An integrative analysis of small molecule transcriptional responses in the human malaria parasite Plasmodium falciparum
© Siwo et al. 2015
Received: 11 March 2015
Accepted: 29 October 2015
Published: 4 December 2015
Transcriptional responses to small molecules can provide insights into drug mode of action (MOA). The capacity of the human malaria parasite, Plasmodium falciparum, to respond specifically to transcriptional perturbations has been unclear based on past approaches. Here, we present the most extensive profiling to date of the parasite’s transcriptional responsiveness to thirty-one chemically and functionally diverse small molecules.
We exposed two laboratory strains of the human malaria parasite P. falciparum to brief treatments of thirty-one chemically and functionally diverse small molecules associated with biological effects across multiple pathways based on various levels of evidence. We investigated the impact of chemical composition and MOA on gene expression similarities that arise between perturbations by various compounds. To determine the target biological pathways for each small molecule, we developed a novel framework for encoding small molecule effects on a spectra of biological processes or GO functions that are enriched in the differentially expressed genes of a given small molecule perturbation.
We find that small molecules associated with similar transcriptional responses contain similar chemical features, and/ or have a shared MOA. The approach also revealed complex relationships between drugs and biological pathways that are missed by most exisiting approaches. For example, the approach was able to partition small molecule responses into drug-specific effects versus non-specific effects.
Our work provides a new framework for linking transcriptional responses to drug MOA in P. falciparum and can be generalized for the same purpose in other organisms.
Malaria continues to take a large toll on the health and economies of some of the world’s poorest nations. Drugs remain the primary option for dealing with malaria infection although there are promising clinical trials that may pave the way for the use of vaccines against the disease . In spite of enormous progress in the fight against the disease, the emergence of drug resistance to artemisinin, the only anti-malarial drug for which clinical resistance is not yet widespread, threatens to reverse the gains [2, 3]. There is an urgent need to fast-track the development of new anti-malarials. Fortunately, high-throughput and phenotypic screens have provided several potential drug leads. Through public and private efforts, nearly six million compounds have been screened leading to the identification of thousands of active compounds [4–6]. The Malaria Box, an open access “pharmacological test kit” has been made freely available to malaria researchers in an effort to spur antimalarial dug development . The malaria drug development pipeline now contains over a dozen new drugs and several new combinations of approved drugs are in various stages of pre-clinical and clinical trials . Approximately half of these new drugs have unknown mechanisms. An understanding of the mode of action (MOA) of these compounds would help prioritize and optimize them while also highlighting potential resistance mechanisms. This could help mitigate the high rate of failure in the drug development pipeline or rapid emergence of drug resistance. Furthermore, even compounds with no potential to be developed into useful therapeutics are valuable as tools for probing the parasite’s biology .
Transcriptional profiling of cells exposed to small molecules has been successfully demonstrated as useful in understanding drug MOA especially in human cell lines. For instance, the Connectivity Map (CMap) [8, 9], a database of gene expression profiles from cancer cell lines exposed to multiple drugs of known MOA has been used to successfully predict the MOA of new drugs [10, 11]. However, chemical perturbation of P. falciparum, which has been widely described as transcriptionally hard-wired [12–14], is thought to provoke little in the way of a specific response. For example, treatment of the parasite with lethal anti-folate drugs for up to 24 hrs did not reproducibly induce up-regulation of genes in the folate pathway or elsewhere in the genome . Exposure to chloroquine (CQ) was reported to have no effect on the transcript levels of genes presumed to be involved in its MOA [13, 14]. However, in other studies, perturbation of the parasite with a polyamine synthesis inhibitor, diflouromethylornithine- DFMO , febrile temperatures , artesunate [17, 18] and an inhibitor of sphingomyelin synthase  were found to provoke transcriptional changes in the expected target biological pathways. These reports suggest that the reported lack of responsiveness to CQ and anti-folate drugs cannot be generalized to all perturbations. In an earlier large-scale perturbation of malaria parasites with 20 drugs , 59 % of genes responded transcriptionally to at least one. Some drugs, including CQ, quinine and colcichine affected fewer than 50 genes, while drugs like apicidin, trichostatin A and staurosporine affected more than 250 genes and, importantly, even for drugs that only modestly affected gene expression, the effects were highly reproducible to those in other experiments within the same study . The ability of the parasite to respond in a reproducible way implies that the response is physiologically relevant and coordinated. Except for the study by Hu et al. , transcriptional studies in the parasite have focused on only one drug at a time, making it difficult to reach general conclusions about the parasite’s capacity to mount specific transcriptional responses [12, 13, 15]. We expand on these previous reports to investigate the extent to which transcriptional responses in P. falciparum can indicate precise small molecule targets and/or broader biological effects. To do this, we devised a 6 step approach that involves: i) performing perturbations with 31 chemically and functionally diverse drugs; ii) minimizing biological variation among samples by leveraging the multiplex exon array developed in our lab; iii) generating perturbations in two genetically and phenotypically distinct lab clones; iv) exposing parasites only briefly (2 hrs) to small molecules to minimize secondary effects; v) minimizing non-specific perturbation effects by normalizing transcript levels relative to all other perturbations rather than to untreated controls as is typically done, and, vi) leveraging multiple independent datasets to cross-validate that transcriptional responses reflect biologically meaningful small molecule relationships.
