Bookmark and Share
BioAssay: AID 743274

Late stage assay provider results from the probe development effort to identify inhibitors of the Plasmodium falciparum M17 Leucine Aminopeptidase (M17LAP): radiolabel-based cell-based assay to identify compounds that inhibit P. falciparum growth in RBCs Set 2

Name: Late stage assay provider results from the probe development effort to identify inhibitors of the Plasmodium falciparum M17 Leucine Aminopeptidase (M17LAP): radiolabel-based cell-based assay to identify compounds that inhibit P. falciparum growth in RBCs Set 2. ..more
_
   
 Tested Compounds
 Tested Compounds
All(37)
 
 
Active(18)
 
 
Inactive(19)
 
 
 Tested Substances
 Tested Substances
All(37)
 
 
Active(18)
 
 
Inactive(19)
 
 
 Related BioAssays
 Related BioAssays
AID: 743274
Data Source: The Scripps Research Institute Molecular Screening Center (P. FALCIPARUM GROWTH_INH_RAD_96_%INH_M17LAP_SET2)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
Deposit Date: 2014-02-05

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 18
Related Experiments
Show more
AIDNameTypeComment
1619Inhibitors of Plasmodium falciparum M17- Family Leucine Aminopeptidase (M17LAP)Confirmatorydepositor-specified cross reference: Dose response primary screen (M17LAP inhibitors in triplicate)
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)
434965Summary of the probe development effort to identify inhibitors of the Plasmodium falciparum M17- Family Leucine Aminopeptidase (M17AAP)Summarydepositor-specified cross reference: Summary (M17LAP inhibitors)
489016Late stage assay provider results from the probe development effort to identify inhibitors of the Plasmodium falciparum M17 Leucine Aminopeptidase (M17LAP): radiolabel-based cell-based dose response assay to identify compounds that inhibit P. falciparum growth in RBCsConfirmatorydepositor-specified cross reference: Dose Response (P. falciparum growth)
492955Late stage assay provider results from the probe development effort to identify inhibitors of the Plasmodium falciparum M17 Leucine Aminopeptidase (M17LAP): radiolabel-based cell-based assay to identify compounds that inhibit P. falciparum growth in RBCsOtherdepositor-specified cross reference: Inhibition (P. falciparum growth)
492973Late stage assay provider results from the probe development effort to identify inhibitors of the Plasmodium falciparum M7 Leucine Aminopeptidase (PfM17LAP): fluorescence-based biochemical assay to identify inhibitors of malaria cell lysateScreeningdepositor-specified cross reference: Inhibition (malaria cell lysate)
492977Late stage assay provider results from the probe development effort to identify inhibitors of the Plasmodium falciparum M7 Leucine Aminopeptidase (PfM17LAP): fluorescence-based assay to identify inhibitors of rPfM17LAPScreeningdepositor-specified cross reference: Inhibition (rPfM17LAP)
588688QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M17 Leucine Aminopeptidase (PFM17LAP)Confirmatorydepositor-specified cross reference: M17 Main Screen
588697Counterscreen for inhibitors of PFM17LAP: QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (PFM18AAP)Confirmatorydepositor-specified cross reference: M18 Counter Screen for M17
588698Counterscreen for inhibitors of PFM17LAP: QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M1AAP (PFM1AAP)Confirmatorydepositor-specified cross reference: M1 Counter Screen for M17
588707Vero Cytoxicity Assay: A Cell Based Secondary Assay To Explore Cytotoxicity of Compounds that Inhibit Plasmodium falciparum M1 Aspartyl Aminopeptidase (PFM17LAP)Confirmatorydepositor-specified cross reference: Cytotox
602214Counterscreen for inhibitors of PFM17LAP: QFRET-based biochemical high throughput dose response assay for inhibitors of the Human M17LAP (HUM17LAP)Confirmatorydepositor-specified cross reference: Human M17 Counter Screen
602216QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M17 Leucine Aminopeptidase (PFM17LAP) (2)Confirmatorydepositor-specified cross reference: M17 Main Screen
602218Counterscreen for inhibitors of PFM17LAP: QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (PFM18AAP) (2)Confirmatorydepositor-specified cross reference: M18 Counter Screen for M17
602223Counterscreen for inhibitors of PFM17LAP: QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M1AAP (PFM1AAP) (2)Confirmatorydepositor-specified cross reference: M1 Counter Screen for M17
602227Vero Cytoxicity Assay: A Cell Based Secondary Assay To Explore Cytotoxicity of Compounds that Inhibit Plasmodium falciparum M17 Leucine Aminopeptidase (PFM17LAP) (2)Confirmatorydepositor-specified cross reference: Cytotox
602309QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M17 Leucine Aminopeptidase (PFM17LAP) (3)Confirmatorydepositor-specified cross reference: Dose Response (PFM17LAP inhibition)
602315Counterscreen for inhibitors of PFM17LAP: QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M1AAP (PFM1AAP) (3)Confirmatorydepositor-specified cross reference: M1 counterscreen for M17
602316Counterscreen for inhibitors of PFM17LAP: QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (PFM18AAP) (3)Confirmatorydepositor-specified cross reference: M18 Counter Screen for M17
602317Vero Cytoxicity Assay: A Cell Based Secondary Assay To Explore Cytotoxicity of Compounds that Inhibit Plasmodium falciparum M17 Leucine Aminopeptidase (PFM17LAP) (3)Confirmatorydepositor-specified cross reference: Cytox (Inhibitors of M17)
602407Name: Counterscreen for inhibitors of PFM17LAP: QFRET-based biochemical high throughput dose response assay for inhibitors of the Human M17LAP (HUM17LAP)Confirmatorydepositor-specified cross reference: Human M17 counterscreen for M17
743273Late stage assay provider results from the probe development effort to identify inhibitors of the Plasmodium falciparum M17 Leucine Aminopeptidase (M17LAP): radiolabel-based cell-based dose response assay to identify compounds that inhibit P. falciparum growth in RBCs Set 2Confirmatorysame 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_%INH_M17LAP_SET2

