Inhibitors of Plasmodium falciparum M1- Family Alanyl Aminopeptidase (M1AAP)
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 more ..
BioActive Compounds: 913
Southern Research Molecular Libraries Screening Center (SRMLSC)
Southern Research Institute (Birmingham, Alabama)
NIH Molecular Libraries Screening Centers Network (MLSCN)
Assay Provider: Dr. Donald Gardiner, Queensland Institute of Medical Research
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.
A simple assay using a fluorogenic peptide substrate (H-Leu-NHMec) was used for quantifying the activity of M1AAP. The rate of hydrolysis of this substrate was measured by monitoring the release of the -NHMec fluorogenic leaving group at an excitation wavelength of 370nm and an emission wavelength of 460nm. A kinetic assay was chosen to minimize interference by compounds that fluoresce at these wavelengths.
A total of 217,208 compounds were initially screened at a final concentration of 30 uM. For follow-up testing, the 1104 most potent inhibitors were tested in a dose response assay at concentrations ranging from 100-1.0 uM.
M1AAP Assay Protocol for 1536-well HT
Purified recombinant M1AAP enzyme was provided by Prof. John Dalton, Institute for the Biotechnology of Infectious Diseases (IBID) University of Technology Sydney.
Compound Dosing/Plating: For the primary screen 15 nl of compounds in DMSO were dispensed into 1536-well black non-binding surface plates. This resulted in a concentration of 30 uM of compound in the assay. For the follow up dose response assays 8 concentrations of each compound ranging from 100-1 uM were used.
Assay Setup: 2.5 uL of M1AAP reagent mix, which included the fluorogenic peptide substrate (H-Leu-NHMec) in assay buffer, was added to each well of the previously compound dosed 1536-well plates. The reaction was initiated with the addition of 2.5 uL of the M1AAP diluted in assay buffer. The final concentrations in the reaction were 0.1 mM H-Leu-NHMec and 50 ug/ml M1AAP diluted in assay buffer (50 mM Tris-HCl (pH 7.5), 0.01% Triton X-100, and 2% DMSO). The test plate was immediately transferred to a Perkin Elmer Envision microplate reader and fluorescence was measured at an excitation wavelength of 370nm and an emission wavelength of 460nm at 35 second intervals for 315 seconds (10 readings). Each plate had 256 control wells in the eight outside columns with 128 wells containing the complete reaction mixture with carrier control (full reaction) and 128 wells in which the M1AAP had been left out (background).
Possible artifacts in this assay include, but are not limited to, compounds that fluoresce at 370/460 nm, that absorb at either 370 or 460 nm, or that precipitate.
The effect of library compounds at 30 uM on the activity of M1AAP was calculated as follows:
% Inhibition = 100*((Med Full Rxn Rate-Med Bkg Rate)-(Cmpd Rate-Med Bkg Rate))/((Med Full Rxn Rate-Med Bkg Rate))
Full Rxn refers to reagent mix and enzyme.
Bkg refers to reagent mix without enzyme.
Rate is calculated as (Slope of test well-median background slope).
In the confirmatory testing, the 1104 most active compounds from the primary screen were screened at 10 concentrations ranging from 1 to 100 uM. Compounds showing >30% Inhibition at any concentration were considered sufficiently active to calculate IC50 data using IDBS XLfit formula 205 with minimum limit=0 and maximum limit = 100, extrapolation was allowed.
Compounds that showed inhibition >23.27% in the primary screen were considered "hits" (the median %inhibition for all experimental compounds [3.68%] plus 3 times the standard deviation [6.53%]). Compounds with % inhibition <23.27% in the primary screen were deemed "Inactive". Conflicting primary data received an outcome of "Inconclusive". Compounds that exceeded the hit cutoff but were not selected for dose response testing were labeled "Inconclusive;" only the 1104 most potent compounds were submitted for dose response testing. Compounds that showed >50% inhibition for at least one concentration in the dose response were defined as "Active". If the inhibition at all doses was <50% in the dose response assay, the compound was defined as "Inactive".
The scoring system used by SRMLSC uses a tiered scale of 0-100. The range applied to primary data of 0-40 is a linear calculation based on % Inhibition of the primary result. Confirmatory concentration response testing is represented by the range 41-80 calculated on an inverse linear scale based on tested values, extrapolated values received a score of 41, inactive compounds receiving the score of 0. The dataset contained within this submission contains both Primary and Confirmatory data and will contain scores ranging from 0-80 based on these criteria. Subsequent concentration response of resynthesized compounds will reflect the top tier range of 81-100 except in cases where a compound confirms inactivity and is assigned a score of 0 at any tier.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)