Overview of the study
Synchronized cultures of two laboratory clones, Dd2 and HB3, were exposed for 2 h to each compound at a single developmental stage (24 h trophozoites) at a concentration (IC50) obtained from literature sources or determined by our laboratory (Table 1). Genome-wide transcript abundances were determined for each drug perturbation by microarrays.
Previous studies in P. falciparum noted only very subtle transcriptional responses, largely attributed to generalized stress responses [12, 13, 20]. Therefore, we undertook additional data processing steps of RMA-normalized (see Methods) signal intensity data. First, for each perturbation, we averaged the signal intensity for each gene across two distinct lab clones (Dd2 and HB3), to enhance signal-to-noise ratio as demonstrated in the CMap project [10, 22]. We further validated that averaging gene expression data across two different strains in this way strengthens the identification of drug MOA (see Additional file 1: section D). Importantly, because the two clones have very different geographical and drug selection histories, they effectively serve as robustly independent replicates for each perturbation. Secondly, we computed a compound-specific response index for each gene by normalizing the gene’s average transcript level following perturbations in the two clones against its average level across all perturbations within the same experimental batch (Additional file 2) to increase signal-to-noise ratio . This normalization procedure uses distinct biological replicates to help mitigate non-specific transcriptional responses associated with many perturbations as well as experimental batch effects. This approach differs significantly from previous studies [12, 13, 20] in P. falciparum that relied on normalization using untreated controls for single perturbations; in this case, widespread non-specific transcriptional responses can obscure perturbation-specific responses (additional evidence in support of this is in Additional file 1: section D).
Global relationships in small molecule transcriptional responses relate to chemical structure
The biological response of cells to small molecules is dependent on chemical structure. Small molecules with similar chemical components have similar biological effects as supported by structure activity relationships (SAR) studies and correlations between gene expression effects of drugs and their chemical structures [24–27]. To better understand the role of chemical structure on the observed dichotomy (Fig. 1), we hierarchically clustered the compounds based on their PubChem substructure fingerprints (Additional file 1: Figure S1; see Additional file 3 for the fingerprints). The PubChem substructure fingerprint clusters to some extent recapitulate the transcription profile clusters (Additional file 1: Figure S1). In particular, 8 of 9 (89 %) Class I compounds (based on transcriptional responses) also cluster together purely based on substructure fingerprints (hypergeometric test P = 0.04, Additional file 1: Figure S1) while 11 out 12 (92 %) Class II compounds are placed in a separate cluster based on substructure fingerprints (hypergeometric test P = 0.006, Additional file 1: Figure S1). Furthermore, we corroborated these observations using an independent method (multidimensional scaling, MDS) to view the pairwise distances between the compounds based on their chemical features. In the 2-dimensional MDS plot (Additional file 1: section B and Additional file 1: Figure S2), Class II compounds are enriched in a distinct quadrant of the plot (hypergeometric test P = 0.002, Additional file 1: Figure S2) demonstrating a strong association between their similarity in the chemical and transcriptional spaces. That is, small molecules that are chemically similar are also more likely to induce similar transcriptional responses.
