AID	Name	Type	Comment
1619	Inhibitors of Plasmodium falciparum M17- Family Leucine Aminopeptidase (M17LAP)	confirmatory	Dose response primary screen (M17LAP inhibitors in triplicate)
2214	Counterscreen for inhibitors of M1 and M17 aminopeptidases: QFRET-based biochemical high throughput dose response assay for inhibitors of the Cathepsin L proteinase (CTSL1).	confirmatory	Dose response counterscreen (CTSL1 inhibitors in triplicate)
2215	Counterscreen 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).	confirmatory	Dose response counterscreen (PFM18AAP inhibitors in triplicate)
434965	Summary of the probe development effort to identify inhibitors of the Plasmodium falciparum M17- Family Leucine Aminopeptidase (M17AAP)	summary	Summary (M17LAP inhibitors)

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_IC50_M17LAP

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 dose response assay to identify compounds that inhibit P. falciparum growth in RBCs.

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, dose response, IC50, 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
nd Institute of Medical Research
External Assay ID: P. FALCIPARUM GROWTH_INH_RAD_96_%INH_M17LAP

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

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

Assay Overview:

The purpose of this assay is to determine the potency of a powder sample of an inhibitor compound identified in a previous assay 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. Compounds were tested in triplicate using a 5-point dilution series starting at a nominal concentration of 25 uM.

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.

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

For each test compound, percent inhibition was plotted against compound concentration. The reported IC50 value was generated from a fitted curve by solving for the X-intercept value at the 50% inhibition level of the Y-intercept value.

PubChem Activity Outcome and Score:

Compounds with an IC50 greater than 10 uM were considered inactive. Compounds with an IC50 equal to or less than 10 uM 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 of the compounds with fitted curves, with the most potent compounds assigned the highest activity scores.

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

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.

Grant Number: 1 R03 MH084103-01