Please review the reference below:

J Med Chem. 2007 Mar 22;50(6):1322-34. Epub 2007 Feb 28.

Novel selective inhibitors of the zinc plasmodial aminopeptidase PfA-M1 as potential antimalarial agents.

Flipo M1, Beghyn T, Leroux V, Florent I, Deprez BP, Deprez-Poulain RF.

Author information

1Inserm, U761, Biostructures and Drug Discovery, Lille F-59006 France.

Abstract

Proteases that are expressed during the erythocytic stage of Plasmodium falciparum are newly explored drug targets for the treatment of malaria. We report here the discovery of potent inhibitors of PfA-M1, a metallo-aminopeptidase of the parasite. These compounds are based on a malonic hydroxamic template and present a very good selectivity toward neutral aminopeptidase (APN-CD13), a related protease in mammals. Structure-activity relationships in these series are described. Further optimization of the best inhibitor yielded a nanomolar, selective inhibitor of PfA-M1. This inhibitor displays good physicochemical and pharmacokinetic properties and a promising antimalarial activity.

Inhibition of APN. Microsomal APN from porcine kidney was

purchased from Sigma, Inc., as an ammonium sulfate suspension

(3.5 M (NH4)2SO4 solution containing 10 mM MgCl2), 10-40 units/

mg protein. The enzyme suspension was diluted 600-fold in Tris-

HCl buffer (25 mM; pH ) 7.4) before use. The assays were

performed in 96-well plates. The compounds were tested at the

concentration of 10 íM. Purified APN (33 íL) was preincubated

10 min at room temperature with 33 íL of the inhibitor (30 íM in

Tris-HCl buffer, 0.3% DMSO). The substrate Leu-pNA (33 íL;

Km ) 0.099 mM), 0.3 mM in Tris-HCl buffer, was then added.

The reaction kinetics performed at room temperature was followed

on a UV-microplate reader MultiskanRC (Labsystems, Finland) at

405 nm. The control activity was determined by incubating the

enzyme in the same conditions without inhibitor. Bestatin was used

as the reference inhibitor (IC50 ) 2.7 íM). The statistical Z¢ factor

for the test was 0.75, allowing activities to be determined with a

single point with a 95% confidence.40 Initial velocities are expressed

in ímolâmin-1. Data were normalized to the controls that represent

Vmax. For the determination of IC50 values, initial velocities were

plotted as a function of inhibitor concentration, using XLfit software

from IDBS.

Parasite Growth Inhibition. P. falciparum strains were maintained

continuously in culture on human erythrocytes, as described

by Trager and Jensen.46 In vitro antiplasmodial activity was

determined using a modification of the semiautomated microdilution

technique of Desjardins et al.47 P. falciparum chloroquine (CQ)-

resistant FcB1 (Colombia) strain (CQ: IC50 ) 115 ( 5 nM) or

chloroquine (CQ)-sensitive F32 (Tanzania) strain (CQ: IC50 ) 21

( 5 nM) was used. Stock solutions of test compounds were prepared

in sterile distilled water and DMSO, respectively. Drug solutions

were serially diluted with culture medium and introduced to

asynchronous parasite cultures (0.5% parasitemia and 1% final

hematocrite) on 96-well plates for 24 h at 37 °C prior to the addition

of 0.5 mCi of [3H]hypoxanthine (1-5 Ci/mmol; Amersham, France)

per well. The growth inhibition of each drug concentration was

determined by comparison of the radioactivity incorporated into

the treated culture with that in the control culture (without drug)

maintained on the same plate. The IC50 value, as obtained from

the drug concentration-response curve, was expressed as the mean

of three independent experiments, SD were

Thank you prof. Amar Safdar.

I have already read this publication, we do not have the facilities  to run the biological testing, we are looking  for someone who can run the testing for us .