Optimisation-based modelling for explainable lead discovery in malaria.

BACKGROUND: The search for new antimalarial treatments is urgent due to growing resistance to existing therapies. The Open Source Malaria (OSM) project offers a promising starting point, having extensively screened various compounds for their effectiveness. Further analysis of the chemical space surrounding these compounds could provide the means for innovative drugs. METHODS: We report an optimisation-based method for quantitative structure-activity relationship (QSAR) modelling that provides explainable modelling of ligand activity through a mathematical programming formulation. The methodology is based on piecewise regression principles and offers optimal detection of breakpoint features, efficient allocation of samples into distinct sub-groups based on breakpoint feature values, and insightful regression coefficients. Analysis of OSM antimalarial compounds yields interpretable results through rules generated by the model that reflect the contribution of individual fingerprint fragments in ligand activity prediction. Using knowledge of fragment prioritisation and screening of commercially available compound libraries, potential lead compounds for antimalarials are identified and evaluated experimentally via a Plasmodium falciparum asexual growth inhibition assay (PfGIA) and a human cell cytotoxicity assay. CONCLUSIONS: Three compounds are identified as potential leads for antimalarials using the methodology described above. This work illustrates how explainable predictive models based on mathematical optimisation can pave the way towards more efficient fragment-based lead discovery as applied in malaria.

Machine learning-based phenotypic imaging to characterise the targetable biology of Plasmodium falciparum male gametocytes for the development of transmission-blocking antimalarials.

Preventing parasite transmission from humans to mosquitoes is recognised to be critical for achieving elimination and eradication of malaria. Consequently developing new antimalarial drugs with transmission-blocking properties is a priority. Large screening campaigns have identified many new transmission-blocking molecules, however little is known about how they target the mosquito-transmissible Plasmodium falciparum stage V gametocytes, or how they affect their underlying cell biology. To respond to this knowledge gap, we have developed a machine learning image analysis pipeline to characterise and compare the cellular phenotypes generated by transmission-blocking molecules during male gametogenesis. Using this approach, we studied 40 molecules, categorising their activity based upon timing of action and visual effects on the organisation of tubulin and DNA within the cell. Our data both proposes new modes of action and corroborates existing modes of action of identified transmission-blocking molecules. Furthermore, the characterised molecules provide a new armoury of tool compounds to probe gametocyte cell biology and the generated imaging dataset provides a new reference for researchers to correlate molecular target or gene deletion to specific cellular phenotype. Our analysis pipeline is not optimised for a specific organism and could be applied to any fluorescence microscopy dataset containing cells delineated by bounding boxes, and so is potentially extendible to any disease model.

Bis-6-amidino-benzothiazole Derivative that Cures Experimental Stage 1 African Trypanosomiasis with a Single Dose.

We designed and synthesized a series of symmetric bis-6-amidino-benzothiazole derivatives with aliphatic central units and evaluated their efficacy against bloodstream forms of the African trypanosome Trypanosoma brucei. Of these, a dicationic benzothiazole compound (9a) exhibited sub-nanomolar in vitro potency with remarkable selectivity over mammalian cells (>26,000-fold). Unsubstituted 5-amidine groups and a cyclohexyl spacer were the crucial determinants of trypanocidal activity. In all cases, mice treated with a single dose of 20 mg kg-1 were cured of stage 1 trypanosomiasis. The compound displayed a favorable in vitro ADME profile, with the exception of low membrane permeability. However, we found evidence that uptake by T. brucei is mediated by endocytosis, a process that results in lysosomal sequestration. The compound was also active in low nanomolar concentrations against cultured asexual forms of the malaria parasite Plasmodium falciparum. Therefore, 9a has exquisite cross-species efficacy and represents a lead compound with considerable therapeutic potential.

A Pyridyl-Furan Series Developed from the Open Global Health Library Block Red Blood Cell Invasion and Protein Trafficking in Plasmodium falciparum through Potential Inhibition of the Parasite's PI4KIIIB Enzyme.

