Research Article

Open-source discovery of chemical leads for next-generation chemoprotective antimalarials

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Science  07 Dec 2018:
Vol. 362, Issue 6419, eaat9446
DOI: 10.1126/science.aat9446
  • A Plasmodium vivax liver-stage schizont on a lawn of hepatocytes.

    The parasite schizont has been stained with antibodies to parasite HSP70 (red) and UIS4 (yellow). Cell (parasite and hepatoma) nuclei are shown in blue. This study identifies compounds that can prevent the development of these liver-stage parasites and may function as chemoprotective drugs for malaria.

  • Fig. 1 Screen workflow.

    (A) The Charles River library (538,273 compounds) was plated into 1603 384-well plates. For the primary Pbluc single-point screen, compounds from four of the 384-well plates were transferred with a pin-tool instrument (50 nl per well) into 1536-well assay plates containing seeded HepG2 cells (3 × 103 cells per well). The next day, P. berghei luciferase–expressing sporozoites were freshly prepared from infected Anopheles stephensi mosquitoes, and ~1000 sporozoites in a 5 μl volume were added to each well. After 48 hours, P. berghei–Luc growth within hepatocytes was measured with bioluminescence. (B) To prepare source plates for the first round of reconfirmation (second round of screening), the 9989 hit compounds were transferred from the original 384-well library with an automated hit-picking system and serially diluted into eight points (1:3 dilutions) for dose-response screening in duplicate. The hit compounds (50 nl per well) in serial dilutions were acoustically transferred into assay wells containing HepG2 cells for Pbluc and HepG2tox assays. In addition, biochemical recombinant luciferase inhibition assay (Ffluc) were also performed. (C) For final reconfirmation (third round), 631 compounds prepared from re-sourced powders and were serially diluted (10 points, 1:3 dilution) and plated into Aurora 1536-well compound plates. Compounds (50 nl per well) were acoustically transferred into 1536-well assay plates. Multiple dose-respose assays such as Pbluc, HepG2tox, Ffluc, and ABS-Sybr were performed to determine IC50 in the third round of screening. A P. vivax liver schizont formation high-content assay in single-point (2X) was also performed.

  • Fig. 2 Cluster analysis.

    (A) For display, 405 compounds from 68 clusters that show a P value ≤ 0.05, cluster size ≥ 4, and hit fraction ≥ 0.75 are presented (data file S2). Most common substructure (MCS) per cluster is identified by using the top three active compounds, and a hierarchical tree was constructed from the MCSs to illustrate the intergroup connection. Compound members were then added to surround MCS nodes. All compound nodes are colored by hit status and shaped by other annotations. Primary hits are orange, reconfirmation hits (Pbluc IC50 < 10 μM) are red, third-round reconfirmation set (631 compounds) is purple, and others are light blue. P. falciparum asexual blood state–active compounds (ABS-Sybr IC50 < 10μM) are indicated by squares, luciferase inhibitors (Ffluc IC50 < Pbluc IC50 / 2) are indicated by triangles, hepatocyte toxic (primary HepG2tox > 50% or HepG2tox CC50 < Pbluc IC50 / 2) are indicated by diamonds, and the rest are shown as circles. (B to G) Active compounds in selected clusters [(B), (C), and (D)] with hit fractions of less than 0.75, as well as examples of singleton hits that are similar to the known antimalarial compounds, (E) cycloguanil, (F) DDD107498 (13), and (G) DSM265 (data file S3) (16).

  • Fig. 3 Target identification studies.

