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Twenty six new phenylurea substituted 2,4-diamino-pyrimidines active against Plasmodium falciparum (Pf) 3D7 are reported.

New antimalarial treatments with novel mechanism of action are needed to tackle Plasmodium falciparum infections that are resistant to first-line therapeutics. Here we report the exploration of MMV692140 (2) from the Pathogen Box, a collection of 400 compounds that was made available by Medicines for Malaria Venture (MMV) in 2015. Compound 2 was profiled in in vitro models of malaria and was found to be active against multiple life-cycle stages of Plasmodium parasites. The mode of resistance, and putatively its mode of action, was identified as Plasmodium falciparum translation elongation factor 2 (PfeEF2), which is responsible for the GTP-dependent translocation of the ribosome along mRNA. The compound maintains activity against a series of drug-resistant parasite strains. The structural motif of the tetrahydroquinoline (2) was explored in a chemistry program with its structure-activity relationships examined, resulting in the identification of an analog with 30-fold improvement of antimalarial asexual blood stage potency.

We utilized synthetic photochemistry to generate novel sp3-rich scaffolds and report the design, synthesis, and biological testing of a diverse series of amides based on the 1-(amino-methyl)-2-benzyl-2-aza-bicyclo[2.1.1]hexane scaffold. Preliminary antimalarial screening of the library provided promising compounds with activity in the 1-5 μM range with an enhanced hit rate. Further evaluation (solubility, drug metabolism and pharmacokinetics (DMPK), and toxicity) of a selected compound (9) suggested that this series represents an excellent opportunity for further optimization with the framework offering multiple opportunities for the addition of uniquely vectorally positioned extra functionality.

The replacement of one chemical motif with another that is broadly similar is a common method in medicinal chemistry to modulate the physical and biological properties of a molecule (i.e., bioisosterism). In recent years, bioisosteres such as cubane and bicyclo[1.1.1]pentane (BCP) have been used as highly effective phenyl mimics. Herein, we show the successful incorporation of a range of phenyl bioisosteres during the open-source optimization of an antimalarial series. Cubane (19) and closo-carborane (23) analogues exhibited improved in vitro potency against Plasmodium falciparum compared to the parent phenyl compound; however, these changes resulted in a reduction in metabolic stability; unusually, enzyme-mediated oxidation was found to take place on the cubane core. A BCP analogue (22) was found to be equipotent to its parent phenyl compound and showed significantly improved metabolic properties. While these results demonstrate the utility of these atypical bioisosteres when used in a medicinal chemistry program, the search to find a suitable bioisostere may well require the preparation of many candidates, in our case, 32 compounds.

The discovery of new antimalarial medicines with novel mechanisms of action is key to combating the problem of increasing resistance to our frontline treatments. The Open Source Malaria (OSM) consortium has been developing compounds ("Series 4") that have potent activity against Plasmodium falciparum in vitro and in vivo and that have been suggested to act through the inhibition of PfATP4, an essential membrane ion pump that regulates the parasite’s intracellular Na+ concentration. The structure of PfATP4 is yet to be determined. In the absence of structural information about this target, a public competition was created to develop a model that would allow the prediction of anti-PfATP4 activity among Series 4 compounds, thereby reducing project costs associated with the unnecessary synthesis of inactive compounds.In the first round, in 2016, six participants used the open data collated by OSM to develop moderately predictive models using diverse methods. Notably, all submitted models were available to all other participants in real time. Since then further bioactivity data have been acquired and machine learning methods have rapidly developed, so a second round of the competition was undertaken, in 2019, again with freely-donated models that other participants could see. The best-performing models from this second round were used to predict novel inhibitory molecules, of which several were synthesised and evaluated against the parasite. One such compound, containing a motif that the human chemists familiar with this series would have dismissed as ill-advised, was active. The project demonstrated the abilities of new machine learning methods in the prediction of active compounds where there is no biological target structure, frequently the central problem in phenotypic drug discovery. Since all data and participant interactions remain in the public domain, this research project “lives” and may be improved by others.