With the resistance increasing to current antimalarial medicines, there is an urgent need to discover new drug targets and to develop new medicines against these targets. We therefore screened the Open Global Health Library of Merck KGaA, Darmstadt, Germany, of 250 compounds against the asexual blood stage of the deadliest malarial parasite Plasmodium falciparum, from which eight inhibitors with low micromolar potency were found. Due to its combined potencies against parasite growth and inhibition of red blood cell invasion, the pyridyl-furan compound OGHL250 was prioritized for further optimization. The potency of the series lead compound (WEHI-518) was improved 250-fold to low nanomolar levels against parasite blood-stage growth. Parasites selected for resistance to a related compound, MMV396797, were also resistant to WEHI-518 as well as KDU731, an inhibitor of the phosphatidylinositol kinase PfPI4KIIIB, suggesting that this kinase is the target of the pyridyl-furan series. Inhibition of PfPI4KIIIB blocks multiple stages of the parasite's life cycle and other potent inhibitors are currently under preclinical development. MMV396797-resistant parasites possess an E1316D mutation in PfPKI4IIIB that clusters with known resistance mutations of other inhibitors of the kinase. Building upon earlier studies that showed that PfPI4KIIIB inhibitors block the development of the invasive merozoite parasite stage, we show that members of the pyridyl-furan series also block invasion and/or the conversion of merozoites into ring-stage intracellular parasites through inhibition of protein secretion and export into red blood cells.

A novel class of sulphonamides potently block malaria transmission by targeting a Plasmodium vacuole membrane protein.

Phenotypic cell-based screens are critical tools for discovering candidate drugs for development, yet identification of the cellular target and mode of action of a candidate drug is often lacking. Using an imaging-based screen, we recently discovered an N-[(4-hydroxychroman-4-yl)methyl]-sulphonamide (N-4HCS) compound, DDD01035881, that blocks male gamete formation in the malaria parasite life cycle and subsequent transmission of the parasite to the mosquito with nanomolar activity. To identify the target(s) of DDD01035881, and of the N-4HCS class of compounds more broadly, we synthesised a photoactivatable derivative, probe 2. Photoaffinity labelling of probe 2 coupled with mass spectrometry identified the 16 kDa Plasmodium falciparum parasitophorous vacuole membrane protein Pfs16 as a potential parasite target. Complementary methods including cellular thermal shift assays confirmed that the parent molecule DDD01035881 stabilised Pfs16 in lysates from activated mature gametocytes. Combined with high-resolution, fluorescence and electron microscopy data, which demonstrated that parasites inhibited with N-4HCS compounds phenocopy the targeted deletion of Pfs16 in gametocytes, these data implicate Pfs16 as a likely target of DDD01035881. This finding establishes N-4HCS compounds as being flexible and effective starting candidates from which transmission-blocking antimalarials can be developed in the future.

Protocols for Plasmodium gametocyte production in vitro: an integrative review and analysis.

BACKGROUND: The production of Plasmodium gametocytes in vitro is a real challenge. Many protocols have been described, but few have resulted in the production of viable and infectious gametocytes in sufficient quantities to conduct research on-but not limited to-transmission-blocking drug and vaccine development. The aim of this review was to identify and discuss gametocyte production protocols that have been developed over the last two decades. METHODS: We analyzed the original gametocyte production protocols published from 2000 onwards based on a literature search and a thorough review. A systematic review was performed of relevant articles identified in the PubMed, Web of Sciences and ScienceDirect databases. RESULTS: A total 23 studies on the production of Plasmodium gametocytes were identified, 19 involving in vitro Plasmodium falciparum, one involving Plasmodium knowlesi and three involving ex vivo Plasmodium vivax. Of the in vitro studies, 90% used environmental stressors to trigger gametocytogenesis. Mature gametocytemia of up to 4% was reported. CONCLUSIONS: Several biological parameters contribute to an optimal production in vitro of viable and infectious mature gametocytes. The knowledge gained from this systematic review on the molecular mechanisms involved in gametocytogenesis enables reproducible gametocyte protocols with transgenic parasite lines to be set up. This review highlights the need for additional gametocyte production protocols for Plasmodium species other than P. falciparum.

