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

Mechanism of action studies reveal an abundance of mitochondrial transport chain inhibitors

To further investigate the mechanism of action of 13 of the most potent ABS hits (Fig. 3A), we acquired additional compound from commercial vendors. Because most, if not all, target identification methods require activity in P. falciparum blood stages, all compounds selected for initial target discovery were active against ABS parasites, with an average IC50 of 465 nM (range of 13 nM to 1.2 μM).

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).

As an initial pass, we first subjected the compounds to metabolic profiling (20). This liquid chromatography–mass spectrometry (LC-MS)–based method measures several hundred metabolites and identifies those that show statistically significant increases or decreases upon parasite compound exposure (data file S5). Whereas three gave ambiguous results, 10 of the 13 analyzed scaffolds gave a metabolic profile signature analogous to that of atovaquone (fig. S3), indicating that these 10 most likely interfere with one or more targets in the mitochondrial electron transport chain (mETC), a known druggable pathway for P. falciparum blood and liver stages (Fig. 3A). The set of 13 compounds represents 11 distinct scaffolds (Fig. 3B), so this degree of functional overlap would not have been predicted by structure alone. To our knowledge, none of the molecules have been previously identified as acting against the mitochondrial electron transport pathway.

To further confirm that these 10 compounds (representing eight chemotypes) inhibited the mETC, we took advantage of a transgenic parasite line that overexpresses the Saccharomyces cerevisisae dihydroorotate dehydrogenase (Dd2-ScDHODH) (21). Unlike the type-2 P. falciparum enzyme that is dependent on cytochrome bc1 for ubiquinone, the cytosolic type-1 yeast enzyme can use fumarate as an electron acceptor. This allows the transgenic parasites to bypass the need for ETC activity to provide ubiquinone to PfDHODH (21). Compounds that target PfDHODH or other enzymes along the mETC lose potency in the Dd2-ScDHODH transgenic cell line. As expected, the Dd2-ScDHODH parasites show marked (24.8 to 1000-fold) resistance to the 10 compounds with the mETC metabolic profile (Fig. 3C and table S3). Furthermore, a variation of this functional assay can distinguish between inhibitors of PfDHODH and cytochrome bc1. Specifically, the addition of proguanil to Dd2-ScDHODH parasites restores the inhibitory capabilities of cytochrome bc1 inhibitors; however, growth is not affected in the case of PfDHODH inhibitors. Three out of six mitochondrial inhibitors tested in these conditions were not inactivated by proguanil, suggesting a profile consistent with PfDHODH inhibition (Fig. 3C and table S4). To further investigate mitochondrial inhibition, and because there are multiple potential targets, we used an in vitro evolution and whole-genome analysis (IViEWGA) approach (22) to further elucidate the molecular target of several of the compounds, including MMV1454442, MMV1432711, and MMV142795. First, three independent lines resistant to MMV1454442 were isolated after growing in sublethal concentrations of compound. The resistant clones showed an average 4.2-fold shift in the IC50 (range of 1.9 to 9.4) (table S5). Whole-genome sequencing of the nine clones (three each from three independent selections), as well as the drug-sensitive parent clone to 78-fold coverage (table S6), revealed that the resistant lines carried either a single-nucleotide variant Phe188Ile (Fig. 3D and data file S6) or a copy number variant (table S8) in P. falciparum dihydroorotate dehydrogenase (PF3D7_0603300), which is a well-validated drug target in P. falciparum (16). This result is consistent with proguanil not affecting growth inhibition in Dd2-ScDHODH parasites (Fig. 3C). MMV1454442, an amino-triazol, although somewhat similar to a pyrrole-based DHODH inhibitor (23), is a previously unidentified chemotype, which would not have been predicted with structural information alone. The Phe188 residue is located in the species-selective inhibitor-binding pocket of PfDHODH (24) and has been shown to be in contact with the known DHODH inhibitor leflunomide (25) and the triazolopyrimidine DSM338 (Fig. 3D) (26). Furthermore, mutation of this residue has been shown to confer resistance to the alkylthiophene inhibitor Genz-669178 (27), suggesting that MMV1454442 likely shares the same space (Fig. 3D).

IViEWGA of MMV1432711-resistant parasites revealed they had acquired one of two nonsynonymous single-nucleotide variants (SNVs) in the gene encoding cytochrome b (data file S7). The amino acid mutations found, Tyr126Cys and Val259Leu, are located within helix C in the ubiquitinol-binding pocket of cytochrome b, a catalytically important subunit of the cytochrome bc1 complex that contains two reaction sites, Q0 (ubiquitinone reduction site) and Qi (ubiquitinol oxidation site). MMV1432711 has a chemical scaffold similar to that of the Qi inhibitors, so we used a homology model of PfCYTb (Fig. 3E) to resolve the mode of binding. Docking into the model showed that MMV1432711 is likely a class II inhibitor. The allele Y126C was previously reported to confer resistance to decoquinate (28) and MMV008149 (29). To our knowledge, allele Val259Leu has not been reported in the literature.

For compound MMV1427995 {2-[(5,6-diphenyl-1,2,4-triazin-3-yl)thio]-1-(pyrrolidin-1-yl)propan-1-one}, in vitro evolution studies yielded two resistant lines that were cloned for further phenotyping and whole-genome sequencing (tables S5 and S6). Clones showed an average 2.6-fold IC50 shift (range of 1.8- to 3.4-fold) in susceptibility to MMV1427995 (table S5). Sequencing revealed that both clones carried the amino acid mutation, Arg95Lys, in cytochrome b, located in the matrix-oriented region of the protein after the second transmembrane domain (Fig. 3E and data file S6). One clone also carried an additional Pro102Thr mutation in cytochrome c oxidase subunit 1. This mutation is located between the second and third transmembrane domain, is not located in any of the iron or copper redox centers, and is, to our knowledge, the first described mutation in cytochrome c oxidase selected for during compound exposure. However, this mutation did not induce a higher resistance level than did the Arg95Lys mutation alone and may thus represent a compensatory mutation. MMV1427995 is a different scaffold family (also overrepresented in the initial set of screening hits) from known P. falciparum cytochrome bc1 inhibitors, and its target would not have been predicted through similarity searching.

Given the high number of mitochondrial inhibitors in the dataset, we further examined the set of 631 compounds (repurchased validation set). All 631 compounds were tested in duplicate in eight-point ABS dose response in two P. falciparum D10–derived lines (21), one of which expresses ScDHODH (fig. S4 and data file S7) in the presence and absence of 1 μM proguanil in duplicate (~80,000 data points). Of the 136 compounds with ABS activity, visual inspection showed that 78 were likely not mitochondrial inhibitors, and 58 showed profiles consistent with mitochondrial inhibition (figs. S4 and S5 and data file S7). Of these, 10 were clear DHODH inhibitors (including the three shown in Fig. 3), one was a potential DHODH inhibitor, and 47 were likely or possible cytochrome bc1 inhibitors (including all nine from Fig. 3 that were tested). Strong nonrandom structure-activity relationships are evident (fig. S5), validating the assay. For example, six of the seven compounds that were more than 55% similar to MMV1042937 (fig. S5, fourth row) in the set of 631 were predicted to be cytochrome bc1 inhibitors (log10 p = –6.09). The seventh, MMV1457596, was missed because the IC50 were all 10 μM, but visual inspection of the curves showed an ~75% reduction in signal at 10 μM for the ScDHODH line relative to the PfDHODH line (data file S7).