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

Abstract

To discover leads for next-generation chemoprotective antimalarial drugs, we tested more than 500,000 compounds for their ability to inhibit liver-stage development of luciferase-expressing Plasmodium spp. parasites (681 compounds showed a half-maximal inhibitory concentration of less than 1 micromolar). Cluster analysis identified potent and previously unreported scaffold families as well as other series previously associated with chemoprophylaxis. Further testing through multiple phenotypic assays that predict stage-specific and multispecies antimalarial activity distinguished compound classes that are likely to provide symptomatic relief by reducing asexual blood-stage parasitemia from those which are likely to only prevent malaria. Target identification by using functional assays, in vitro evolution, or metabolic profiling revealed 58 mitochondrial inhibitors but also many chemotypes possibly with previously unidentified mechanisms of action.

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 ml 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-Laspartate (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).