The Novel bis-1,2,4-Triazine MIPS-0004373 Demonstrates Rapid and Potent Activity against All Blood Stages of the Malaria Parasite.

Novel bis-1,2,4-triazine compounds with potent in vitro activity against Plasmodium falciparum parasites were recently identified. The bis-1,2,4-triazines represent a unique antimalarial pharmacophore and are proposed to act by a novel but as-yet-unknown mechanism of action. This study investigated the activity of the bis-1,2,4-triazine MIPS-0004373 across the mammalian life cycle stages of the parasite and profiled the kinetics of activity against blood and transmission stage parasites in vitro and in vivo. MIPS-0004373 demonstrated rapid and potent activity against P. falciparum, with excellent in vitro activity against all asexual blood stages. Prolonged in vitro drug exposure failed to generate stable resistance de novo, suggesting a low propensity for the emergence of resistance. Excellent activity was observed against sexually committed ring stage parasites, but activity against mature gametocytes was limited to inhibiting male gametogenesis. Assessment of liver stage activity demonstrated good activity in an in vitro P. berghei model but no activity against Plasmodium cynomolgi hypnozoites or liver schizonts. The bis-1,2,4-triazine MIPS-0004373 efficiently cleared an established P. berghei infection in vivo, with efficacy similar to that of artesunate and chloroquine and a recrudescence profile comparable to that of chloroquine. This study demonstrates the suitability of bis-1,2,4-triazines for further development toward a novel treatment for acute malaria.

Identification and Profiling of a Novel Diazaspiro[3.4]octane Chemical Series Active against Multiple Stages of the Human Malaria Parasite Plasmodium falciparum and Optimization Efforts.

A novel diazaspiro[3.4]octane series was identified from a Plasmodium falciparum whole-cell high-throughput screening campaign. Hits displayed activity against multiple stages of the parasite lifecycle, which together with a novel sp3-rich scaffold provided an attractive starting point for a hit-to-lead medicinal chemistry optimization and biological profiling program. Structure-activity-relationship studies led to the identification of compounds that showed low nanomolar asexual blood-stage activity (<50 nM) together with strong gametocyte sterilizing properties that translated to transmission-blocking activity in the standard membrane feeding assay. Mechanistic studies through resistance selection with one of the analogues followed by whole-genome sequencing implicated the P. falciparum cyclic amine resistance locus in the mode of resistance.

The antimalarial efficacy and mechanism of resistance of the novel chemotype DDD01034957.

New antimalarial therapeutics are needed to ensure that malaria cases continue to be driven down, as both emerging parasite resistance to frontline chemotherapies and mosquito resistance to current insecticides threaten control programmes. Plasmodium, the apicomplexan parasite responsible for malaria, causes disease pathology through repeated cycles of invasion and replication within host erythrocytes (the asexual cycle). Antimalarial drugs primarily target this cycle, seeking to reduce parasite burden within the host as fast as possible and to supress recrudescence for as long as possible. Intense phenotypic drug screening efforts have identified a number of promising new antimalarial molecules. Particularly important is the identification of compounds with new modes of action within the parasite to combat existing drug resistance and suitable for formulation of efficacious combination therapies. Here we detail the antimalarial properties of DDD01034957-a novel antimalarial molecule which is fast-acting and potent against drug resistant strains in vitro, shows activity in vivo, and possesses a resistance mechanism linked to the membrane transporter PfABCI3. These data support further medicinal chemistry lead-optimization of DDD01034957 as a novel antimalarial chemical class and provide new insights to further reduce in vivo metabolic clearance.

Transmission of Artemisinin-Resistant Malaria Parasites to Mosquitoes under Antimalarial Drug Pressure.

