotease inhibitors using statistical response-surface modelling. Antimicrob Agents Chemother. 2001 Nov;45(11):3175-81.
12. Nankya-Kitaka MF, Curley GP, Gavigan CS, Bell A, Dalton JP. Plasmodium chabaudi chabaudi and P. falciparum: inhibition of aminopeptidase and parasite growth by bestatin and nitrobestatin. Parasitol Res. 1998 Jul;84(7):552-8.

Keywords:

late stage, late stage AID, assay provider, purchased, synthesized, powders, growth, M1, M1AAP, alanyl, AAP, aminopeptidase, malaria, parasite, Plasmodium falciparum, inhibitor, inhibition, hypoxanthine, radiolabel, 3H, University of Kansas, University of Kansas Specialized Chemistry Center, KUSCC, KU, Scripps, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN
Protocol
Assay Overview:

The purpose of this assay is to determine the ability of powder samples of inhibitor compounds identified in previous assays to inhibit the growth of Plasmodium falciparum in its asexual erythrocytic stage. In this assay, compounds are incubated with red blood cells (RBC) and P. falciparum parasites in hypoxanthine-free media. 3H-hypoxanthine is added, cells incubated 48 hours, and incorporation of 3H determined. As designed, compounds that inhibit the growth of P. falciparum in RBC will decrease the level of 3H incorporated.

Protocol Summary:

Prior to the start of the assay, 200 uL of PBS was added to the outside wells of a 96 well flat-bottomed microtiter plate. 100 uL hypoxanthine-free RPMI 1640 media plus 10% serum was added to all other wells except those that contained diluted compound or vehicle control. An appropriate volume of compound (10 uM) or vehicle control (DMSO ? 1%) in hypoxanthine-free RPMI 1640 media plus 10% serum was added to remaining wells (each test added in six to nine wells). A parasite suspension (1% parasitemia, 2% hematocrit) was prepared in hypoxanthine-free RPMI media containing 10% serum and added to all test wells (except RBC controls) before the addition of 0.5 Ci/well of 3H-hypoxanthine (10 uL). Uninfected RBC controls (in triplicate) were included on each assay plate. Plates were incubated at 37 degrees C for 48 hours in an atmosphere of 90% N2, 5% CO2, and 5% O2. A cell harvester and Beta counter were used to determine the amount of hypoxanthine incorporated for each well. Six to nine replicates at 10 uM were performed for each compound tested.

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

% Inhibition = 100 - % Growth

% Growth = ( Test - Background_Control ) / ( Positive_Control - Background Control ) * 100

Where:

Test = count (3H-hypoxanthine incorporation) by parasites treated with test compound
Background_Control = mean count of uninfected RBC control tests
Positive_Control = mean count (3H-hypoxanthine incorporation) by untreated parasites exposed to vehicle only

PubChem Activity Outcome and Score:

Compounds achieving >= 50% growth inhibition were considered active; compounds achieving < 50% growth inhibition were considered inactive.

The reported PubChem Activity Score has been normalized to 100% observed inhibition. Negative % inhibition values are reported as activity score zero.

The PubChem Activity Score range for inactive compounds is 100-0. There are no active compounds.

List of Reagents:

Plasmodium falciparum clone 3D7
Hypoxanthine Mono-Hydrochloride, [3H(G)]-(Perkin Elmer code Net177)
Plate Microtiter 96 well flat bottom Sterile (Costar, catalog 3595)
Special Gas Mix (5% CO2 5% O2 90% N2) (BOC Gas)
Culture media (85% RPMI, 4% Sodium Bicarbonate, 10% human sera) (GIBCO, catalog 31800-089)
Comment
This assay was performed by the assay provider, and submitted to PubChem by the Scripps Research Institute Molecular Screening Center (SRIMSC). Compounds tested in this assay were purchased and/or synthesized by the University of Kansas Specialized Chemistry Center.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Average Inhibition at 10 uM (10μM**)The normalized value for inhibition of parasite growth at 10 uM.Float%
2Standard DeviationStandard deviation derived from the normalized percent inhibition of the 9 replicates for each compound.Float
3Inhibition at 10 uM [1] (10μM**)The value for inhibition of parasite growth at 10 uM; replicate one.Float%
4Inhibition at 10 uM [2] (10μM**)The value for inhibition of parasite growth at 10 uM; replicate two.Float%
5Inhibition at 10 uM [3] (10μM**)The value for inhibition of parasite growth at 10 uM; replicate three.Float%
6Inhibition at 10 uM [4] (10μM**)The value for inhibition of parasite growth at 10 uM; replicate four.Float%
7Inhibition at 10 uM [5] (10μM**)The value for inhibition of parasite growth at 10 uM; replicate five.Float%
8Inhibition at 10 uM [6] (10μM**)The value for inhibition of parasite growth at 10 uM; replicate six.Float%
9Inhibition at 10 uM [7] (10μM**)The value for inhibition of parasite growth at 10 uM; replicate seven.Float%
10Inhibition at 10 uM [8] (10μM**)The value for inhibition of parasite growth at 10 uM; replicate eight.Float%
11Inhibition at 10 uM [9] (10μM**)The value for inhibition of parasite growth at 10 uM; replicate nine.Float%

** Test Concentration.
Additional Information
Grant Number: 1 R03 MH082342-01A1

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