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Definition
Malaria aspartyl proteinase substrate is a peptide substrate for a continuous fluorescence-based assay of the malaria aspartyl proteinase.

Discovery
In 1987, Rosenthal et al., identified three P. Falciparum proteases by gel electrophoresis, two of these had an active site cysteine. In 1994, two aspartyl proteases, plasmepsins I  and II, were isolated from the P. falciparum food vacuole and shown to perform the first cleavage of hemoglobin. Plasmepsin II has been shown to cleave other erythrocyte proteins 1,2,3. Jiang et al., in 2001 characterized a new class of small nonpeptidyl compounds blocks Plasmodium falciparum development in vitro by Inhibiting plasmepsins 4.

Structural Characteristics
Malarial parasites rely on aspartic proteases called plasmepsins to digest hemoglobin during the intraerythrocytic stage. Plasmepsins from Plasmodium falciparum and Plasmodium vivax have been cloned and expressed for a variety of structural and enzymatic studies. Recombinant plasmepsins possess kinetic similarity to the native enzymes, indicating their suitability for target-based antimalarial drug development. Authors developed an automated assay of P. falciparum plasmepsin II and P. vivax plasmepsin to quickly screen compounds in the Walter Reed chemical database. A low-molecular-mass (0.35 kDa) diphenylurea derivative (WR268961) was found to inhibit plasmepsins. WR268961 is a small nonpeptidyl compound with relatively high solubility in aqueous solution. Structurally, it belongs to a class of compounds containing a diphenylurea moiety [1-(4-amidinophenyl)-3-(4- phenoxyphenyl) urea] 4.

Mode of Action
WR268961 significantly abolished parasite proliferation, with IC50 values ranging from 0.03 (chloroquine-resistant W2 strain) to 0.16 (chloroquine-sensitive D6-strain) mg/ml. WR268961 general toxicity to mammalian cells was also tested. Cultured neuronal cells and macrophages were 25 to 100 times less sensitive to WR268961 than the parasites. One concern about protease inhibitors is that they often inhibit closely related mammalian proteases, such as cathepsin D, as well as the intended target protease. WR268961 displayed almost no inhibition for human liver cathepsin D. WR268961 does not inhibit the initial step of hemoglobin degradation, but instead blocks the further processing of partially digested hemoglobin. Diphenylurea with a phenoxyl side chain substrate may be the functional structure that is responsible for plasmepsin inhibition.4.

Functions
Malarial parasites invade human erythrocytes in the asexual stage of infection. While residing in erythrocytes, the parasites rely on human hemoglobin as a food source, digesting it with a series of proteases. The aspartic proteases, plasmepsins, are critical for hemoglobin degradation and are thus logical targets for antimalarial drug development 5,6. Nine new inhibitors were tested in vitro in drug susceptibility assay to determine whether they were capable of interrupting parasite growth. Despite their ability to inhibit plasmepsin, all of these compounds displayed weak potency in blocking P. falciparum growth, with IC50 values greater than 6 mg/ml.  WR268961 is specific for plasmepsins versus mammalian aspartic proteases and is specific for malaria parasites versus mammalian cells. These nine WR268961 analogues inhibit the plasmepsins 17 to 1,000 times better than human cathepsin D, demonstrating specificity between parasite and mammalian proteases 4. All nine compounds contain an acidic sulfonic acid group, whereas WR268961 contains a basic amidine group. Docking experiments suggest that the diphenylurea core of WR268961 interacts specifically with the plasmepsin active site, whereas the amidine group does not.  The pKa values of the plasmepsin inhibitors may have an impact on the in vitro inhibition of parasite growth. Although all nine compounds inhibit plasmepsin with similar values, only the basic WR268961 is a potent inhibitor of parasite growth. Several peptide-like compounds, such as pepstatin, SC-50083, and Ro40–4388, display potent inhibition of plasmepsins, but they are much less effective at inhibiting parasite growth, perhaps due to the difficulty these large compounds have gaining access to the parasitic food vacuoles in which hemoglobin degradation takes place 7,8.


References

1.     Hill J, Tyas L, Phylip LH, Kay J, Dunn BM, Berry C (1994). High level expression and characterisation of Plasmepsin II, an aspartic proteinase from Plasmodium falciparum. Febs Lett., 352:155-158.

2.     Le Bonniec S, Deregnaucourt C, Redeker V, Banerjee R, Grellier P, Goldberg DE, Schrével J (1999). Plasmepsin II, an acidic hemoglobinase from the Plasmodium falciparum food vacuole, is active at neutral pH on the host erythrocyte membrane skeleton. J. Biol. Chem., 274:14218-14223.

3.     Joachimiak MP, Chang C, Rosenthal PJ, Cohen FE (2001). The Impact of Whole Genome Sequence Data on Drug Discovery—A Malaria Case Study. Molecular Medicine, 7(10):698–710.

4.     Jiang S, Prigge ST, Wei L, Gao Y, Hudson TH, Gerena L, Dame JB, Kyle DE (2001). New Class of Small Nonpeptidyl Compounds Blocks Plasmodium falciparum Development In Vitro by Inhibiting Plasmepsins. Antimicrob Agents Chemother., 45(9):2577–2584.

5.     Rosenthal PJ (1998). Proteases of malaria parasites: new targets for chemotherapy.       Emerg. Infect Dis., 4:49-57.

6.     Gluzman IY, Francis SE, Oksman A, Smith CE, Duffin KL, Goldberg DE (1994). Order and specificity of the Plasmodium falciparum hemoglobin degradation pathway. J. Clin. Investig., 93:1602-1608.

7.     Francis SE, Gluzman IY, Oksman A, Knickerbocker A, Mueller R, Bryant ML, Sherman DR, Russell DG, Goldberg DE (1994). Molecular characterization and inhibition of Plasmodium falciparum aspartic hemoglobinase. EMBO J., 13:306-317.

8.     Moon RP, Tyas L, Certa U, Rupp K, Bur D, Jacquet C, Matile H (1997). Expression and characterization of plasmepsin I from Plasmodium falciparum. Eur. J. Biochem., 244:552–560.

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