A major cause of the paucity of new starting points for drug discovery is the lack of interaction between academia and industry. Much of the global resource in biology is present in universities, whereas the focus of medicinal chemistry is still largely within industry. Open source drug discovery, with sharing of information, is clearly a first step towards overcoming this gap. But the interface could especially be bridged through a scale-up of open sharing of physical compounds, which would accelerate the finding of new starting points for drug discovery. The Medicines for Malaria Venture Malaria Box is a collection of over 400 compounds representing families of structures identified in phenotypic screens of pharmaceutical and academic libraries against the Plasmodium falciparum malaria parasite. The set has now been distributed to almost 200 research groups globally in the last two years, with the only stipulation that information from the screens is deposited in the public domain. This paper reports for the first time on 236 screens that have been carried out against the Malaria Box and compares these results with 55 assays that were previously published, in a format that allows a meta-analysis of the combined dataset. The combined biochemical and cellular assays presented here suggest mechanisms of action for 135 (34%) of the compounds active in killing multiple life-cycle stages of the malaria parasite, including asexual blood, liver, gametocyte, gametes and insect ookinete stages. In addition, many compounds demonstrated activity against other pathogens, showing hits in assays with 16 protozoa, 7 helminths, 9 bacterial and mycobacterial species, the dengue fever mosquito vector, and the NCI60 human cancer cell line panel of 60 human tumor cell lines. Toxicological, pharmacokinetic and metabolic properties were collected on all the compounds, assisting in the selection of the most promising candidates for murine proof-of-concept experiments and medicinal chemistry programs. The data for all of these assays are presented and analyzed to show how outstanding leads for many indications can be selected. These results reveal the immense potential for translating the dispersed expertise in biological assays involving human pathogens into drug discovery starting points, by providing open access to new families of molecules, and emphasize how a small additional investment made to help acquire and distribute compounds, and sharing the data, can catalyze drug discovery for dozens of different indications. Another lesson is that when multiple screens from different groups are run on the same library, results can be integrated quickly to select the most valuable starting points for subsequent medicinal chemistry efforts.

Malaria leads to the loss of over 440,000 lives annually; accelerating research to discover new candidate drugs is a priority. Medicines for Malaria Venture (MMV) has distilled over 25,000 compounds that kill malaria parasites in vitro into a group of 400 representative compounds, called the "Malaria Box". These Malaria Box sets were distributed free-of-charge to research laboratories in 30 different countries that work on a wide variety of pathogens. Fifty-five groups compiled >290 assay results for this paper describing the many activities of the Malaria Box compounds. The collective results suggest a potential mechanism of action for over 130 compounds against malaria and illuminate the most promising compounds for further malaria drug development research. Excitingly some of these compounds also showed outstanding activity against other disease agents including fungi, bacteria, other single-cellular parasites, worms, and even human cancer cells. The results have ignited over 30 drug development programs for a variety of diseases. This open access effort was so successful that MMV has begun to distribute another set of compounds with initial activity against a wider range of infectious agents that are of public health concern, called the Pathogen Box, available now to scientific labs all over the world ( www.PathogenBox.org ).

Competing interests: The following commercial organizations employ or employed some of the authors, which might be considered a conflict of interest by some readers: BBD BioPhenix SL—BIOBIDE: Aintzane Alday PhD, Ainhoa Alzualde PhD, and, Arantza Muriana; Celia Quevedo PhD; SCYNEXIS, Inc.: Tana Bowling, Audrey Burton, Luke Mercer, and, Bakela Nare PhD; GlaxoSmithKline: Francisco Javier Gamo, PhD, Maria Jose Lafuente, PhD, and Sarah Prats; Eisai Co., Ltd.: Takaaki Horii Ph.D. and Nao-aki Watanabe Ph.D.; Novartis Inc.: Mark Baker PhD MSc (med), and David M. Plouffe; Definiens AG: Andreas Spitzmüller PhD; and, Merck Serono Inc: Thomas Spangenberg PhD.

