Development of a Novel High-Density [ ³ H]Hypoxanthine Scintillation Proximity Assay To Assess Plasmodium falciparum Growth

Abstract

The discovery and development of new antimalarial drugs are becoming imperative because of the spread of resistance to current clinical treatments. The lack of robustly validated antimalarial targets and the difficulties with the building in of whole-cell activity in screening hits are hampering target-based approaches. However, phenotypic screens of structurally diverse molecule libraries are offering new opportunities for the identification of novel antimalarials. Several methodologies can be used to determine the whole-cell in vitro potencies of antimalarial hits. The [ 3 H]hypoxanthine incorporation assay is considered the "gold standard" assay for measurement of the activity of antimalarial compounds against intraerythrocytic forms of Plasmodium falciparum. However, the method has important limitations, as the assay is not amenable for high-throughput screening since it remains associated with the 96-well plate format. We have overcome this drawback by adapting the [ 3 H]hypoxanthine incorporation method to a 384-well high-density format by coupling a homogeneous scintillation proximity assay (SPA) and thus eliminating the limiting filtration step. This SPA has been validated using a diverse set of 1,000 molecules, including both a representative set from the Tres Cantos Antimalarial Set (TCAMS) of compounds and molecules inactive against whole cells. The results were compared with those from the P. falciparum lactate dehydrogenase whole-cell assay, another method that is well established as a surrogate for parasite growth and is amenable for high-throughput screening. The results obtained demonstrate that the SPA-based [ 3 H]hypoxanthine incorporation assay is a suitable design that is adaptable to high-throughput antimalarial drug screening and that maintains the features, robustness, and reliability of the standard filtration hypoxanthine incorporation method.

Evaluation of 4-Amino 2-Anilinoquinazolines against Plasmodium and Other Apicomplexan Parasites In Vitro and in a P. falciparum Humanized NOD- scid IL2Rγ null Mouse Model of Malaria

“…1) were previously evaluated against P. falciparum (8) using the lactate dehydrogenase (LDH)-based assay format (23). To ensure the consistency of EC 50 values obtained by the different assay technologies, we determined the EC 50 values of the lead compounds against P. falciparum in three previously described assay formats: the LDH (10) and hypoxanthine (24) assays and SYBR green fluorescence flow cytometry (25). Within each assay, compounds 1 to 4 displayed similar EC 50 values ( Table 1).…”

“…P. falciparum hypoxanthine incorporation assay. The hypoxanthine incorporation assay was adapted from the procedure described by de Cozar et al (24). A culture of red blood cells parasitized with strain 3D7 at 0.5% parasitemia and 2% hematocrit in RPMI 1640 medium, AlbuMAX, and 5 M hypoxanthine was exposed to 9-step 3-fold serial dilutions of the compounds.…”

Identification of Small Molecules Disrupting the Ubiquitin Proteasome System in Malaria

“…The parasite growth inhibition assay was performed using the standard H-hypoxanthine incorporation method. This assay relies on parasite incorporation of labeled hypoxanthine that is proportional to P. falciparum growth (40). were taken of Giemsa-stained thin blood smears.…”

Identification and characterization of novel chemical scaffolds to overcome drug resistance in malaria

“…In silico screenings provide cost-effective ways of large-scale testing, although it only serves as a decent springboard onto in vitro validation [216,217]. Targetbased approaches are typically limited by the poor understanding of the parasite's unique biology, while cellular phenotypic assays are the most direct form of screening assays with the ability to be applied in a mid-to high-throughput fashion [218][219][220][221]. A simple and classical approach would be to determine parasite growth inhibition, where quantification is based on a cell counting assay through a reporter dye/protein [220,221].…”

“…Targetbased approaches are typically limited by the poor understanding of the parasite's unique biology, while cellular phenotypic assays are the most direct form of screening assays with the ability to be applied in a mid-to high-throughput fashion [218][219][220][221]. A simple and classical approach would be to determine parasite growth inhibition, where quantification is based on a cell counting assay through a reporter dye/protein [220,221]. Libraries consisting of a variety of novel chemical scaffolds typically have the biggest potential for identifying compounds which fit the profile of an ideal antimalarial, with examples of breakthroughs derived from such sources [185,222,223].…”