Lipophilic Mediated Assays for β-Hematin Inhibitors

Abstract

The growing drug resistance of Plasmodia spp. to current antimalarial agents in the quinine and artemisinin families further asserts the need for novel drug classes to combat malaria infection. One approach to the discovery of new antimalarials is the screening of natural product extracts for activity against the formation of hemozoin, a biomineral essential to parasite survival. By mimicking the in vivo lipid-water interface at which native hemozoin is found, hemozoin can be synthesized outside the parasite. In this work, a variety of lipophilic mediators was used to determine the optimal platform for in vitro hemozoin formation and then tested for efficacy in preliminary screens containing crude natural product extracts. The complete optimization and validation of a NP-40 detergent-mediated assay provide a screening template with an expedited 4-hour incubation time and identical IC50 values to those measured from the parasite’s native lipid component.

Synthesis and <i>in Vitro</i> Testing of Antimalarial Activity of Non-natural-Type Neocryptolepines: Structure–Activity Relationship Study of 2,11- and 9,11-Disubstituted 6-Methylindolo[2,3-<i>b</i>]quinolines

“…The β-haematin formation inhibition assay method reported by Carter et al 32,33) was modified for manual liquid delivery as previously described. 34) The formation of haemozoin by P. falciparum, the details and relevance of which has been previously described, [34][35][36] is a suitable parasite-specific drug target for compounds able to cross the red blood cell membrane, the parasitophorous vacuole membrane, the P. falciparum plasma membrane and the acidic digestive vacuole (DV) membrane.…”

“…Detergent Mediated Assay for β-Haematin Inhibition The β-haematin formation inhibition assay method described by Carter et al 46,47) was modified for manual liquid delivery. Three stock solutions of the samples were prepared by dissolving the pre-weighed compound in DMSO and after sonication, diluting with DMSO to give 20 mM, 2 mM and 0.4 mM solutions of each sample.…”

High-Throughput Screening and Prediction Model Building for Novel Hemozoin Inhibitors Using Physicochemical Properties

“…The core library, which contains 9,600 structurally diverse compounds, was from the Open Innovation Center for Drug Discovery, University of Tokyo (Tokyo, Japan). The detergent NP-40 served as a mediator for ␤-hematin formation due to its stability, low cost, and low IC 50 , beside its similarity to the natural lipid particles of the parasite's vacuole (21,43). Dimethyl sulfoxide (DMSO), from Wako Pure Chemicals, Osaka, Japan, was chosen as the negative control because of its proven inability to inhibit the heme crystallization reaction (44).…”

“…Following the transfer of compounds, a Multidrop combi dispenser (Thermo Fisher Scientific) was used to distribute 20 l of hemin solution (10 mM heme in DMSO and 100 mM acetate buffer, pH 4.8) and 10 l of detergent NP-40 into each well of the plates. The assay mixture was incubated at 37°C for 250 min (43). Afterwards, pyridine solution was added and the mixture shaken for 10 min.…”

Biomimetic Approaches to Understanding the Mechanism of Haemozoin Formation

“…This suggests that detergents are most efficient at mediating -haematin formation below their cmc values. Indeed, Carter et al (2010) have investigated a series of detergents as mediators of -haematin with yields ranging between 7 and 74% all well below their cmc values. NP 40, Tween 20 and Tween 80 were found to give the highest yields, ranging between 69 and 74%, while SDS, Triton X-100 and Chaps were found to be inefficient with yields between 7 and 10%.…”