Funding: Thanks to the UK DFID and the Bill and Melinda Gates Foundation Grand Challenges Explorations for providing funding for testing of the Malaria Box and funding the support of individual groups including: Medicines for Malaria Venture MMV Challenge Grant, Grant Numbers MMV 12/0048 and MMV 12/0076 (to JHA), the Australian Research Council (FT10100185 to SAP; FT0991213 to KTA and LP120200557 awarded to VMA), Bill & Melinda Gates Foundation Grant OPP1040394 to PA, OPP1040399 to DAF and VMA and OPP1086189 to KKH, OPP1069393 and OPP1119049 to ML, OPP1024029 to CN, the Bloomberg Family Foundation (JBr), JHMRI for a predoctoral fellowship, the US NIH for the CBI training grant T32GM080189 (to LEB), R01GM104486 (to PAW & WS), R01AI117017 (to JHA) the National Science Foundation Graduate Research Fellowship Program Grant No.DGE-1232825 (DDCL), the South African Medical Research Council Strategic Health Innovation Partnerships (grant V6YBT51 to DM) and the Council for Scientific and Industrial Research (grant V1YTB95, to DM), and the French ANR program Mammamia (ANR-12-BS07-0020-01). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Since 2007, over 6 million compounds were screened against asexual-stage Plasmodium falciparum, at two pharmaceutical companies (GlaxoSmithKline [ 1 ] and Novartis [ 2 ]), and two academic centers (St. Jude, Memphis [ 3 ], and Eskitis, Australia [ 4 ]), resulting in over 20,000 compounds active in the low- to sub-micromolar range. The structures of the 20,000 anti-malaria hits were made available in ChEMBL ( www.ebi.ac.uk/chembl ), but discussions with biology groups had underlined the importance of access to the compounds themselves for testing. Cluster analysis and commercial availability reduced this to a set of 400 representative compounds, the ‘Malaria Box’, which was distributed freely to researchers who provided a rationale for screening [ 5 ]. This paper presents a summary and analysis of the collected results of the Malaria Box screening from 55 groups who performed a wide variety of assays, the large majority of which are presented in this paper. The collective results are greater than the sum of the individual assays, because each compound can be queried for activity, pharmacokinetic, and safety data to gauge its suitability as a starting point for subsequent medicinal chemistry optimization efforts.

Preclinical development for drugs in neglected diseases remains a slow process due to a lack of access to compounds, and legal complications over intellectual property ownership. One way to accelerate drug discovery is to provide open access to bioactive molecules with public disclosure of the resulting biological data. The data from open access of bioactive molecules can help prioritize which compounds to investigate further through medicinal chemistry for the original indication and can also uncover other indications for compound development. It was in this spirit of providing open access of malaria-bioactive compounds, and disseminating the results in the public domain, that the Malaria Box project was initiated by the Medicines for Malaria Venture.

Results

The Heat Map (S1 Table) reports the data from over 290 assays run on the Malaria Box compounds; a snapshot is shown in Fig 1. The results are color coded, where the compounds with the highest activity are coded red and those with relative inactivity green. In the center of the box in S1 Table, the numerical value for the compound is given. It can be seen immediately that some compounds have activities in several biological assays across multiple species and these tend to have activity against mammalian cells as well, whereas other compounds have a rather limited spectrum of activity and are less toxic to mammalian cells.

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larger image TIFF original image Download: Fig 1. Malaria Box Heatmap. Shown are selected data from the HeatMap (S1 Table) for the 400 Malaria Box compounds. Each column represents an assay (grouped by category), compounds are represented in rows. The red-green gradient represents higher to lower activity. Favorable PK activities are scored green. Pf: Plasmodium falciparum, Pb: Plasmodium berghei, PK: pharmacokinetics, sol.: solubility, hERG: human ether-a-go-go channel inhibition, DDI: drug-drug interactions (predicted). https://doi.org/10.1371/journal.ppat.1005763.g001

The data demonstrated in S1 Table are provided by 55 groups who have performed 291 assays to screen the Malaria Box. The vast majority of the data are presented for the first time in this paper. In supplementary data S1 Table, note that columns with data presented for the first time in this paper, representing 236 assays, are colored pink on the top row; published /in press data columns, 55, are grey, with citations provided. Presenting the combined dataset provides insights into the hit rates in these various assays while allowing rapid access to the data by the wider scientific community.

The Heat Map (S1 Table) presents the Malaria Box chemicals grouped by chemical relatedness. Of the 400 compounds, over 100 are closely-related paired molecules so immediate structure-activity-relationships (SAR) can often be seen from hits with these pairs. The Heat Map identified obvious correlations in chemistry and biology between compounds (both Mechanism-of-Action and phenotypic activity). Some biological assays are relatively similar; for example, there were a large number of different P. falciparum gametocyte assays (S1 Table, columns AV-CB), which also cluster, although not perfectly. As such, the aggregate screening data help overcome inter-laboratory bias and identify outstanding activities. For example, compounds that were active in multiple gametocyte assays represent more solid positives than a compound that was active in only one screening assay. However, the gametocyte assays were often performed using different techniques and screening concentrations (see S1 Methods and Results, for details) and one assay may be preferred over another to select compounds with gametocyte activity. Thus having the aggregate data presented together with the individual protocols is more valuable than just having each individual data set to look at sequentially.

