A heterogeneous enzymatic assay for quantification of Plasmepsin II activity and the evaluation of its inhibitors

• Published in: Journal of pharmaceutical and biomedical analysis 2004-03, Vol.34 (4), p.833-840
• Main Authors: Salas, Emir, Ramı́rez, Angel, Otero-Bilbao, Anabel, Vázquez, Rayner, Reyes, Osvaldo, Mendiola, Judith, Duarte, Carlos A, Otero-González, Anselmo, Gutiérrez, Omar A, Chávez, Marı́a A
• Format: Article
• Language: English
• Subjects: Animals; Aspartic Acid Endopeptidases - chemistry; Aspartic Acid Endopeptidases - metabolism; Aspartic Acid Endopeptidases - pharmacology; Catalysis; Clinical Enzyme Tests - economics; Clinical Enzyme Tests - methods; Cuba; Dose-Response Relationship, Drug; Drug Delivery Systems; Drug Evaluation, Preclinical - methods; Enzyme-Linked Immunosorbent Assay - methods; Evaluation Studies as Topic; Hemoglobins - drug effects; Hemoglobins - metabolism; Heterogeneous enzymatic assay; Malaria protease; Molecular Sequence Data; Molecular Weight; Pepstatins - pharmacology; Peptide Fragments - chemical synthesis; Plasmepsin II inhibitors; Plasmodium falciparum; Plasmodium falciparum - drug effects; Plasmodium falciparum - enzymology; Protease Inhibitors - chemistry; Protease Inhibitors - pharmacology; Protozoan Proteins; Solid-phase
• ISSN: 0731-7085; 1873-264X; 1873-264X
• DOI: 10.1016/S0731-7085(03)00566-1

The emergence and worldwide spreading of Plasmodium falciparum strains that shown to be resistant to traditional drugs is considered a very serious health problem, given the high mortality and morbidity rate of Malaria. In the search for new drugs against this parasite, Hb hydrolyzing enzymes, such as Plasmepsin II (Plm II), have been classified as very promising targets for therapeutic attacks. In this work, it is developed a cheap and high-throughput heterogeneous enzymatic assay for measuring Plasmepsin II activity in order to use it as a tool in the discovery of new inhibitors of this enzyme. In this assay, Plasmepsin II acts upon a solid-phase bound synthetic peptide (DU2) whose sequence comprises the cleavage site F 33-L 34 present in Hb α-chain. The peptide surface density is quantified by means of a classical ELISA-based procedure. In order to estimate the kinetic constants of the system and to quantify both, enzymatic and inhibitory activity, it was used a model for the kinetics of enzyme quasi-saturable systems previously developed by our group, that fitted very well to the experimental data. It was used Pepstatin as a model inhibitor of Plasmepsin II and the resulting dose–response relation agreed with the expected behavior for the Pepstatin–Plasmepsin II pair under the employed experimental conditions.