Drug relationships depend on both chemical structure and MOA
Small molecules induce transcriptional responses in expected target pathways
The small molecule-GO networks predict complex relationships between drugs and biological pathways
Unlike previous work on drug-drug networks in other species , the small molecule-GO network provides hypotheses for why two drugs are biologically related. For example, the small molecule-GO network confirms that the drugs CQ, TQ and E64 are related through the GO process ‘proteolysis involved in cellular catabolic processes’, consistent with the effect of these drugs on hemoglobin catabolism. In contrast, curcumin and apicidin- drugs that interfere with regulation of gene expression through inhibition of histone acetylation and deactylation, respectively- are both connected to the biological process ‘regulation of gene expression’ while the drugs methotrexate and 5-FU which interfere with pyrimidine synthesis are connected to the function ‘nucleobase-containing compound metabolic process’. The connection between 5-FU and this biological function is unexpected in P. falciparum because the parasite lacks a pyrimidine salvage pathway which is required for the activation of 5-FU to an inhibitor of thymidylate synthase (TS) . Nevertheless, 5-FU is a weak competitive inhibitor of the P. falciparum orotate phosphoribosyl transferase (pfOPRT), an essential step in pyrimidine biosynthesis . Inhibition of pfOPRT could impact the synthesis of pyrimidines and provide a connection between methotrexate and 5-FU activity. To validate the connection between these two drugs, we measured dose responses to 5-FU across the Dd2 × HB3 genetic cross (Additional file 7) and explored potential genetic determinants of its effects through QTL. A direct apriori test of the genetic locus containing the pfOPRT gene (chromosome 5, cM 31.5) shows that parental alleles in this locus are significantly associated with 5-FU dose response with a higher IC50 for progeny inheriting this locus from the Dd2 parent (LOD = 1.90, P = 0.002). A genome-wide QTL scan demonstrates that in addition to this locus, dose response variation to the drug is associated with 3 other loci on chromosomes 8 (cM 91.8, LOD = 2.98), 11 (cM 143.2, LOD = 2.3) and 14 (cM 189.4, LOD = 1.80). The chromosome 8 QTL region includes the Rad54 gene (PF3D7_0803400) encoding a DNA repair protein, providing additional support for the connection between 5-FU and the GO function ‘nucleobase-containing metabolic process’. Thus, the small molecule-GO network provides a framework for identifying drug similarities while at the same time identifying the biological processes underlying the similarities.
The network approach reveals that while some biological processes are widely perturbed or affected by many small molecules, others are only perturbed by only one or a few small molecules (Fig. 4c). For example, the biological categories ‘translocation of peptides into host’ (GO:0042000), ‘respiratory electron transport chain’ (GO:0022904) and ‘antigenic variation’ (GO:0020033) were up-regulated by many compounds (13, 12 and 10, respectively). These processes may reflect generalized stress responses that are associated with subsets of small molecules. In contrast, categories that are up-regulated by only one small molecule include: ‘protein import into nucleus’ (GO:0000059)- perturbed by rapamycin, ‘N-terminal protein amino acid acetylation’ (GO:0006474)- perturbed by MMS and ‘transposition’ (GO:0032196)- perturbed by 5-FU.
Transcriptional insights into artemisinin’s MOA
Predicting MOA of novel compounds by integration of QTL
Public and private screening efforts have recently led to the identification of thousands of compounds with cytotoxic activity in P. falciparum [4, 5]. Techniques for predicting MOA of these compounds will be valuable for their characterization and prioritization for further development . Among the compounds profiled in our study, five (SJ194935, SJ119930, SJ140722, SJ292024 and SJ77572) were derived from a high-throughput screening effort by the St Jude’s children’s hospital . To ascertain the biological effects of these compounds, we determined the coherence of biological functions predicted from transcriptional perturbations with those encoded in QTL associated with dose response variation to the compounds. The small molecule-GO network demonstrates that a single compound can perturb more than one biological process. However, some functions are perturbed by a single or just a few compounds and yield predictions of higher specificity. Therefore, we prioritized the links between these compounds and each biological function by considering the specificity of each function.