Herein we describe the optimization of a phenotypic hit against Plasmodium falciparum based on an aminoacetamide scaffold. This led to N‐(3‐chloro‐4‐fluorophenyl)‐2‐methyl‐2‐{[4‐methyl‐3‐(morpholinosulfonyl)phenyl]amino}propanamide (compound 28) with low‐nanomolar activity against the intraerythrocytic stages of the malaria parasite, and which was found to be inactive in a mammalian cell counter‐screen up to 25 μm. Inhibition of gametes in the dual gamete activation assay suggests that this family of compounds may also have transmission blocking capabilities. Whilst we were unable to optimize the aqueous solubility and microsomal stability to a point at which the aminoacetamides would be suitable for in vivo pharmacokinetic and efficacy studies, compound 28 displayed excellent antimalarial potency and selectivity; it could therefore serve as a suitable chemical tool for drug target identification., New roads to novel chemotypes: Compound selection from the TCAMS library (GSK), followed by iterative rounds of inhibitor design and synthesis afforded a single‐digit nanomolar inhibitor of P. falciparum (3D7), based on an aminoacetamide core. Compound 28 is a potent antimalarial and displayed excellent selectivity in a mammalian counter‐screen. This compound could be used as a suitable chemical tool for drug target identification or as a lead compound for further optimization.

Twenty eight new N

Global health concern regarding malaria has increased since the first report of artemisinin-resistant Plasmodium falciparum (Pf) two decades ago. The current therapies suffer various drawbacks such as low efficacy and significant side effects, alarming for an urgent need of more effective and less toxic drugs with higher patient compliance. Chemical entities with natural origins become progressively attractive as new drug leads due to their structural diversity and bio-compatibility. This study initially aimed at the targeted isolation of hydroxyquinoline derivatives following our published genomics and metabolomics study of Pantoea agglomerans (Pa). Fermentation of Pa on a pre-selected medium followed by chromatographic isolation, NMR and HRMS analyses led to the characterisation of one new hydroxyquinoline alkaloid together with another six known congeners and two known hydroxyquinolone derivatives. When screened for their antimalarial activity by high throughput screening against asexual blood-stage parasites, almost all compounds showed potent and selective sub-micromolar activities. Computational investigation was performed to identify the antiplasmodial potential targets. Ligand-based similarity search predicted the tested compounds to act as hemozoin inhibitors. Computational target identification results were further validated by competitive hemozoin inhibitory properties of hydroxyquinoline and hydroxyquinolone derivatives in vitro. The overall results suggest this natural scaffold is of potential to be developed as antimalarial drug lead.

[Image: see text] We utilized synthetic photochemistry to generate novel sp(3)-rich scaffolds and report the design, synthesis, and biological testing of a diverse series of amides based on the 1-(amino-methyl)-2-benzyl-2-aza-bicyclo[2.1.1]hexane scaffold. Preliminary antimalarial screening of the library provided promising compounds with activity in the 1–5 μM range with an enhanced hit rate. Further evaluation (solubility, drug metabolism and pharmacokinetics (DMPK), and toxicity) of a selected compound (9) suggested that this series represents an excellent opportunity for further optimization with the framework offering multiple opportunities for the addition of uniquely vectorally positioned extra functionality.

Twenty eight new N2,N4-diphenylpyrimidine-2,4-diamines have been prepared in order to expand our understanding of the anti-malarial SAR of the scaffold. The aim of the study was to make structural modifications to improve the overall potency, selectivity and solubility of the series by varying the anilino groups attached to the 2- and 4-position. We evaluated the activity of the compounds against Plasmodium falciparum (Pf) 3D7, cytotoxicity against HepG2, % inhibition at a panel of 10 human kinases, solubility, permeability and lipophilicity, and human and rat in vitro clearance. 11 was identified as a potent anti-malarial with an IC50 of 0.66 µM at the 3D7 strain and a selectivity (SI) of ~ 40 in terms of cytotoxicity against the HepG2 cell line. It also displayed low experimental logD7.4 (2.27), reasonable solubility (124 µg/ml), good metabolic stability, but low permeability. A proteo-chemometric workflow was employed to identify putative Pf targets of the most promising compounds. Ligand-based similarity searching of the ChEMBL database led to the identification of most probable human targets. These were then used as input for sequence-based searching of the Pf proteome. Homology modelling and molecular docking were used to evaluate whether compounds could indeed bind to these targets with valid binding modes. In vitro biological testing against close human analogs of these targets was subsequently undertaken. This allowed us to identify potential Pf targets and human anti-targets that could be exploited in future development.

