Use of the NP-40 Detergent-Mediated Assay in Discovery of Inhibitors of β-Hematin Crystallization

Abstract

The protozoan parasite responsible for malaria affects over 500 million people each year. Current antimalarials have experienced decreased efficacy due to the development of drug-resistant strains of Plasmodium spp., resulting in a critical need for the discovery of new antimalarials. Hemozoin, a crystalline by-product of heme detoxification that is necessary for parasite survival, serves as an important drug target. The quinoline antimalarials, including amodiaquine and chloroquine, act by inhibiting the formation of hemozoin. The formation of this crystal does not occur spontaneously, and recent evidence suggests crystallization occurs in the presence of neutral lipid particles located in the acidic digestive vacuole of the parasite. To mimic these conditions, the lipophilic detergent NP-40 has previously been shown to successfully mediate the formation of ␤-hematin, synthetic hemozoin. Here, an NP-40 detergent-based assay was successfully adapted for use as a high-throughput screen to identify inhibitors of ␤-hematin formation. The resulting assay exhibited a favorable Z of 0.82 and maximal drift of less than 4%. The assay was used in a pilot screen of 38,400 diverse compounds at a screening concentration of 19.3 M, resulting in the identification of 161 previously unreported ␤-hematin inhibitors. Of these, 48 also exhibited >90% inhibition of parasitemia in a Plasmodium falciparum whole-cell assay at a screening concentration of 23 M. Eight of these compounds were identified to have nanomolar 50% inhibitory concentration values near that of chloroquine in this assay.

Identification and Mechanistic Evaluation of Hemozoin-Inhibiting Triarylimidazoles Active against Plasmodium falciparum

“…13−16 Most recently, a total of 144,330 compounds from the Vanderbilt University (VU) chemical compound library were screened for their ability to inhibit βH formation using an NP-40 based assay described previously and the ferrihemochrome method of detection. 8,15,16 Of the 530 confirmed βH inhibitors, 171 inhibited parasite growth by ≥90% at 23 μM (32.3% of the 530 βH inhibitors) and 25 compounds exhibited favorable IC 50 values below 1 μM. Furthermore, cell fractionation studies successfully validated Hz formation as the mechanism of action in parasites for a subset of tested compounds, which represented 12 of the 14 novel scaffolds identified.…”

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

“…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.…”

“…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.…”

Antimalarial benzoheterocyclic 4-aminoquinolines: Structure–activity relationship, in vivo evaluation, mechanistic and bioactivation studies

“…The β-hematin formation inhibition assay method described by Carter et al was modified for manual liquid delivery 38,39 . Two stock solutions of the samples were prepared by dissolving the pre-weighed compound in DMSO with sonication to give 20 mM and 2 mM solutions of each sample.…”