Prediction of MOA of novel compounds from St Jude using QTL and small molecule-GO network
Coherent functions in QTL and unique connections in network
14 cM 106.1
8 cM 83.2
7 cM 66.1
5 cM 65.9
Drug transmembrane transport
Similarly, to predict potential MOA of the other novel compounds, we compared the functional coherence between their unique connections in the small molecule-GO network and the functions of genes located within their QTL. For each small molecule we considered QTL peaks with at least a LOD score of 2 (Table 3; see Additional file 7 for dose responses used in QTL). The St Jude compound SJ119930 had no unique biological process connections in the up-/ down-regulated small molecule-GO networks; hence predictions could not be made at 100 % specificity. Two functions were predicted as its targets at 94 % specificity in the up-regulated network: ‘respiratory complex IV assembly’ and ‘autophagic vacuole assembly’. QTL mapping revealed a suggestive peak on chromosome 8 (cM 83.2) but we could not identify genes whose functions are coherent to those predicted in the network. For SJ140722, a single biological process (‘dolichol biosynthetic process’) was uniquely connected to it in the up-regulated network and no unique connections were observed in the down-regulated network. We did not find any gene with this function in the QTL region associated with variation in dose response to the compound. Another compound, SJ292024, was uniquely connected to the biological process ‘ribosomal large subunit biogenesis’ in the up-regulated network. Nine processes were connected to this compound at 100 % specificity in the down-regulated network: ‘protein glycosylation’, ‘respiratory chain IV complex assembly’, ‘intein mediated protein splicing’, ‘drug transmembrane transport’, ‘phosphorylation’, ‘catabolic process’, ‘DNA strand elongation involved in DNA replication’, ‘signal peptide processing’ and ‘terpenoid biosynthetic pathway’. The QTL peak associated with this compound (chromosome 5, cM 65.9) includes the gene encoding the multi-drug resistance tansporter, pfMDR1 coherent with prediction connection between the drug and the GO function ‘drug transmembrane transport’.
Drug responses are complex: perturbation of a single protein can lead to direct effects on its multiple functions and indirect effects can be perpetuated through the cellular network. In addition, broad chemical properties of compounds, for example hydrophobicity, can affect multiple biological processes. Therefore, understanding the nature of transcriptional responses to specific perturbations and filtering non-specific effects requires knowledge of how cells respond to a range of chemical perturbations. Without this knowledge, it may not be possible to identify transcriptional responses specifically induced by a perturbation vs. those resulting from general stress. This may be one reason that the parasite’s transcriptional responses to perturbations have been considered to be non-specific [12, 13]. Our study design utilizes many small molecule perturbations in the same experiment and provides a novel way to disentangle transcriptional responses.
The results presented in this work demonstrate that transcriptional responses can point to drug MOA in P. falciparum. The observation that small molecules containing similar chemical substructures also tend to show similar transcriptional responses (Fig. 2 and Additional file 1: Figure S1) imply that these responses are specific to the perturbations. The clustering of the 31 small molecules into two broad classes (Fig. 1) coupled with the observation that a single chemical substructure (Fig. 2) can distinguish these classes implies that a large component of transcriptional responses are due to specific chemical features, most prominently rings (Fig. 2 and Additional file 1: Figure S2). Even though this study has profiled the largest set of small molecule transcriptional responses in P. falciparum to date, it is important to note that the 31 small molecules represent a small proportion of chemical diversity. Our study provides a foundation for more extensive studies in future.
To capture the complexity of small molecule relationships, we developed a novel framework for encoding small molecule effects into binary fingerprints of biological categories that are enriched in their perturbed gene sets (Fig. 4a, b and c). We have demonstrated that this representation allows a detailed view of complex relationships that can exist between small molecules in which some biological functions are affected by multiple compounds while others are specifically affected by a smaller number of compounds (Fig. 4c). The resulting small molecule-GO network has enabled us to tease apart small molecule-specific effects from non-specific responses, thereby providing a reliable approach for predicting small molecule MOA (Table 2 and Additional file 1: Table S1). From this network, each small molecule can be queried to determine the biological processes that are uniquely connected to it as well as those that it shares with one or more other compounds (Fig. 4c). This approach can be applied in predicting drug MOA, understanding cross-resistance between drugs, drug-drug interactions and off-target effects.
