Chemoprotective antimalarials identified through quantitative high-throughput screening of Plasmodium blood and liver stage parasites

Abstract

The spread of Plasmodium falciparum parasites resistant to most first-line antimalarials creates an imperative to enrich the drug discovery pipeline, preferably with curative compounds that can also act prophylactically. We report a phenotypic quantitative high-throughput screen (qHTS), based on concentration-response curves, which was designed to identify compounds active against Plasmodium liver and asexual blood stage parasites. Our qHTS screened over 450,000 compounds, tested across a range of 5 to 11 concentrations, for activity against Plasmodium falciparum asexual blood stages. Active compounds were then filtered for unique structures and drug-like properties and subsequently screened in a P. berghei liver stage assay to identify novel dual-active antiplasmodial chemotypes. Hits from thiadiazine and pyrimidine azepine chemotypes were subsequently prioritized for resistance selection studies, yielding distinct mutations in P. falciparum cytochrome b, a validated antimalarial drug target. The thiadiazine chemotype was subjected to an initial medicinal chemistry campaign, yielding a metabolically stable analog with sub-micromolar potency. Our qHTS methodology and resulting dataset provides a large-scale resource to investigate Plasmodium liver and asexual blood stage parasite biology and inform further research to develop novel chemotypes as causal prophylactic antimalarials.

Figure 1

Implementation of a quantitative high-throughput (qHTS) screen against P. falciparum asexual blood stage parasites. (A) Overview of qHTS primary and secondary screens of compound libraries assayed against P. falciparum ABS parasites cultured in human RBCs. In our second major screen, growth inhibition was monitored via quantification of SYBR Green fluorescence, which detects parasite DNA in human DNA-deficient RBCs. Alternatively, inhibition of parasite proliferation was quantified by measuring luciferase activity (not shown). (B) Screening pipeline for identifying candidate causal chemoprophylactic antimalarials. Two large qHTS campaigns used either SYBR Green or luciferase to test for inhibition of parasite growth, assaying a combined total of 456,817 compounds. Following qHTS, manual triage selected 4253 synthetically tractable compounds for validation of antiplasmodial activity and mammalian toxicity. Compounds were then evaluated in vitro against P. berghei liver stage parasites at 3 μM and 1 μM concentrations. Selected compounds were further evaluated in a 12-point dose–response against P. berghei liver stage parasites, yielding 46 with submicromolar AC₅₀ values.

Figure 2

Stage-specific activity and chemical relatedness of compounds active against P. falciparum ABS and P. berghei liver stage parasites. (A) Multidimensional scatterplot of P. berghei liver stage activity compared to P. falciparum asexual stage inhibition. P. berghei liver stage percent survival (Y-axis) was assessed at a single 1 μM concentration. The color denotes the survival in the liver stage assay at a single 3 μM concentration (red, 0% survival; black 100% survival). P. falciparum asexual activity AC₅₀ value (X-axis) was determined from qHTS 72 h in vitro growth proliferation assays. (B) Uniform manifold approximation and projection (UMAP) of structural similarity, based on the Tanimoto coefficient, for the 994 compounds active against P. falciparum ABS parasites and screened for activity against P. berghei liver stages. Clusters 16 and 27 are highlighted with red arrowheads. (C,D) Depiction of clusters 16 (including the thiadiazine hit NCGC00473217) and 27 (including the pyrimidine azepine hit NCGC00374598). The compounds are clustered by Tanimoto similarity, with heat map representation of the ABS activity against P. falciparum Dd2-B2 (shown as Log [M] concentration values), activity against P. berghei liver stages (Pb LS), and toxicity against HepG2 cells, tested at both 3 μM and 1 μM and represented as percentage viability. Heat maps were generated on the UMAP data projection using the software DBSCAN (version 0.24.0; https://scikit-learn.org/stable/modules/generated/sklearn.cluster.DBSCAN.html; see “Methods” section).

Figure 3

Antiplasmodial activity, toxicity and stability profiles of the prioritized hits NCGC00374598 and NCGC00473217. Compound activity profiles for NCGC00374598 (compound 1) and NCGC00473217 (compound 2) against P. falciparum asexual blood stage parasites (black line, circle), as well as P. berghei liver stages in either laboratory A (blue line, squares) or laboratory B (purple line, triangles). Percent inhibition is shown on the Y-axis and compound concentration (Log [M]) is shown on the X-axis. Also shown are activities against P. falciparum Dd2 ABS parasites, in vitro inhibition activity in the P. berghei liver stage development assay (ILSDA), toxicity against mammalian HepG2 cells, and half-life rat liver microsome stability (RLM) values. Mean ± SD values were derived from of at least three independent replicates.

Figure 4

Resistance to pyrimidine azepine and thiadiazine hit compounds results in mutations in P. falciparum cytochrome b. (A) Resistance selection studies (left) with the pyrimidine azepine NCGC00374598 and the thiadiazine NCGC00473217 result in several cytochrome b mutations. Ribbon model (right) of the Saccharomyces cerevisiae cytochrome b subunit, generated using CCP4mg (version 2.10, CCP4 Molecular Graphics; https://www.ccp4.ac.uk/MG/index.html). The positions homologous to the mutations found in this study (colored spheres) cluster within or near the quinone oxidation site Qo (green), and are distal to the quinone reduction site Qi (light blue). Cytochrome b subunits form a dimer within the multi-component cytochrome bc₁ complex, a mitochondrial integral membrane protein complex required for mitochondrial respiration. (B) Comparative activity of the lead compounds NCGC00374598 and NCGC00473217 against P. falciparum Dd2 (B2 clone; black) and drug-selected resistant isolates (V259L (orange), A122D (blue) and F123L (purple) cytochrome b mutants were selected under NCGC00473217 pressure; cytochrome b G131S (brown) was selected under NCGC00374598 pressure). The F123L variant showed no susceptibility to NCGC00473217 up to the highest concentration tested (− 4.54 Log [M] or 29 µM). Also shown are the susceptibility responses to atovaquone, a clinically used antimalarial that targets cytochrome b and DSM265 a clinical candidate that targets the parasite dihydroorotate dehydrogenase enzyme. The y-axis denotes the AC₅₀ value (shown as mean ± SD) in Log molar concentration (left axis). The right y-axis shows the AC₅₀ value in nanomolar concentration. Responses to chloroquine, amodiaquine, mefloquine, ELQ-300 and artemether were similar across all parasite lines (Supplementary Table 6). Statistical variance of AC₅₀ compared to Dd2 (clone B2) parental line by Student’s t-test; p < 0.05, *; p < 0.01, **; p < 0.001, ***. (C) P. falciparum parasite asexual response to NCGC00374598 or NCGC00473217 in the transgenic Dd2 strain expressing the S. cerevisiae dihydroorotate dehydrogenase (Dd2-attB-ScDHODH), which confers resistance to mETC and DHODH inhibitors, or the transgenic parental line Dd2-attB.