Visceral leishmaniasis (VL), caused by the protozoan parasites Leishmania donovani and L. infantum, is responsible for ∼30 000 deaths annually. Available treatments are inadequate, and there is a pressing need for new therapeutics. N-Myristoyltransferase (NMT) remains one of the few genetically validated drug targets in these parasites. Here, we sought to pharmacologically validate this enzyme in Leishmania. A focused set of 1600 pyrazolyl sulfonamide compounds was screened against L. major NMT in a robust high-throughput biochemical assay. Several potent inhibitors were identified with marginal selectivity over the human enzyme. There was little correlation between the enzyme potency of these inhibitors and their cellular activity against L. donovani axenic amastigotes, and this discrepancy could be due to poor cellular uptake due to the basicity of these compounds. Thus, a series of analogues were synthesized with less basic centers. Although most of these compounds continued to suffer from relatively poor antileishmanial activity, our most potent inhibitor of LmNMT (DDD100097, Ki of 0.34 nM) showed modest activity against L. donovani intracellular amastigotes (EC50 of 2.4 μM) and maintained a modest therapeutic window over the human enzyme. Two unbiased approaches, namely, screening against our cosmid-based overexpression library and thermal proteome profiling (TPP), confirm that DDD100097 (compound 2) acts on-target within parasites. Oral dosing with compound 2 resulted in a 52% reduction in parasite burden in our mouse model of VL. Thus, NMT is now a pharmacologically validated target in Leishmania. The challenge in finding drug candidates remains to identify alternative strategies to address the drop-off in activity between enzyme inhibition and in vitro activity while maintaining sufficient selectivity over the human enzyme, both issues that continue to plague studies in this area.

Crystallography has guided the hybridization of two series of Trypanosoma brucei N-myristoyltransferase (NMT) inhibitors, leading to a novel highly selective series. The effect of combining the selectivity enhancing elements from two pharmacophores is shown to be additive and has led to compounds that have greater than 1000-fold selectivity for TbNMT vs HsNMT. Further optimization of the hybrid series has identified compounds with significant trypanocidal activity capable of crossing the blood-brain barrier. By using CF-1 mdr1a deficient mice, we were able to demonstrate full cures in vivo in a mouse model of stage 2 African sleeping sickness. This and previous work provides very strong validation for NMT as a drug target for human African trypanosomiasis in both the peripheral and central nervous system stages of disease.

Visceral leishmaniasis is a neglected tropical disease with significant health impact. The current treatments are poor, and there is an urgent need to develop new drugs. Primary screening assays used for drug discovery campaigns have typically used free-living forms of the Leishmania parasite to allow for high-throughput screening. Such screens do not necessarily reflect the physiological situation, as the disease-causing stage of the parasite resides inside human host cells. Assessing the drug sensitivity of intracellular parasites on scale has recently become feasible with the advent of high-content screening methods. We describe here a 384-well microscopy-based intramacrophage Leishmania donovani assay and compare it to an axenic amastigote system. A panel of eight reference compounds was tested in both systems, as well as a human counterscreen cell line, and our findings show that for most clinically used compounds both axenic and intramacrophage assays report very similar results. A set of 15,659 diverse compounds was also screened using both systems. This resulted in the identification of seven new antileishmanial compounds and revealed a high false-positive rate for the axenic assay. We conclude that the intramacrophage assay is more suited as a primary hit-discovery platform than the current form of axenic assay, and we discuss how modifications to the axenic assay may render it more suitable for hit-discovery.