We observed that some small molecules perturb a wide array of functions, while others have a specific target pathway. Small molecules have to cross at least one membrane to reach their expected target. Broad chemical properties of small molecules such as their hydrophobicity, molecular weight, solubility and pKa could indirectly interfere with multiple biological processes. Once a small molecule reaches its target, the direct inhibition of a target enzyme, for example, can result in metabolic changes that stimulate downstream biological processes. Furthermore, drugs that bind to distinct molecular targets within the same pathway can lead to similar biological effects. It is important to identify all these levels at which a drug can have a biological effect because the therapeutic success of a drug is determined by factors such as drug transport, metabolism and resistance that in many cases are distinct from molecular targets of the drug. Most approaches for the prediction of drug MOA in P. falciparum rely on the identification of single targets . Understanding the full spectrum of effects of a small molecule could aid in minimizing off-target effects and enhance the design of highly specific drugs.
Our study uncovers some potential challenges of using transcriptional perturbations for predicting drug MOA and reveals limitations that will need to be addressed in future studies. First, a large library of perturbations is needed in order to filter non-specific effects of small molecules. Secondly, predicting the MOA of small molecules that target biological processes that are non-specifically associated with numerous perturbations will pose a bigger challenge than those of small molecules that affect pathways that respond to only very few compounds. For example, the biological process “respiratory electron transport chain” was connected to 12 compounds in the small molecule-GO network, increasing the FDR for predicting the MOA of compounds affecting this process. However, as more compounds are profiled, the FDR for such compounds could be reduced. The small molecule-GO network provides a way to identify these non-specific effects and is generalizable for the prediction of drug MOA in other organisms.
We have shown the utility of transcriptional perturbations in predicting drug MOA and provide novel biological insights. Similarities between transcriptional responses of small molecules depend on their chemical composition and MOA. In particular, small molecules containing similar chemical constituents induce similar transcriptional responses. Furthermore, we determine that the presence/absence of rings in a compound dominate transcriptional similarities between compounds. We further show that small molecules with the same MOA induce similar transcriptional responses. Using a novel network representation (the ‘small molecule-GO network’), we demonstrate that some biological processes are affected by many compounds, potentially reflecting generalized stress responses, while others are affected by only one or few compounds. This network approach provides a hypothesis-testing framework for explaining similarities between any two or more small molecules based on the biological processes to which they are connected in the network. The small molecule-GO network correctly identifies the MOA of 72 % of compounds at a specificity of 80 %, establishing that this approach can be applied for predicting MOA. The representation of transcriptional responses as a small molecule-GO network is a novel approach that can be generalizable to other organisms.
Parasite clones Dd2 and HB3 were cultured using standard methods in human red blood cells (Indiana Regional Blood Center, Indianapolis, Indiana) suspended in complete medium (CM) containing RPMI 1640 with L-glutamine (Invitrogen Corp.), 50 mg/L hypoxanthine (Sigma-Aldrich), 25 mM HEPES (Cal Biochem), 0.5 % Albumax II (Invitrogen Corp.), 10 mg/L gentamicin (Invitrogen Corp.) and 0.225 % NaHCO3 (Biosource) at 5 % hematocrit. Cultures were grown separately in sealed flasks at 37 °C under an atmosphere of 5 % CO2, 5 % O2, and 90 % N2. Small molecules were purchased from Sigma-Aldrich with the exception of JQ1 (provided by Dr. James Bradner, Harvard Medical School) and the compounds SJ194935, SJ119930, SJ77572, SJ292024 and SJ140722 (provided by Dr. Kiplin Guy, St Jude’s Children’s hospital). Sorbitol double-synchronized cultures of the two clones were exposed briefly for 2 h to each compound at a single developmental stage (24 h trophozoites) at a concentration (IC50) obtained from literature sources or determined by our laboratory (Table 1).
