title: Imaging flow cytometry for the screening of compounds that disrupt the Plasmodium falciparum digestive vacuole.
abstract: Malaria, despite being one of the world's oldest infectious diseases, remains difficult to eradicate because the parasite is rapidly developing resistance to frontline chemotherapies. Previous studies have shown that the parasite exhibits features resembling programmed cell death upon treatment with drugs that disrupt its digestive vacuole (DV), providing a phenotypic readout that can be detected using the imaging flow cytometer. Large compound collections can thus be screened to identify drugs that are able to disrupt the DV of the malaria parasite using this high-content high-throughput screening platform. As a proof-of-concept, 4440 compounds were screened using this platform in 4months and 254 hits (5.7% hit rate) were obtained. Additionally, 25 compounds (0.6% top hit rate) were observed to retain potent DV disruption activity that was comparable to the canonical DV disruptive drug chloroquine when tested at a ten-fold lower concentration from the original screen. This pilot study demonstrates the robustness and high-throughput capability of the imaging flow cytometer and we report herein the methodology of this screening assay.
authors.affiliation: Department of Microbiology and Immunology, National University of Singapore, Singapore.
authors.forename: Wan Ni
authors.initials: WN
authors.lastname: Chia
authors.affiliation: Department of Microbiology and Immunology, National University of Singapore, Singapore; NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore.
authors.forename: Yan Quan
authors.initials: YQ
authors.lastname: Lee
authors.affiliation: Department of Microbiology and Immunology, National University of Singapore, Singapore; NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore. Electronic address: mictank@nus.edu.sg.
authors.forename: Kevin Shyong-Wei
authors.initials: KS
authors.lastname: Tan
mesh_terms: D000814:Aniline Compounds / Q000737:chemistry; D000962:Antimalarials / Q000737:chemistry / Q000494:pharmacology; D001562:Benzimidazoles / Q000737:chemistry; D002232:Carbocyanines / Q000737:chemistry; D002478:Cells, Cultured; D004912:Erythrocytes / Q000187:drug effects / Q000469:parasitology* / Q000648:ultrastructure; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D057166:High-Throughput Screening Assays; D006801:Humans; D019044:Image Cytometry / Q000379:methods*; D008018:Life Cycle Stages / Q000187:drug effects* / Q000502:physiology; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000254:growth & development / Q000648:ultrastructure; D054852:Small Molecule Libraries / Q000737:chemistry / Q000494:pharmacology; D013194:Staining and Labeling / Q000379:methods; D014617:Vacuoles / Q000187:drug effects* / Q000648:ultrastructure; D014966:Xanthenes / Q000737:chemistry
chemical_list: D000814:Aniline Compounds; D000962:Antimalarials; D001562:Benzimidazoles; D002232:Carbocyanines; C409648:Fluo 4; D005456:Fluorescent Dyes; D054852:Small Molecule Libraries; D014966:Xanthenes; C068624:5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine; C017807:bisbenzimide ethoxide trihydrochloride
keywords: Digestive vacuole; Drug discovery; High-content high-throughput screening; Imaging flow cytometry; Malaria; Plasmodium falciparum