Resistance to artemisinin-based combination therapy (ACT) in the Plasmodium falciparum parasite is threatening to reverse recent gains in reducing global deaths from malaria. While resistance manifests as delayed parasite clearance in patients, the phenotype can only spread geographically via the sexual stages and mosquito transmission. In addition to their asexual killing properties, artemisinin and its derivatives sterilize sexual male gametocytes. Whether resistant parasites overcome this sterilizing effect has not, however, been fully tested. Here, we analyzed P. falciparum clinical isolates from the Greater Mekong Subregion, each demonstrating delayed clinical clearance and known resistance-associated polymorphisms in the Kelch13 (PfK13var) gene. As well as demonstrating reduced asexual sensitivity to drug, certain PfK13var isolates demonstrated a marked reduction in sensitivity to artemisinin in an in vitro male gamete formation assay. Importantly, this same reduction in sensitivity was observed when the most resistant isolate was tested directly in mosquito feeds. These results indicate that, under artemisinin drug pressure, while sensitive parasites are blocked, resistant parasites continue transmission. This selective advantage for resistance transmission could favor acquisition of additional host-specificity or polymorphisms affecting partner drug sensitivity in mixed infections. Favored resistance transmission under ACT coverage could have profound implications for the spread of multidrug-resistant malaria beyond Southeast Asia.

Inhibition of Resistance-Refractory P. falciparum Kinase PKG Delivers Prophylactic, Blood Stage, and Transmission-Blocking Antiplasmodial Activity.

The search for antimalarial chemotypes with modes of action unrelated to existing drugs has intensified with the recent failure of first-line therapies across Southeast Asia. Here, we show that the trisubstituted imidazole MMV030084 potently inhibits hepatocyte invasion by Plasmodium sporozoites, merozoite egress from asexual blood stage schizonts, and male gamete exflagellation. Metabolomic, phosphoproteomic, and chemoproteomic studies, validated with conditional knockdown parasites, molecular docking, and recombinant kinase assays, identified cGMP-dependent protein kinase (PKG) as the primary target of MMV030084. PKG is known to play essential roles in Plasmodium invasion of and egress from host cells, matching MMV030084's activity profile. Resistance selections and gene editing identified tyrosine kinase-like protein 3 as a low-level resistance mediator for PKG inhibitors, while PKG itself never mutated under pressure. These studies highlight PKG as a resistance-refractory antimalarial target throughout the Plasmodium life cycle and promote MMV030084 as a promising Plasmodium PKG-targeting chemotype.

Structure-Activity Relationship Studies of a Novel Class of Transmission Blocking Antimalarials Targeting Male Gametes.

Malaria is still a leading cause of mortality among children in the developing world, and despite the immense progress made in reducing the global burden, further efforts are needed if eradication is to be achieved. In this context, targeting transmission is widely recognized as a necessary intervention toward that goal. After carrying out a screen to discover new transmission-blocking agents, herein we report our medicinal chemistry efforts to study the potential of the most robust hit, DDD01035881, as a male-gamete targeted compound. We reveal key structural features for the activity of this series and identify analogues with greater potency and improved metabolic stability. We believe this study lays the groundwork for further development of this series as a transmission blocking agent.

Screen of traditional soup broths with reported antipyretic activity towards the discovery of potential antimalarials.