Name: Late stage assay provider results from the probe development effort to identify inhibitors of the Plasmodium falciparum M17 Leucine Aminopeptidase (M17LAP): radiolabel-based cell-based assay to identify compounds that inhibit P. falciparum growth in RBCs Set 2.

Description:

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 (1). 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 (2). 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 (3-5). Thus, P. falciparum M17-family leucine aminopeptidase (M17LAP) is an attractive chemotherapeutic target.

References:

1. 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.
2. 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.
3. 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.
4. 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.
5. 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, M17, M7LAP, leucine, LAP, aminopeptidase, malaria, parasite, Plasmodium falciparum, inhibitor, inhibition, hypoxanthine, radiolabel, 3H, University of Kansas, University of Kansas Specialized Chemistry Center, KUSCC, KU, 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 assay is to determine the ability of powder samples of inhibitor compounds 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. 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 uCi/well of 3H-hypoxanthine (10 uL). Uninfected RBC controls (in triplicate) were included on each assay plate. Plates were incubated at 37 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. Three replicates at 10 uM were performed for each compound tested in each experiment, and each compound was tested in two experiments.

The % inhibition for each well was 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 resulting in >= 50% inhibition for any replicate at 10 uM concentration were considered "active". Compounds resulting in < 50% inhibition for all replicates at 10 uM concentration 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 active compounds is 100-41, and for inactive compounds 34-0.

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) on behalf of the University of Kansas Specialized Chemistry Center. Compounds tested in this assay were purchased and/or synthesized by the University of Kansas Specialized Chemistry Center.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Cell-based
Assay Cell Type: RBCs
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Average % Inhibition (Expt. 1) (10μM**)The value for average percent inhibition of parasite growth at 10 uM compound; experiment one.Float%
2Average % Inhibition (Expt. 2) (10μM**)The value for average percent inhibition of parasite growth at 10 uM compound; experiment two.Float%

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

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