Graphical abstract, Quinols have been developed as a class of potential anti-cancer compounds. They are thought to act as double Michael acceptors, forming two covalent bonds to their target protein(s). Quinols have also been shown to have activity against the parasite Trypanosoma brucei, the causative organism of human African trypanosomiasis, but they demonstrated little selectivity over mammalian MRC5 cells in a counter-screen. In this paper, we report screening of further examples of quinols against T. brucei. We were able to derive an SAR, but the compounds demonstrated little selectivity over MRC5 cells. In an approach to increase selectivity, we attached melamine and benzamidine motifs to the quinols, because these moieties are known to be selectively concentrated in the parasite by transporter proteins. In general these transporter motif-containing analogues showed increased selectivity; however they also showed reduced levels of potency against T. brucei.

N-Myristoyltransferase (NMT) represents a promising drug target for human African trypanosomiasis (HAT), which is caused by the parasitic protozoa Trypanosoma brucei. We report the optimization of a high throughput screening hit (1) to give a lead molecule DDD85646 (63), which has potent activity against the enzyme (IC(50) = 2 nM) and T. brucei (EC(50) = 2 nM) in culture. The compound has good oral pharmacokinetics and cures rodent models of peripheral HAT infection. This compound provides an excellent tool for validation of T. brucei NMT as a drug target for HAT as well as a valuable lead for further optimization.

Screening of the Sigma-Aldrich Library of Pharmacologically Active Compounds (LOPAC) against cultured Trypanosoma brucei, the causative agent of African sleeping sickness, resulted in the identification of a number of compounds with selective antiproliferative activity over mammalian cells. These included (+)-(1R,2R)-U50488, a weak opioid agonist with an EC(50) value of 59 nM as determined in our T. brucei in vitro assay reported previously. This paper describes the modification of key structural elements of U50488 to investigate structure-activity relationships (SAR) and to optimise the antiproliferative activity and pharmacokinetic properties of this compound.

African sleeping sickness or human African trypanosomiasis, caused by Trypanosoma brucei spp., is responsible for approximately 30,000 deaths each year. Available treatments for this disease are poor, with unacceptable efficacy and safety profiles, particularly in the late stage of the disease when the parasite has infected the central nervous system. Here we report the validation of a molecular target and the discovery of associated lead compounds with the potential to address this lack of suitable treatments. Inhibition of this target-T. brucei N-myristoyltransferase-leads to rapid killing of trypanosomes both in vitro and in vivo and cures trypanosomiasis in mice. These high-affinity inhibitors bind into the peptide substrate pocket of the enzyme and inhibit protein N-myristoylation in trypanosomes. The compounds identified have promising pharmaceutical properties and represent an opportunity to develop oral drugs to treat this devastating disease. Our studies validate T. brucei N-myristoyltransferase as a promising therapeutic target for human African trypanosomiasis.

Graphical abstract Phenotypic screening of the LOPAC library identified several potent and selective inhibitors of African trypanosomes. The κ-opioid agonist (+)-U50,488 represents a novel lead for drug discovery against sleeping sickness., A resazurin-based cell viability assay was developed for phenotypic screening of the LOPAC 1280 ‘library of pharmacologically active compounds’ against bloodstream forms of Trypanosoma brucei in vitro identifying 33 compounds with EC50 values 700-fold) compounds being suramin and pentamidine. These are well-known antitrypanosomal drugs which demonstrate the robustness of the resazurin cell viability assay. The most selective novel inhibitor was (+)-trans-(1R,2R)-U50,488 having an EC50 value of 60 nM against T. brucei and 270-fold selectivity over human fibroblasts. Interestingly, (−)-U50,488, a known CNS-active κ-opioid receptor agonist and other structurally related compounds were >70-fold less active or inactive, as were several μ- and κ-opioid antagonists. Although (+)-U50,488 was well tolerated by the oral route and displayed good pharmaceutical properties, including high brain penetration, the compound was not curative in the mouse model of infection. Nonetheless, the divergence of antinociceptive and antitrypanosomal activity represents a promising start point for further exploratory chemistry. Bioinformatic studies did not reveal any obvious candidate opioid receptors and the target of this cytostatic compound is unknown. Among the other potent, but less selective screening hits were compound classes with activity against protein kinases, topoisomerases, tubulin, as well as DNA and energy metabolism.