    (A) Chemical structures and IC50 of select antimalarial compounds identified as hits. Tanimoto clustering demonstrates that most molecules are structurally distinct, although some share similar scaffolds. (B) Metabolomic analysis reveals that 10 of the 13 compounds likely target the mETC and pyrimidine biosynthesis pathways. Robust increases in pyrimidine biosynthesis precursors N-carbamoyl-l-aspartate (CA) and dihydroorotate (DHO) are signatures of metabolic disruption of de novo pyrimidine biosynthesis. The metaprints for MMV1068987 and MMV1431916 are similar to the metaprint of the PfATP4 inhibitor KAE609, whereas the metaprint for MMV011772 is inconclusive. The numbers below each compound name indicate the Pearson correlation with an atovaquone profile (fig. S3). (C) IC50 of each compound in Dd2 cells expressing S. cerevisiae DHODH normalized to parent. The transgenic Dd2-ScDHODH strain expresses the cytosolic type 1 DHODH from S. cerevisiae (ScDHODH) and is resistant to P. falciparum mETC inhibitors. Ablation of compound activity in this cell line relative to its parent indicates inhibition of DHODH or downstream effectors in the mETC such as Cytbc1. Atovaquone, a known Cytb inhibitor, was included as a positive control, whereas other licensed antimalarials with mETC-independent mechanisms of action serve as negative controls. (D) Location of Phe188Ile mutation found in whole-genome sequences of MMV1454442-resistant parasites by using a crystal structure of PfDHODH (4ORM) (27). Amino acid residue 188 is highlighted in magenta. The structure shows a known PfDHODH inhibitor, DSM338 (27), cocrystalized with the protein. (E) Homology model of PfCytb (from PDB 1BE3) (35) with Tyr126Cys and Val259Leu mutations (highlighted in magenta) from MMV1432711-resistant parasites. The Arg95 has previously been implicated in atovaquone binding and resistance (36).

Supplementary Materials

  • Open-source discovery of chemical leads for next-generation chemoprotective antimalarials

    Yevgeniya Antonova-Koch, Stephan Meister, Matthew Abraham, Madeline R. Luth, Sabine Ottilie, Amanda K. Lukens, Tomoyo Sakata-Kato, Manu Vanaerschot, Edward Owen, Juan Carlos Jado Rodriguez, Steven P. Maher, Jaeson Calla, David Plouffe Yang Zhong, Kaisheng Chen, Victor Chaumeau, Amy J. Conway, Case W. McNamara, Maureen Ibanez, Kerstin Gagaring, Fernando Neria Serrano, Korina Eribez, Cullin McLean Taggard, Andrea L. Cheung, Christie Lincoln, Biniam Ambachew, Melanie Rouillier, Dionicio Siegel, François Nosten, Dennis E. Kyle, Francisco-Javier Gamo, Yingyao Zhou, Manuel Llinás, David A. Fidock, Dyann F. Wirth, Jeremy Burrows, Brice Campo, Elizabeth A. Winzeler

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S4
    • Tables S1 to S8
    • References
    Data File S1
    Compounds previously identified in high content imaging assays (9). The 197 compounds shared with this study were initially identified in an P. falciparum ABS assay (ABS-Sybr) and then retested against Plasmodium. yoelii hepatic stages (9). The file shows data from the previous publication, including whether the compound was a "hit" in the P. yoelii high content liver stage assay, whether it was selected for powder-resupply dose response testing in the high content P. yoelii assay (based on percent inhibition and compound availability), as well as the primary Pbluc and HepG2tox percent inhibition data from this study.
    Data File S2
    Clustering results. The file contains names, structures and cluster number for all compounds that are structurally similar to compounds with >83% inhibition in Pbluc. Also included are structure displays for 405 compounds from 68 clusters that show a p-value <= 0.05, cluster size >= 4 and hit fraction >= 0.75 are which are presented in Fig. 2.
    Data File S3
    Reconfirmation Set. File contains names, structures, and % inhibition in primary screen as well as dose response in secondary tests
    Data File S4
    Repurchased Validation Set (631 Compounds). Compounds selected for third round characterization
    Data File S5
    Metabolomic data. The file contains raw metabolomic data used to create metaprints.
    Data File S6
    Sequencing Data. The file contains whole genome sequencing variant detection files
    Data File S7
    Mitochondrial inhibition data. Dose response curves for compounds active in the D10-ScDHODH high-throughput (1536-well) assay. The attached Excel spreadsheet shows values for the 631 compounds (Repurchased Validation Set) plus two controls (atovaquone and artemisinin)

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