Malaria Box safety and pharmacokinetic data Early safety data were obtained by testing all compounds against 73 human cell lines at 10 μM or above, and developing zebrafish embryos were exposed at 5 μM, providing further clues on potential safety issues. A frequent cardiotoxicity safety concern is QTc prolongation, and all compounds were screened for hERG inhibition [6], which is a proxy for this risk (S1 Table column GI). The efficacy and safety of anti-malarial compounds could be altered in endemic regions when administered to patients who are also treated for HIV (Human Immunodeficiency Virus) or TB (tuberculosis), due to drug-drug interactions in the liver. To flag such interactions, we employed two recent breakthrough models: a bioengineered microscale human liver in a high-throughput assay format that accurately captures human drug-drug interactions not detectable in animals or cell lines [7] and a custom-made, robotic high-throughput Luminex bead-based method for profiling the expression of 83 human liver drug-metabolizing enzymes [8]. Combining these tools, we profiled the Malaria Box compounds for induction or inhibition of drug-metabolizing pathways (S1 Table, columns GL-HA) and thereby ranked compounds for potential for drug interactions with existing HIV and TB regimens, to enhance selection of compounds with the lowest safety risks. We also scored the Malaria Box compounds for acute hepatoxicity by monitoring morphology and daily albumin and urea secretion from hepatocytes (S1 Table, columns FQ-FS). G protein-coupled receptors (GPCRs) represent the largest human drug target class [9]; they affect neurological and cardiovascular physiology and are included in routine safety pharmacology panels [10]. Therefore, in vitro affinity determinations on 23 selected human off-target GPCRs were performed on a subset (10%) of MMV compounds (S1 Table, columns HC-HZ). One of the most severe GPCR-related adverse effects is cardiac valvulopathy linked to 5-HT 2B activation [11, 12]. Therefore, some of the MMV compounds with significant binding affinity for the 5-HT 2B receptor were also tested on the corresponding functional assay to determine a potential agonistic effect. In addition, predictions of compound glutathione reactivity and epoxidation potential were calculated for each of the Malaria Box compounds (S1 Table, columns IB-IC). These combined safety results alert us to compounds with issues that hopefully can be resolved in subsequent medicinal chemistry programs. Prior to in vivo pharmacology evaluation it is important to know that an effective plasma concentration can be reached; this exposure was measured in rodents for all compounds, from a single high oral dose (140 μmol/kg). Around one third of the compounds generated high plasma C max (>1 μg/ml) and/or high overall exposure (S1 Table, columns GD-GE). This is a higher than expected percentage of compounds with measureable oral bioavailability than if compounds were randomly selected, and probably reflects the large number of drug-like leads selected for the Malaria Box. The combination of in vitro potency and bioavailability provides a rough dosing estimate, informing subsequent decision-making around selection of development leads. The combined analysis of all of these safety and pharmacokinetic data allows selection of the most promising compounds to advance to medicinal chemistry, and which parameters should be monitored and improved during a medicinal chemistry program.

New insights into malaria The activity of Malaria Box compounds against the asexual, erythrocytic stage of P. falciparum was confirmed by five laboratories on seven different P. falciparum strains. There were sometimes 5-10-fold differences in the effective concentration that caused a 50% reduction in growth (EC 50 ) in each assay, and these may have been due to variations in the readouts for the screening assays (LDH release, MitoTracker or Sybr Green dye incorporation, hypoxanthine incorporation, DAPI imaging assay), variations in the protein concentration in the assay medium (affecting the free compound concentration), the time the compound incubated time, or other differences. However, usually the results were consistent and strain-independent. We have documented which sub-stage of the asexual lifecycle the compounds acted upon (S1 Table, columns AA-AE). This information is important in identifying compounds that may overcome existing resistance against artemisinin and other antimalarials. For instance, compounds that target early ring stage intra-erythrocytic parasites and have fast-killing dynamics are sought after because, like artemisinins, they kill parasites rapidly and may reduce patient mortality. Table 1 shows compounds that also target liver stages of the parasite’s life cycle. PPT PowerPoint slide

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larger image TIFF original image Download: Table 1. Malaria Box compounds with activity in biological assays (malaria, helminths, Wolbachia, and cancer cells) and lacking toxicity at therapeutic levels. Selectivity Index, SI, is toxicity level/activity level; p, probe-like; d, drug-like. https://doi.org/10.1371/journal.ppat.1005763.t001

