Late stage assay provider results from the probe development effort to identify inhibitors of the Plasmodium falciparum M18 Alanyl Aminopeptidase (PfM18AAP): fluorescence-based biochemical assay to identify inhibitors of rPfM18AAP
Name: Late stage assay provider results from the probe development effort to identify inhibitors of the Plasmodium falciparum M18 Alanyl Aminopeptidase (PfM18AAP): fluorescence-based biochemical assay to identify inhibitors of rPfM18AAP. ..more
BioActive Compounds: 22
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 MH084103-01
Grant Proposal PI: John Dalton, Queensland Institute of Medical Research
External Assay ID: rPfM18AAP_INH_FLUO_96_1X%INH_ENZYME
Name: Late stage assay provider results from the probe development effort to identify inhibitors of the Plasmodium falciparum M18 Alanyl Aminopeptidase (PfM18AAP): fluorescence-based biochemical assay to identify inhibitors of rPfM18AAP.
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). PfM18AAP is the sole aspartyl aminopeptidase (AAP) present in the genome of the malaria parasite Plasmodium falciparum (8). It exhibits exopeptidase activity exclusively against the N-terminal acidic amino acids glutamate and aspartate (9-11), is found in all intra-erythrocytic stages of the parasite (9), and functions to complete the hydrolysis of host hemoglobin into amino acids for use in de novo protein synthesis by the parasite (12, 13). Studies demonstrating that genetic knockdown of PfM18AAP results in a lethal parasite phenotype (9), and that inhibitors of methionine (14) and leucine (12, 15) aminopeptidases prevent malaria growth in culture and hemoglobin degradation, suggest that these enzymes are essential for parasite survival. As a result, the identification of selective inhibitors of PfM18AAP would elucidate this enzyme's role in the P. falciparum lifecycle, and serve as potential therapeutic agents to control malaria infection.
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. Wilk, S., Wilk, E., and Magnusson, R.P., Purification, characterization, and cloning of a cytosolic aspartyl aminopeptidase. J Biol Chem, 1998. 273(26): p. 15961-70.
9. Teuscher, F., Lowther, J., Skinner-Adams, T.S., Spielmann, T., Dixon, M.W., Stack, C.M., Donnelly, S., Mucha, A., Kafarski, P., Vassiliou, S., Gardiner, D.L., Dalton, J.P., and Trenholme, K.R., The M18 aspartyl aminopeptidase of the human malaria parasite Plasmodium falciparum. J Biol Chem, 2007. 282(42): p. 30817-26.
10. Gyang, F.N., Poole, B., and Trager, W., Peptidases from Plasmodium falciparum cultured in vitro. Mol Biochem Parasitol, 1982. 5(4): p. 263-73.
11. Vander Jagt, D.L., Baack, B.R., and Hunsaker, L.A., Purification and characterization of an aminopeptidase from Plasmodium falciparum. Mol Biochem Parasitol, 1984. 10(1): p. 45-54.
12. Nankya-Kitaka, M.F., Curley, G.P., Gavigan, C.S., Bell, A., and Dalton, J.P., Plasmodium chabaudi chabaudi and P. falciparum: inhibition of aminopeptidase and parasite growth by bestatin and nitrobestatin. Parasitol Res, 1998. 84(6): p. 552-8.
13. Lauterbach, S.B. and Coetzer, T.L., The M18 aspartyl aminopeptidase of Plasmodium falciparum binds to human erythrocyte spectrin in vitro. Malar J, 2008. 7: p. 161.
14. Chen, X., Chong, C.R., Shi, L., Yoshimoto, T., Sullivan, D.J., Jr., and Liu, J.O., Inhibitors of Plasmodium falciparum methionine aminopeptidase 1b possess antimalarial activity. Proc Natl Acad Sci U S A, 2006. 103(39): p. 14548-53.
15. Stack, C.M., Lowther, J., Cunningham, E., Donnelly, S., Gardiner, D.L., Trenholme, K.R., Skinner-Adams, T.S., Teuscher, F., Grembecka, J., Mucha, A., Kafarski, P., Lua, L., Bell, A., and Dalton, J.P., Characterization of the Plasmodium falciparum M17 leucyl aminopeptidase. A protease involved in amino acid regulation with potential for antimalarial drug development. J Biol Chem, 2007. 282(3): p. 2069-80.
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The purpose of this assay is to determine inhibitory activity of powder samples of compounds for recombinant P. falciparum M18AAP. In this assay, a fluorogenic peptide substrate (H-Glu-NHMec) that binds to the active site of rPfM18AAP was used to quantify the activity of rPfM18AAP 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 rPfM18AAP 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.
Prior to the start of the assay, 5 uL of rPfM18AAP 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-Glu-NHMec) in assay buffer was added to each well. The final concentrations in the reaction were 50 uM H-Glu-NHMec, 1 ug/ml rPfM18AAP, 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 rPfM18AAP 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
Test = RFU in wells with rPfM18AAP treated with test compound.
Background_Control = mean count of wells containing substrate in assay buffer only.
Positive_Control = mean count of wells containing rPfM18AAP with substrate but no compound added.
PubChem Activity Outcome and Score:
Compounds resulting in greater than 50% inhibition of rPfM18AAP 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 100-55, and for inactive compounds 54-0.
List of Reagents:
rPfM18AAP (Assay Provider)
H-Glu-NHMec (Bachem Chemical Co., catalog I-1180)
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.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)