Identification of novel compounds with prophylactic activity against hypnozoites using a Plasmodium cynomolgi in vitro model

Abstract

This study aimed to identify new compounds with prophylactic activity against Plasmodium liver stages, particularly hypnozoites. A small, focused set of 568 compounds from the GSK open-access TCAMS library was tested in vitro against Plasmodium cynomolgi liver stages. Among these compounds, 12 showed IC₅₀ values below 2 µM against hypnozoites, with the most active compound displaying nanomolar activity. These active compounds could be assigned to/grouped into six chemical clusters. Some compounds could be associated with potential known modes of action such as cytochrome bc1 inhibition or kinase inhibition, whereas the mode of action of other hit compounds is currently unknown. The high hit rate in this screening may be attributed to preselecting compounds based on their activity against gametocytes and liver stages in other Plasmodium species. Based on our findings, we suggest that this preselection methodology is appropriate for finding compounds that are active against hypnozoites.

Keywords: GSK; TCAMS; drugs assay; hypnozoite; image-based screening; in vitro liver stage assay; malaria; phenotypic screening; plasmodium cynomolgi; prophylactic.

Fig 1

Individual dose-response curves of 12 compounds (arranged by compound cluster) with sub-micromolar activity against hypnozoites in the prophylactic (drug treatment from d0-6pi) in vitro P. cynomolgi liver stage assay. Parasites were fixed on d6 pi, and stained with anti-P. cynomolgi Hsp70 antibodies and Alexa568-labeled secondary antibodies and analyzed using a high-content screening microscope (Operetta, Perkin Elmer). Curves represent the mean ± SD of triplicate measurements. Activity against P. cynomolgi (left Y-axis, %inhibition) liver schizonts (blue lines, squares) and hypnozoites (red lines, triangles) was determined. Dose-dependent reduction of the nuclei counts (right Y-axis) hints toward the cytotoxicity of the compound (evident in R10489/74/4).

Fig 2

Confirmed chemotypes with P. cynomolgi liver stage activity. Cluster 1: aminoindoles; cluster 2: thiazoles; cluster 3: quinolones; cluster 4/5: 7-aza-indoles; and cluster 15: furan-carboxamide. Structures of selected compounds are available in the supplementary data (Fig. S1).