The inhibition of hepatic glycogen-associated protein phosphatase-1 (PP1-G(L)) by glycogen phosphorylase a prevents the dephosphorylation and activation of glycogen synthase, suppressing glycogen synthesis when glycogenolysis is activated. Here, we show that a peptide ((280)LGPYY(284)) comprising the last five amino acids of G(L) retains high-affinity interaction with phosphorylase a and that the two tyrosines play crucial roles. Tyr284 deletion abolishes binding of phosphorylase a to G(L) and replacement by phenylalanine is insufficient to restore high-affinity binding. We show that a phosphorylase inhibitor blocks the interaction of phosphorylase a with the G(L) C-terminus, suggesting that the latter interaction could be targeted to develop an anti-diabetic drug.

A screen of a focused kinase inhibitor library against Trypanosoma brucei rhodesiense led to the identification of seven series, totaling 121 compounds, which showed >50 % inhibition at 5 μm. Screening of these hits in a T. b. brucei proliferation assay highlighted three compounds with a 1H-imidazo[4,5-b]pyrazin-2(3H)-one scaffold that showed sub-micromolar activity and excellent selectivity against the MRC5 cell line. Subsequent rounds of optimisation led to the identification of compounds that exhibited good in vitro drug metabolism and pharmacokinetics (DMPK) properties, although in general this series suffered from poor solubility. A scaffold-hopping exercise led to the identification of a 1H-pyrazolo[3,4-b]pyridine scaffold, which retained potency. A number of examples were assessed in a T. b. brucei growth assay, which could differentiate static and cidal action. Compounds from the 1H-imidazo[4,5-b]pyrazin-2(3H)-one series were found to be either static or growth-slowing and not cidal. Compounds with the 1H-pyrazolo[3,4-b]pyridine scaffold were found to be cidal and showed an unusual biphasic nature in this assay, suggesting they act by at least two mechanisms.

Contains fulltext : 155278.pdf (Publisher’s version ) (Closed access) There is an urgent need for new drugs to treat malaria, with broad therapeutic potential and novel modes of action, to widen the scope of treatment and to overcome emerging drug resistance. Here we describe the discovery of DDD107498, a compound with a potent and novel spectrum of antimalarial activity against multiple life-cycle stages of the Plasmodium parasite, with good pharmacokinetic properties and an acceptable safety profile. DDD107498 demonstrates potential to address a variety of clinical needs, including single-dose treatment, transmission blocking and chemoprotection. DDD107498 was developed from a screening programme against blood-stage malaria parasites; its molecular target has been identified as translation elongation factor 2 (eEF2), which is responsible for the GTP-dependent translocation of the ribosome along messenger RNA, and is essential for protein synthesis. This discovery of eEF2 as a viable antimalarial drug target opens up new possibilities for drug discovery.

The bifunctional methylerythritol 4-phosphate cytidylyltransferase methylerythritol 2,4-cyclodiphosphate synthase (IspDF) is unusual in that it catalyzes nonconsecutive reactions in the 1-deoxy-d-xylulose 5-phosphate (DOXP) pathway of isoprenoid precursor biosynthesis. The crystal structure of IspDF from the bacterial pathogen Campylobacter jejuni reveals an elongated hexamer with D3 symmetry compatible with the dimeric 2C-methyl-d-erythritol-4-phosphate cytidylyltransferase and trimeric 2C-methyl-d-erythritol-2,4-cyclodiphosphate synthase monofunctional enzymes. Complex formation of IspDF with 4-diphosphocytidyl-2C-methyl-d-erythritol kinase (IspE), the intervening enzyme activity in the pathway, has been observed in solution for the enzymes from C. jejuni and Agrobacterium tumefaciens. The monofunctional enzymes (2C-methyl-d-erythritol-4-phosphate cytidylyltransferase, IspE, and 2C-methyl-d-erythritol-2,4-cyclodiphosphate synthase) involved in the DOXP biosynthetic pathway of Escherichia coli also show physical associations. We propose that complex formation of the three enzymes at the core of the DOXP pathway can produce an assembly localizing 18 catalytic centers for the early stages of isoprenoid biosynthesis.