RNA extraction and cDNA synthesis
Total RNA was extracted from 20mls of culture using TriZol reagent (Invitrogen, Carlsbad, CA) as described previously . Quantity of RNA and protein/organic contamination were determined using Nanodrop (NanoDrop Technologies). 300 ng of RNA was used as starting material for cDNA synthesis using the Sigma WTA2 whole transcriptome amplification kit (Sigma Aldrich, St Louis, MO). The cDNA synthesis reaction was performed in two steps: library synthesis and library amplification. To synthesize the cDNA library, 300 ng of sample RNA was incubated with reverse transcriptase and non-self-complimentary primers that contain a quasi-random 3’ end and a universal 5’ end. Primer extension was then performed using WTA2 polymerase to generate an OmniPlex cDNA library consisting of random, overlapping 100 to 1000 base fragments flanked by a universal end sequence. Amplification was then performed using primers targeting the universal 5’ ends. cDNA cleanup was performed using 3 M sodium acetate and ethanol.
1 μg of cDNA was labeled with Cy3 dye using 65 % AT rich pre-labeled random hexamers as primers for cDNA synthesis by Klenow fragment of DNA polymerase I. All samples were hybridized to a custom Nimblegen 12-plex microarray containing 128,179 probes, approximately 22 probes per annotated gene (PlasmoDB v6.3) with an average of 5 probes per exon (see Additional file 1: section C and Additional file 1: Figure S3 for microarray validation information). Hybridizations were performed for 22 h followed by washing of the arrays as described according to standard protocols (Roche NimbleGen Inc., Madison, WI). The microarray image was obtained using a 2uM scanner and probe intensity values extracted using NimbleScan software (Roche NimbleGen Inc., Madison, WI).
Microarray data processing
Probe intensities were normalized using robust multichip average (RMA) method . This normalization was performed across all samples hybridized on a single chip. Transcript level for each gene was obtained by averaging the processed signal intensity of all the probes across its exons as follows. Exon signal intensity for each gene was obtained by averaging the intensities of all probes within each exon. To determine a significance threshold for exon expression levels, a background distribution of signal intensities from a set of 10,000 negative control probes with no sequence matches to the P. falciparum genome was generated. A threshold corresponding to the 95th percentile (5 % FDR) of the signal distribution of the negative control probes was then applied . To determine gene expression levels, exons that passed the 5 % significance threshold were subjected to an additional threshold derived from intensities of 1000 simulated exons each consisting of 20 randomly sampled negative control probes. Intensities of exons that passed a 5 % FDR threshold based on this background distribution were averaged to obtain an average transcript level for each gene.
Analysis of pairwise relationships between compounds using genome-wide response profiles
For each perturbation, we averaged the signal intensity for each gene across two lab clones (Dd2 and HB3). The resulting data was then used to obtain a compound-specific response index for each gene by normalizing the gene’s average transcript level following perturbations in the two clones against its average level across all perturbations within the same experimental batch to obtain a gene specific response index. The global transcriptional response to a small molecule was then represented as a vector where each element represents a gene specific response index. This vector is referred to as the genome-wide response index. Small molecule global transcriptional relationships were determined using Pearson correlations between their genome-wide response indices. Clustering and visualization of the correlation matrix was performed in R. To determine the components of variation in the global correlations between small molecule responses, PCA was performed in R using the PCA function in the package FactoMineR.
Analysis of small molecule relationships based on chemical fingerprints
Small molecule relationships in the chemical space were determined by first converting each small molecule into a binary fingerprint of 881 elements using the PubChem 2-dimensional substructure fingerprints . Each position in the binary fingerprint encodes the presence/absence of a substructure such as a specific chemical element, a type of ring system, atom pairing, or atom environment, etc. A full description of the fingerprints can be found at .
We downloaded chemical structure files (SDF) for each small molecule from PubChem. Transformation of chemical structures into substructure fingerprints was performed in the R statistical package ChemmineR . Small molecule relationships based on the fingerprints were then visualized by hierarchical clustering. To determine whether chemical structure accounts for the observed major clustering of small molecules into two main clusters (named Class I and Class II), small molecules were projected onto a two-dimensional surface using multidimensional scaling (MDS) in the MASS package  of the R statistical software. Chemical substructures that discriminate Class I from Class II compounds were identified using a rule induction algorithm (OneR) in the WEKA machine learning package . The OneR algorithm produces a single ‘If-Then’ rule that identifies a single predictor variable that differentiates between two outcomes . The ability of the rule to discriminate Class I and II compounds was evaluated by testing the enrichment of Class I and II molecules when the rule is applied to the substructure fingerprints of all the small molecules. The enrichment P-values were obtained by hypergeometric tests.