OBJECTIVE: The global impact of artemisinin-based combination therapies on malaria-associated mortality and their origins in ancient Chinese medicine has heightened interest in the natural discovery of future antimalarials. METHODS: A double-blind study to identify potential ingredients with antimalarial activity from traditional remedies with reported antipyretic properties. Recipes of clear broths, passed down by tradition in families of diverse ethnic origin, were sourced by school children. Broths were then tested for their ability to arrest malaria parasite asexual growth or sexual stage development in vitro. Clear broth extract was incubated with in vitro cultures of Plasmodium falciparum asexual or mature sexual stage cultures and assayed for parasite viability after 72 hours. RESULTS: Of the 56 broths tested, 5 were found to give >50% in vitro growth inhibition against P. falciparum asexual blood stages, with 2 having comparable inhibition to that seen with dihydroartemisinin, a leading antimalarial. Four other broths were found to have >50% transmission blocking activity, preventing male parasite sexual stage development. After unblinding, two active broths were found to be from siblings from different classes, who had brought in the same vegetarian soup, demonstrating assay robustness. CONCLUSIONS: This screening approach succeeded in finding broths with activity against malaria parasite in vitro growth, arising from complex vegetable and/or meat-based broths. This represented a successful child education exercise, in teaching about the interface between natural remedies, traditional medicine and evidence-based drug discovery.

Fueling Open Innovation for Malaria Transmission-Blocking Drugs: Hundreds of Molecules Targeting Early Parasite Mosquito Stages.

BACKGROUND: Despite recent successes at controlling malaria, progress has stalled with an estimated 219 million cases and 435,000 deaths in 2017 alone. Combined with emerging resistance to front line antimalarial therapies in Southeast Asia, there is an urgent need for new treatment options and novel approaches to halt the spread of malaria. Plasmodium, the parasite responsible for malaria propagates through mosquito transmission. This imposes an acute bottleneck on the parasite population and transmission-blocking interventions exploiting this vulnerability are recognized as vital for malaria elimination. METHODS: 13,533 small molecules with known activity against Plasmodium falciparum asexual parasites were screened for additional transmission-blocking activity in an ex vivo Plasmodium berghei ookinete development assay. Active molecules were then counterscreened in dose response against HepG2 cells to determine their activity/cytotoxicity window and selected non-toxic representative molecules were fully profiled in a range of transmission and mosquito infection assays. Furthermore, the entire dataset was compared to other published screens of the same molecules against P. falciparum gametocytes and female gametogenesis. RESULTS: 437 molecules inhibited P. berghei ookinete formation with an IC50 < 10 µM. of which 273 showed >10-fold parasite selectivity compared to activity against HepG2 cells. Active molecules grouped into 49 chemical clusters of three or more molecules, with 25 doublets and 94 singletons. Six molecules representing six major chemical scaffolds confirmed their transmission-blocking activity against P. falciparum male and female gametocytes and inhibited P. berghei oocyst formation in the standard membrane feeding assay at 1 µM. When screening data in the P. berghei development ookinete assay was compared to published screens of the same library in assays against P. falciparum gametocytes and female gametogenesis, it was established that each assay identified distinct, but partially overlapping subsets of transmission-blocking molecules. However, selected molecules unique to each assay show transmission-blocking activity in mosquito transmission assays. CONCLUSION: The P. berghei ookinete development assay is an excellent high throughput assay for efficiently identifying antimalarial molecules targeting early mosquito stage parasite development. Currently no high throughput transmission-blocking assay is capable of identifying all transmission-blocking molecules.

Kinesin-8B controls basal body function and flagellum formation and is key to malaria transmission.

Eukaryotic flagella are conserved microtubule-based organelles that drive cell motility. Plasmodium, the causative agent of malaria, has a single flagellate stage: the male gamete in the mosquito. Three rounds of endomitotic division in male gametocyte together with an unusual mode of flagellum assembly rapidly produce eight motile gametes. These processes are tightly coordinated, but their regulation is poorly understood. To understand this important developmental stage, we studied the function and location of the microtubule-based motor kinesin-8B, using gene-targeting, electron microscopy, and live cell imaging. Deletion of the kinesin-8B gene showed no effect on mitosis but disrupted 9+2 axoneme assembly and flagellum formation during male gamete development and also completely ablated parasite transmission. Live cell imaging showed that kinesin-8B-GFP did not co-localise with kinetochores in the nucleus but instead revealed a dynamic, cytoplasmic localisation with the basal bodies and the assembling axoneme during flagellum formation. We, thus, uncovered an unexpected role for kinesin-8B in parasite flagellum formation that is vital for the parasite life cycle.