An established culture of Aspergillus fumigatus MBC-F1-10 proved to be very receptive to external stimuli and reacted with the production of secondary metabolites which were undetectable when the fungus was grown under standard conditions. Firstly, co-cultivation with the type strain of Streptomyces bullii, an isolate from hyper-arid Atacama desert soil, led to the isolation of ergosterol 1, seven metabolites belonging to the diketopiperazine alkaloids; brevianamide F 2, spirotryprostatin A 3, 6-methoxy spirotryprostatin B 4, fumitremorgin C and its 12,13-dihydroxy derivative (5–6), fumitremorgin B 7, and verruculogen 8, in addition to 11-O-methylpseurotin A 9 and its new isomer 11-O-methylpseurotin A210. In an independent experiment, addition of N-butyryl-DL-homoserine lactone to the culture medium led to the production of emestrins A and B (11–12). Neither microbe produced these compounds when cultured alone. The structures of all compounds were elucidated using NMR spectroscopic techniques and mass spectrometric analysis. The isolated compounds were tested for their potential antibacterial and antiprotozoal activities.

Nature 522, 315–320 (2015); doi:10.1038/nature14451 In this Article, Torsten Luksch was inadvertently omitted from the author list. He is affiliated with the Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK. His contribution was the analysis of initial screening data alongside author A.

Sole reliance on one drug, Praziquantel, for treatment and control of schistosomiasis raises concerns about development of widespread resistance, prompting renewed interest in the discovery of new anthelmintics. To discover new leads we designed an automated label-free, high content-based, high throughput screen (HTS) to assess drug-induced effects on in vitro cultured larvae (schistosomula) using bright-field imaging. Automatic image analysis and Bayesian prediction models define morphological damage, hit/non-hit prediction and larval phenotype characterization. Motility was also assessed from time-lapse images. In screening a 10,041 compound library the HTS correctly detected 99.8% of the hits scored visually. A proportion of these larval hits were also active in an adult worm ex-vivo screen and are the subject of ongoing studies. The method allows, for the first time, screening of large compound collections against schistosomes and the methods are adaptable to other whole organism and cell-based screening by morphology and motility phenotyping., Author Summary Schistosomiasis is a severe helminth infection affecting an estimated 600 million people. The one drug widely available, praziquantel (PZQ), is not ideal. PZQ kills the adult worms but not the developing juveniles so the treated patient may not be cured long-term. In addition, use of repeated mass treatment campaigns with PZQ to control morbidity raises concerns about the development of drug resistance. Our work is aimed at providing starting points for drug discovery programs for schistosomiasis by screening large compound libraries against whole organisms. Praziquantel and several other known anti-schistosomal drugs are also active in vitro against the adult worms and the larval stages, schistosomula. The latter are ideal for novel drug screening as they can be produced in large numbers in vitro, are small and so are amenable to screening in microwell plates. Drug activity can be assessed visually but this is subjective and laborious. We have built an automated system for assessing drug action involving the collection of images of the larvae and the development of computer algorithms to analyze their morphology and motility, defining them as "hits" or "nonhits." The method is reliable, consistent and efficient, making it feasible, for the first time, to screen large compound collections.

The oxirane (1R,2S)-1,2-epoxypropylphosphonic acid (fosfomycin) is a natural product antibiotic produced in Streptomyces wedmorensis by the metal-ion-dependent (S)-2-hydroxypropylphosphonic acid epoxidase. This epoxidase is highly unusual since it has no requirement for a haem prosthetic group. The gene encoding the enzyme, fom4, has been cloned and a highly efficient recombinant source of the enzyme established. Two different crystal forms, tetragonal and hexagonal, have been obtained. The hexagonal form displays symmetry consistent with space group P6(1/5)22 and unit-cell parameters a = 86.44, c = 221.56 A, gamma = 120 degrees. The Matthews coefficient, VM, of 2.7 A3 Da(-1) corresponds to two subunits, each of approximate weight 21.4 kDa, in the asymmetric unit with 55% solvent content. These crystals diffract to high resolution and experimental phases are being sought to determine the structure.