Encoding small molecule effects into binary biological process fingerprints
GO enrichment analysis was performed on the most responsive genes in the transcriptome (the top 100 [~2 %] most up- and down-regulated genes) and enriched biological processes (hypergeometric test FDR corrected P < 0.05) were determined using the online platform MADIBA . For each small molecule, we then represented the transcriptional response as a binary vector whose elements are biological process categories that have a value of 1 (when the process is enriched following the perturbation) and 0 (when the process is not enriched).
Construction of small molecule-GO networks
To determine the relationships between small molecules to specific biological processes, we constructed small molecule-GO bipartite networks in which nodes were either small molecules or biological processes. Small molecules were connected by an edge to a biological process if perturbations by the small molecule were associated with an enrichment (hypregeometric test P < 0.05) of the biological process in the most responsive genes to the perturbation (top 100 up- and down-regulated genes).
Analysis of the effect of small molecules on their expected target pathways
The effect of small molecules on their expected target pathways was determined by querying the small molecule-GO network in the network visualization software Cytoscape . The expected target pathway for each small molecule was determined from literature and the enzyme database BRENDA. For each small molecule, we determined whether the biological processes it is connected to in the small molecule-GO network includes its expected target pathway. An estimate of the FDR for the association between a small molecule and its expected biological pathway was computed as the proportion of small molecules that were also connected to that category in the network. A small molecule was regarded as specifically connected to its expected target pathway if the FDR was less or equal than 10 %.
Dose response assays and QTL
We performed standard dose response assays across the Dd2 × HB3 recombinant clones cultured under varying concentrations of the drugs 5-FU, methotrexate, SJ194935, SJ119930, SJ140722 and SJ292024. Dose response assays were performed as previously described  using 4 to 6 replicates of parasite cultures of 37 recombinant clones of the Dd2 × HB3 genetic cross, including the parental lines. Quantitative trait locus (QTL) analysis for the dose responses was performed using previously published statistical methods in Pseudomarker v 2.04  and the Dd2 × HB3 genetic cross microsatellite linkage maps. The statistical significance of the obtained log odds scores (LOD) were obtained from a chi-square distribution, P = 1 - chi2cdf (2 × LOD score × Log10, degree of freedom = 1) where chi2cdf is the Matlab chi-square cumulative distribution function. Gene candidates were considered as those lying within the region bounded by the physical location of the genetic marker reported with the highest LOD score and the nearest markers in the region.
Prediction of MOA of novel compounds
The MOAs of five novel compounds (SJ194935, SJ119930, SJ140722, SJ292024 and SJ77572) emerging from a recent phenotypic screen  were predicted by combining predictions from the small molecule GO network and QTL analysis. To predict MOA from the small molecule-GO network, the network was queried in Cytoscape  and biological process connections that are uniquely connected to a given small molecule of interest selected as potential targets. Separately, dose response assays were determined for each of the compounds in 37 parasite lines in the Dd2 × HB3 genetic cross followed by QTL analysis. Gene candidates for each small molecule at a given QTL locus was then determined as genes located upstream and downstream of the region lying between the physical location of the QTL marker and the nearest marker.
The gene candidates at the QTL for a given novel compound were then examined for any functions that are related to those predicted as uniquely connected to the compound in the small molecule-GO network. The functions shared between the candidate genes from QTL and those from the small molecule-GO network were then regarded as potential targets of the compound.
Availability of supporting data
The raw data set supporting the results of this article is available in the NCBI Gene Expression Omnibus repository, [GEO:GSE67127, http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE67127]. The processed data sets supporting the results of this article are included within the article (and its additional file (s)).
GS was supported by an Eck Institute for Global Health Fellowship. This work was supported by NIH grants AI105657 to MTF. The Dana-Farber Cancer Institute provided the experimental compound JQ1 and Dr. Kiplin Guy at Saint Jude’s children’s hospital provided the SJ series of experimental compounds. We would like to thank Lindsey B. Turnbull for help with data annotation and curation.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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