Targeting disease-relevant malaria stages P. berghei liver stage (LS) inhibition, using parasite-encoded luciferase activity as a readout of infection in HepG2 cells, was independently determined by two groups at very different screening concentrations (Hanson: 5 μM, Winzeler: 50 μM). Forty-three compounds, roughly 10% of the compound library, inhibited infection by at least 50% at 5 μM and 90% at 50 μM (referred to as LS double actives). HepG2 cell toxicity, (50% or greater reduction in HepG2 abundance based on direct or indirect readouts) was observed with 63% of Malaria Box compounds at 50 μM, while only 10% were toxic at the 5 μM concentration. After excluding those that showed significant toxicity in HepG2 cells at both 5 and 50 μM, Malaria Box compounds were stratified by potential mode-of-action annotation (S1 Table, column M). Five potential modes of action stood out as enriched in LS double actives: (i) cysteine protease inhibitors (cruzain, rhodesain): 1.8% of all Malaria Box compounds (7/400) and 4.7% (2/43) of LS double actives; (ii) possible respiratory-dependent targets (Δ IC 50 in low oxygen vs. normal oxygen): 0.8% (3/400) of all Malaria Box compounds and 4.7% (2/43); (iii) targeting yeast respiration: 3.5% of all Malaria Box compounds (14/400) and 9.3% of LS double actives (4/43); (iv) suspected or known PfDHODH (dihydroorotate dehydrogenase) inhibitors: 2.5% of all Malaria Box compounds (10/400) and 9.3% of LS double active (4/43); and (v) suspected or known cytochrome bc1 inhibitors: 4.3% of all Malaria Box compounds (17/200) and 16.3% of LS double actives (7/43). Compounds with activity against PfATP4, now the most common intra-erythrocytic asexual target seen in phenotypic screens, were not found amongst the LS double actives. There is a great need for antimalarials that kill dormant, liver-stage P. vivax (hypnozoites), but there is a lack of assays that measure this activity. Only nine compounds (Table 1) show simultaneous activity against gametocytes, liver, and asexual stages, whilst lacking evidence of toxicity in zebrafish and broad cytotoxicity to mammalian cells. These would be compounds to prioritize for in vitro and in vivo screening against P. vivax hypnozoites and would benefit from additional MoA studies. Gametocytocidal drugs would block transmission from the human to the mosquito and break the parasite’s life cycle. The data shown in Table 1 include series with activities on both gametocyte and liver stages, and some of the data intriguingly challenges existing assumptions. For instance, MMV007116 in this category is a mitochondrial (bc1) inhibitor (S1 Table, column M, line 168) and has activity in a number of gametocytocidal assays, but other bc1 inhibitors are not generally gametocytocidal, suggesting another MoA for this compound. We also see 4-aminoquinolines as inhibitors of some gametocyte assays, although the parent 4-aminoquinoline compound chloroquine is known not to be gametocytocidal for P. falciparum. Again, this may imply a different MoA for some 4-aminoquinoline compounds or perhaps multiple modes of action for certain compounds. These findings re-emphasize the strength of looking at assay data in a wider context in Open Source drug discovery.

Screening on yeast to suggest MoAs Four groups carried out screens on S. cerevisiae strains engineered to help elucidate the MoA of test compounds. One screen established that 35 Malaria Box compounds were active on a multiple ABC-transporter deficient strain (also known as the ‘monster strain’) S. cerevisiae [34]. Since yeasts are generally resistant to compound inhibition due to transporters, this monster strain can now be analyzed for MoA of inhibition by these 35 compounds. A second study measured selective growth inhibition of S. cerevisiae using different carbon sources. Growth was measured in three different growth media: rich or minimal media using dextrose as a carbon source, or minimal media using ethanol and glycerol as carbon sources. Compounds affecting growth in a media-specific manner may represent inhibitors of key metabolic pathways. A third group used a yeast strain expressing the Pf phosphoethanolamine methyltransferase (PfPMT) to screen for phosphocholine (PC) synthesis inhibitors. This screen relies on the incapability of this yeast strain to synthesize PC in the absence of exogenous choline, and thus depends on the malaria PfPMT for survival. Screening the Malaria Box compounds, and a variety of controls including wild-type PMT and choline supplemented media, led to the identification of MMV007384, MMV007041 MMV396736, MMV396723, MMV000304, MMV000570, MMV000704, MMV666071, MMV000445, MMV667491, and MMV666080 as possible PfPMT inhibitors. Finally, a fourth group screened S. cerevisiae grown either on ethanol-containing media requiring respiration or glucose-fermentative media not requiring respiration, and identified 12 compounds that gave superior inhibition on ethanol media suggesting that these compounds inhibit a respiratory target. Seven of these were not associated with any other targets; the others were potential inhibitors of DHODH (3, 49), bc1, and IspD.