Galactose metabolism is essential in bloodstream form Trypanosoma brucei and is initiated by the enzyme UDP-Glc 4′-epimerase. Here, we show that the parasite epimerase is a homodimer that can interconvert UDP-Glc and UDP-Gal but not UDP-GlcNAc and UDP-GalNAc. The epimerase was localized to the glycosomes by immunofluorescence microscopy and subcellular fractionation, suggesting a novel compartmentalization of galactose metabolism in this organism. The epimerase is encoded by the TbGALE gene and procyclic form T. brucei single-allele knockouts, and conditional (tetracycline-inducible) null mutants were constructed. Under non-permissive conditions, conditional null mutant cultures ceased growth after 8 days and resumed growth after 15 days. The resumption of growth coincided with constitutive re-expression epimerase mRNA. These data show that galactose metabolism is essential for cell growth in procyclic form T. brucei. The epimerase is required for glycoprotein galactosylation. The major procyclic form glycoproteins, the procyclins., were analyzed in TbGALE single-allele knockouts and in the conditional null mutant after removal of tetracycline. The procyclins contain glycosylphosphatidylinositol membrane anchors with large poly-N-acetyl-lactosamine side chains. The single allele knockouts exhibited 30% reduction in procyclin galactose content. This example of haploid insufficiency suggests that epimerase levels are close to limiting in this life cycle stage. Similar analyses of the conditional null mutant 9 days after the removal of tetracycline showed that the procyclins were virtually galactose-free and greatly reduced in size. The parasites compensated, ultimately unsuccessfully, by expressing 10-fold more procyclin. The implications of these data with respect to the relative roles of procyclin polypeptide and carbohydrate are discussed.

The development of new antimalarial compounds remains a pivotal part of the strategy for malaria elimination. Recent large-scale phenotypic screens have provided a wealth of potential starting points for hit-to-lead campaigns. One such public set is explored, employing an open source research mechanism in which all data and ideas were shared in real time, anyone was able to participate, and patents were not sought. One chemical subseries was found to exhibit oral activity but contained a labile ester that could not be replaced without loss of activity, and the original hit exhibited remarkable sensitivity to minor structural change. A second subseries displayed high potency, including activity within gametocyte and liver stage assays, but at the cost of low solubility. As an open source research project, unexplored avenues are clearly identified and may be explored further by the community; new findings may be cumulatively added to the present work., Starting from hits identified by a pharmaceutical company, new antimalarials have been discovered using an “open source” approach that discloses data in real time and eschews secrecy.

Improved control of Plasmodium vivax malaria can be achieved with the discovery of new antimalarials with radical cure efficacy, including prevention of relapse caused by hypnozoites residing in the liver of patients. We screened several compound libraries against P. vivax liver stages, including 1565 compounds against mature hypnozoites, resulting in one drug-like and several probe-like hits useful for investigating hypnozoite biology. Primaquine and tafenoquine, administered in combination with chloroquine, are currently the only FDA-approved antimalarials for radical cure, yet their activity against mature P. vivax hypnozoites has not yet been demonstrated in vitro. By developing an extended assay, we show both drugs are individually hypnozonticidal and made more potent when partnered with chloroquine, similar to clinically relevant combinations. Post-hoc analyses of screening data revealed excellent performance of ionophore controls and the high quality of single point assays, demonstrating a platform able to support screening of greater compound numbers. A comparison of P. vivax liver stage activity data with that of the P. cynomolgi blood, P. falciparum blood, and P. berghei liver stages reveals overlap in schizonticidal but not hypnozonticidal activity, indicating that the delivery of new radical curative agents killing P. vivax hypnozoites requires an independent and focused drug development test cascade.