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

Salas, Emir; Ramirez, Angel R; Otero-Bilbao, Anabel; Vázquez, Rayner; Reyes, Osvaldo; Mendiola, Judith; Duarte, Carlos A.; Otero‐González, Anselmo J.; Gutiérrez, Omar A.; Chávez, María Á.

doi:10.1016/s0731-7085(03)00566-1

Cited by 6 publications

(9 citation statements)

References 20 publications

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“…Secondly, we set the concentration of FP2 substrate around the K M value, as it equalizes the chances to identify competitive, non-competitive and uncompetitive inhibitors [57,58]. This was not necessary for Plm II assay, since E-I equilibrium is not affected by the immobilized substrate DU2 [31,59]. …”

Section: Resultsmentioning

confidence: 99%

“…A heterogeneous immuno-enzymatic Plm II assay [31] was used for the primary screening of 35 clarified aqueous extracts (Table 1), given its high-throughput compatible format. P. homomalla (heat), S. helianthus (heat and TCA), P. nigra (heat and TCA) and X. muta (heat) extracts, previously used as reference extracts to validate the strategy, were included in the screening to evaluate the consistency of our results.…”

Section: Resultsmentioning

confidence: 99%

“…A heterogeneous immunoenzymatic assay was used for the primary screening of Plm II inhibitors in clarified extracts using the biotinylated peptide DU2 (Biotin-βAβALERTF→LSFPRQSTPIGLGQALYTT-COOH) as substrate ( K M : 1.6 μM; k cat : 3.5 × 10 −3 s −1 ; k cat /K M : 2.2 × 10 3 M −1 ·s −1 ; Signal sensitivity: 4.4 × 10 13 AU∙mol −1 ) [31]. In brief, 96-well microtiter plates are coated with streptavidin.…”

Section: Methodsmentioning

confidence: 99%

“…Despite this fact, a considerable amount of biochemical knowledge and research tools were generated around both enzymes during the last two decades. These include: efficient recombinant expression systems [24,25], crystallographic structures bound to different ligands [26,27], specific substrates and inhibitors [28,29], different kinds of High-Throughput Screening enzymatic assays [30,31,32], computational models for the virtual screening of compounds [28,33] and biophysical techniques for their characterization. This makes Plm II and FP2 exceptionally well characterized model enzymes for virtually any kind of scientific investigation.…”

Section: Introductionmentioning

confidence: 99%

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Identification of Tight-Binding Plasmepsin II and Falcipain 2 Inhibitors in Aqueous Extracts of Marine Invertebrates by the Combination of Enzymatic and Interaction-Based Assays

et al. 2017

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Natural products from marine origin constitute a very promising and underexplored source of interesting compounds for modern biotechnological and pharmaceutical industries. However, their evaluation is quite challenging and requires specifically designed assays to reliably identify the compounds of interest in a highly heterogeneous and interfering context. In the present study, we describe a general strategy for the confident identification of tight-binding protease inhibitors in the aqueous extracts of 62 Cuban marine invertebrates, using Plasmodium falciparum hemoglobinases Plasmepsin II and Falcipain 2 as model enzymes. To this end, we first developed a screening strategy that combined enzymatic with interaction-based assays and then validated screening conditions using five reference extracts. Interferences were evaluated and minimized. The results from the massive screening of such extracts, the validation of several hits by a variety of interaction-based assays and the purification and functional characterization of PhPI, a multifunctional and reversible tight-binding inhibitor for Plasmepsin II and Falcipain 2 from the gorgonian Plexaura homomalla, are presented.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Identification of Tight-Binding Plasmepsin II and Falcipain 2 Inhibitors in Aqueous Extracts of Marine Invertebrates by the Combination of Enzymatic and Interaction-Based Assays

et al. 2017

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“…Malaria is the most serious parasitic disease in the world with mortality and morbidity rates only exceeded by those of tuberculosis (Salas et al, 2004). Today approximately 40% of the world population is at risk of malaria, mostly those living in the poorest countries (Guerin et al, 2002).…”

Section: Introductionmentioning

confidence: 99%

Design, synthesis, and antimalarial evaluation of thiazole-derived amino acids

et al. 2008

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A series of thiazole-derived N-Boc amino acids were synthesized and evaluated as targeted potential antimalarials against plasmepsins II enzyme of malaria parasite Plasmodium falciparum. All the compounds showed moderate to good activity. Compounds 3f and 3g were found to have highest the 50% inhibitory concentration (IC 50 ) values (3.45 lM and 4.89 lM, respectively) against Plasmodium falciparum. The compounds docked to the active site of plasmepsin II. Most of the compounds were found to interact with the catalytic amino acids ASP34 and ASP214 of plasmepsin II. A good correlation was observed between binding energy and antiparasitic activity of the thiazole derivatives.

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