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_%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 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 or three 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 100-100, and for inactive compounds 46-0.

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)

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