Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: John Dalton and Donald Gardiner, Queensland Institute of Medical Research, Australia
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R03 MH084103-01
Grant Proposal PI: John Dalton and Donald Gardiner, Queensland Institute of Medical Research, Australia
External Assay ID: PFM18AAP_INH_QFRET_1536_3XIC50 CSDRUN

Name: 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).

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 (M1AAP) and an M17-family leucine aminopeptidase (M17LAP), in the terminal stages of host hemoglobin digestion. 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. 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. Thus, Plasmodium falciparum M1-family alanyl aminopeptidase (M1AAP) 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 Plasmodium falciparum.

PFM18AAP (M18; M18AAP) 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. This enzyme serves as a relevant counterscreen target for the M1 and M17 enzymes described above.

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. 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.

Keywords:

M1, M1AAP, alanyl, AAP, M17, M17LAP, leucyl, LAP, M18, PFM18AAP, M18AAP, rPFAAP, aspartyl, aminopeptidase, malaria, parasite, plasmodium falciparum, exopeptidase, dose response, counterscreen, HTS, high throughput screen, 1536, inhibitor, inhibition, fluorescence, QFRET, FLINT, peptide, cleavage, 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 M18 dose response curves for compounds identified as active in the following set of experiments entitled, "Inhibitors of Plasmodium falciparum M1- Family Alanyl Aminopeptidase (M1AAP)" (AID 1445), or "Inhibitors of Plasmodium falciparum M17- Family Leucine Aminopeptidase (M17LAP)" (AID 1619). This assay serves as a counterscreen to determine whether compounds are nonselective due to inhibition of M18. In this biochemical assay, a commercially available fluorogenic peptide substrate (H-Glu-NHMec) is incubated with purified recombinant PFM18AAP enzyme (rPFM18AAP) in the presence of test compounds. Cleavage of the substrate by rPFM18AAP enzyme liberates the NHMec leaving group from the peptide, leading to increased well fluorescence. As designed, compounds that inhibit PFM18AAP will block rPFM18AAP-mediated cleavage of H-Glu-NHMec and liberation of the NHMec leaving group from the substrate, resulting in decreased well fluorescence as measured at 340 nm excitation and 450 nm emission. Test compounds were assayed in triplicate in a 10-point 1:3 dilution series starting at a nominal test concentration of 73.5 micromolar.

Protocol Summary:

Prior to the start of the assay, 2.5 microliters of assay buffer (50mM Tris HCl pH7.5, 4mM CoCl2, 0.1% BSA) containing 5micrograms/mL rPFM18AAP were dispensed into a 1536 microtiter plate. Next, 37 nL of test compound in DMSO, ZnCl2 (2mM final concentration), or DMSO alone (0.74% final concentration) were added to the appropriate wells. The plates were then incubated for 30 minutes at 25 degrees Celsius.

The assay was started by dispensing 2.5 microliters of 100 micromolar H-Glu-NHMec substrate in buffer (50 mM Tris HCl, pH 8.8) into all wells. Well fluorescence was read immediately (T0) on the Viewlux (Perkin-Elmer) and again after 90 minutes (T90) of incubation at 25 degrees Celsius.

Prior to further calculations, T0 was subtracted from T90 for each individual well. The difference between RFU values read at T0 (RFU_T0) and T90 (RFU_T90), named delta RFU, was calculated as follows:

delta RFU = RFU_T90 - RFU_T0

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

Percent inhibition = ( test_compound_delta RFU - negative_control_delta RFU ) / ( positive_control_delta RFU - negative_control_delta RFU ) * 100

Where:

Test_Compound is defined as wells containing test compound.
Negative_Control is defined as the median of the wells containing rPFM18AAP.
Positive_Control is defined as the median of the wells containing ZnCl2.

For each test compound, percent inhibition was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (Symyx Technologies Inc). The reported IC50 values were generated from fitted curves by solving for the X-intercept value at the 50% inhibition level of the Y-intercept value. In cases where the highest concentration tested (i.e. 73.5 micromolar) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 73.5 micromolar. Compounds with an IC50 greater than 10 micromolar were considered inactive. Compounds with an IC50 equal to or less than 10 micromolar 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, with the most potent compounds assigned the highest activity scores.

The activity score range of inactive compounds is 100-0. There are no actives.

List of Reagents:

rPFM18AAP enzyme (supplied by Assay Provider)
H-Glu-NHMec substrate (Bachem, part I-1180)
1536-well plates (Greiner, part 789176)
Tris (Amresco, part 0497)
CoCl2 6H20 (Univar, part D3247)
ZnCl2 (Sigma, part 208086)
BSA (Calbiochem, part 126609)

Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. In this assay, ZnCl2 had an IC50 of approximately 0.910 micromolar. 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 fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.

Grant Number: 1 R03 MH084103-01