Substituted Aminoacetamides as Novel Leads for Malaria Treatment.

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.

Lysyl-tRNA synthetase as a drug target in malaria and cryptosporidiosis.

Malaria and cryptosporidiosis, caused by apicomplexan parasites, remain major drivers of global child mortality. New drugs for the treatment of malaria and cryptosporidiosis, in particular, are of high priority; however, there are few chemically validated targets. The natural product cladosporin is active against blood- and liver-stage Plasmodium falciparum and Cryptosporidium parvum in cell-culture studies. Target deconvolution in P. falciparum has shown that cladosporin inhibits lysyl-tRNA synthetase (PfKRS1). Here, we report the identification of a series of selective inhibitors of apicomplexan KRSs. Following a biochemical screen, a small-molecule hit was identified and then optimized by using a structure-based approach, supported by structures of both PfKRS1 and C. parvum KRS (CpKRS). In vivo proof of concept was established in an SCID mouse model of malaria, after oral administration (ED90 = 1.5 mg/kg, once a day for 4 d). Furthermore, we successfully identified an opportunity for pathogen hopping based on the structural homology between PfKRS1 and CpKRS. This series of compounds inhibit CpKRS and C. parvum and Cryptosporidium hominis in culture, and our lead compound shows oral efficacy in two cryptosporidiosis mouse models. X-ray crystallography and molecular dynamics simulations have provided a model to rationalize the selectivity of our compounds for PfKRS1 and CpKRS vs. (human) HsKRS. Our work validates apicomplexan KRSs as promising targets for the development of drugs for malaria and cryptosporidiosis.

The antimalarial screening landscape-looking beyond the asexual blood stage.

In recent years, the research agenda to tackle global morbidity and mortality from malaria disease has shifted towards innovation, in the hope that efforts at the frontiers of scientific research may re-invigorate gains made towards eradication. Discovery of new antimalarial drugs with novel chemotypes or modes of action lie at the heart of these efforts. There is a particular interest in drug candidates that target stages of the malaria parasite lifecycle beyond the symptomatic asexual blood stages. This is especially important given the spectre of emerging drug resistance to all current frontline antimalarials. One approach gaining increased interest is the potential of designing novel drugs that target parasite passage from infected individual to feeding mosquito and back again. Action of such therapeutics is geared much more at the population level rather than just concerned with the infected individual. The search for novel drugs active against these stages has been helped by improvements to in vitro culture of transmission and pre-erythrocytic parasite lifecycle stages, robotic automation and high content imaging, methodologies that permit the high-throughput screening (HTS) of compound libraries for drug discovery. Here, we review recent advances in the antimalarial screening landscape, focussed on transmission blocking as a key aim for drug-treatment campaigns of the future.

Transmission-blocking Effects of Primaquine and Methylene Blue Suggest Plasmodium falciparum Gametocyte Sterilization Rather Than Effects on Sex Ratio.

Gametocyte density and sex ratio can predict the proportion of mosquitoes that will become infected after feeding on blood of patients receiving nongametocytocidal drugs. Because primaquine and methylene blue sterilize gametocytes before affecting their density and sex ratio, mosquito feeding experiments are required to demonstrate their early transmission-blocking effects.

8-Aminoquinolines with an Aminoxyalkyl Side Chain Exert in vitro Dual-Stage Antiplasmodial Activity.

A series of novel 8-aminoquinolines (8-AQs) with an aminoxyalkyl side chain were synthesized and evaluated for in vitro antiplasmodial properties against asexual blood stages, liver stages, and sexual stages of Plasmodium falciparum. 8-AQs bearing 2-alkoxy and 5-phenoxy substituents on the quinoline ring system were found to be the most promising compounds under study, exhibiting potent blood schizontocidal and moderate tissue schizontocidal in vitro activity.