Late stage assay provider results from the probe development effort to identify inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (M18AAP): radiolabel-based cell-based dose response 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 M18 Aspartyl Aminopeptidase (M18AAP): radiolabel-based cell-based dose response assay to identify compounds that inhibit P. falciparum growth in RBCs, Set2. ..more
BioActive Compounds: 32
Depositor Specified Assays
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_M18AAP_SET_2
Name: Late stage assay provider results from the probe development effort to identify inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (M18AAP): radiolabel-based cell-based dose response assay to identify compounds that inhibit P. falciparum growth in RBCs, Set2.
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.
late stage, late stage AID, assay provider, purchased, synthesized, powders, growth, M18, M8AAP, aspartyl, AAP, 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
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.
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
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 Log10[IC50] of the compounds, with the most potent compounds assigned the highest activity scores.
The PubChem Activity Score range for active compounds is 100-18, and for inactive compounds 18-0.
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.
* Activity Concentration.
Data Table (Concise)