AID	Name	Type	Comment
1445	Inhibitors of Plasmodium falciparum M1- Family Alanyl Aminopeptidase (M1AAP)	confirmatory	Dose response primary screen (M1AAP 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)
434966	Summary of the probe development effort to identify inhibitors of the Plasmodium falciparum M1- Family Alanyl Aminopeptidase (M1AAP)	summary	Summary (M1AAP 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: rPfM1AAP_INH_FLUOR_96_1X%INH_ENZYME

Name: Late 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 rPfM1AAP.

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 (PfM1AAP) and an M17-family leucine aminopeptidase (PfM17LAP), 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 (PfM1AAP) 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, M1, M1AAP, rPfM1AAP, alanyl, AAP, aminopeptidase, malaria, parasite, Plasmodium falciparum, inhibitor, inhibition, fluorescence, fluorogenic peptide, H-Leu-NHMec, 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

Assay Overview:

The purpose of this assay is to determine inhibitory activity of powder samples of compounds for recombinant P. falciparum M1AAP. In this assay, a fluorogenic peptide substrate (H-Leu-NHMec) that binds to the active site of rPfM1AAP was used to quantify the activity of rPfM1AAP in the presence of inhibitor compounds. The rate of hydrolysis of this substrate in the presence of 5 uM inhibitor compounds was measured by monitoring the release of the -NHMec fluorogenic leaving group at an excitation wavelength of 370 nm and an emission wavelength of 460 nm. As designed, compounds that bind to rPfM1AAP will compete with binding of the fluorogenic peptide substrate, resulting in a decrease in the release of the fluorogenic leaving group and a decrease in well fluorescence.

Protocol Summary:

Prior to the start of the assay, 5 uL of rPfM1AAP were dispensed into 96-well black non-binding surface plates. This was followed by 90 uL of assay buffer (25 mM Tris HCl, pH 7.5) and then 5 uL of compound. After 10 minutes, 100 uL of 100 uM fluorogenic peptide substrate (H-Leu-NHMec) in assay buffer was added to each well. The final concentrations in the reaction were 50 uM H-Leu-NHMec, 1 ug/ml rPfM1AAP, 25 mM Tris-HCl (pH 7.5), and 5 uM compound. The test plate was immediately transferred to a Synergy HT fluorimeter with microplate reader and fluorescence was measured at an excitation wavelength of 370 nm and an emission wavelength of 460 nm at 60-second intervals for 30 minutes (30 readings). Each plate had 4 control wells in the eight outside columns with 2 wells containing the complete reaction mixture with carrier control (positive control) and 2 wells in which the rPfM1AAP had been left out (background).

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

% Inhibition = 100 - % Enzyme Activity

% Enzyme Activity = ( Test - Background_Control ) / ( Positive_Control - Background_Control ) * 100

Where:

Test = RFU in wells with rPfM1AAP treated with test compound.
Background_Control = mean count of wells containing substrate in assay buffer only.
Positive_Control = mean count of wells containing rPfM1AAP with substrate but no compound added.

PubChem Activity Outcome and Score:

Compounds resulting in 45% or greater inhibition of rPfM1AAP were considered active.

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 100-65, and for inactive compounds 55-0.

List of Reagents:

rPfM1AAP (Assay Provider)
H-Leu-NHMec (Bachem Chemical Co., catalog I-1240)
Tris buffer (Supplier name, BioShop Canada, catalog TRS001-1)
96-well plates (Costar Incoporated USA, catalog 3904)

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 luminescence. 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