Activity against protozoa other than Plasmodium The Malaria Box was screened against 16 additional protozoa, all of which are of medical or veterinary significance. Compounds with activity against three or more protozoa were usually toxic for the zebrafish or non-cancer mammalian cell lines, underlining the need to limit the concentrations used in assays, to avoid meaningless positives. Table 2 lists compounds with activity against protozoa that were nontoxic to zebrafish and most mammalian cells. In the Cryptosporidium parvum assay there were numerous active compounds, but none were completely devoid of toxicity for zebrafish and mammalian cell lines. MMV665917 had a >20-fold Selectivity Index (SI) for C. parvum over mammalian cells. Trypanosoma cruzi actives were non-overlapping between groups, and are listed separately, but T. brucei actives overlapped extensively with other screens and are presented together. There were seven non-toxic hits that were active against extracellular amastigotes of Leishmania infantum, but no non-toxic compounds were active on intracellular macrophage growth of L. infantum. There were five non-toxic Malaria Box compounds active against T. gondii (MMV666095, MMV007363, MMV007791, MMV007881 and MMV006704). Many of the compounds that were active on Neospora caninum raised no toxicity flags on the accompanying host cell fibroblast screen, but many were toxic at 10 μM or below for mammalian cells and zebrafish. The remaining non-toxic N. caninum actives that bear further investigation include: MMV019670, MMV000911 and MMV006309. Most compounds active against Entamoeba histolytica, Naegleria fowleri, or exflagellation of Chromera velia were toxic. An exception was MMV665979, an outstanding hit for Naegleria fowleri, with limited toxicity elsewhere in the dataset. With respect to screening Babesia and Theileria, ten novel anti-Babesia and anti-Theileria hits with nanomolar IC 50 s were identified: MMV666093, MMV396794, MMV006706, MMV665941, MMV085203, MMV396693, MMV006787, MMV073843, MMV007092 and MMV665875. The most interesting hits were MMV396693, MMV073843, MMV666093, MMV665875 and MMV006706 with mean SIs greater than 230 and IC 50 s ranging from 43 to 750 nM for both bovine Babesia and equine Babesia and Theileria parasites. Additionally, 64, 45 and 49 Malaria Box compounds exhibited IC 50 s lower than those of diminazene aceturate (the most widely used antibabesial drug) against the in vitro growth of B. bovis, B. bigemina and T. equi, respectively. PPT PowerPoint slide

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larger image TIFF original image Download: Table 2. Antiprotozoal Malaria Box compounds with activity in biological assays and lacking toxicity at therapeutic levels. Selectivity Index, SI, is toxicity level/activity level; p, probe-like; d, drug-like. https://doi.org/10.1371/journal.ppat.1005763.t002 In vitro screening of Open Access Malaria Box compounds against Babesia bovis, B. bigemina, Theileria equi and B. caballi has led to the discovery of 10 novel potent anti-babesial hits exhibiting submicromolar potency against both bovine Babesia and equine Babesia and Theileria. In vitro follow up of the many of the hits identified in this study for B. bovis, B. bigemina, B. caballi, and T. equi parasites, revealed IC 50 s lower than that obtained with the previously described drug-leads luteolin, pyronaridine, nimbolide, gedunin and enoxacin [35]. The ten potent hits for bovine Babesia and equine Babesia and Theileria identified in this study exhibited IC 50 s lower than that obtained with the apicoplast-targeting antibacterials (ciprofloxacin, thiostrepton, and rifampin), miltefosine, fusidic acid or allicin [36–39].

Activity on helminths, mycobacteria, and bacteria Many Malaria Box compounds were active on helminths at 10 μM, but most of these were also toxic for mammalian cells or zebrafish. The remaining non-toxic compounds had activity against Brugia malayi (lymphatic filariasis) and Ancylostoma ceylanicum (hookworm; Table 1). But no non-toxic compounds were found with consistent activity against Schistosoma mansoni, Strongyloides stercoralis, Trichuris muris, Haemonchus contortus, or Onchocerca linenalis. There remains the possibility that some of the toxic hits against these species can be addressed by medicinal chemistry. With respect to activity against mycobacteria and bacteria, although every screen delivered actives, the majority were again discarded because of a toxicity signal against zebrafish and/or mammalian cells. The exceptions were non-toxic Malaria Box compounds that were active against Wolbachia (Table 1). Wolbachia bacteria are targeted as anti-filarials in order to deprive nematodes causing river blindness and elephantiasis from essential nutrients provided by this bacterium [40].