Oxidative stress changes the effectiveness of artemisinin in Plasmodium falciparum.

Malaria parasites have adaptive mechanisms to modulate their intracellular redox status to tolerate the enhanced oxidizing effects created by malaria fever, hemoglobinopathies and other stress conditions, including antimalaria drugs. Emerging artemisinin (ART) resistance in Plasmodium falciparum is a complex phenotype linked to the parasite's tolerance of the activated drug's oxidative damage along with changes in vesicular transport, lipid metabolism, DNA repair, and exported proteins. In an earlier study, we discovered that many of these metabolic processes are induced in P. falciparum to respond to the oxidative damage caused by artemisinin, which exhibited a highly significant overlap with the parasite's adaptive response mechanisms to survive febrile temperatures. In addition, there was a significant overlap with the parasite's survival responses to oxidative stress. In this study, we investigated these relationships further using an in vitro model to evaluate if oxidative stress and heat-shock conditions could alter the parasite's response to artemisinin. The results revealed that compared to ideal culture conditions, the antimalarial efficacy of artemisinin was significantly reduced in parasites growing in intraerythrocytic oxidative stress but not in heat-shock condition. In contrast, heat shock significantly reduced the efficacy of lumefantrine that is an important ART combination therapy partner drug. We propose that prolonged exposure to intraerythrocytic microenvironmental oxidative stress, as would occur in endemic regions with high prevalence for sickle trait and other hemoglobinopathies, can predispose malaria parasites to develop tolerance to the oxidative damage caused by antimalarial drugs like artemisinin. IMPORTANCE: Emerging resistance to the frontline antimalarial drug artemisinin represents a significant threat to worldwide malaria control and elimination. The patterns of parasite changes associated with emerging resistance represent a complex array of metabolic processes evident in various genetic mutations and altered transcription profiles. Genetic factors identified in regulating P. falciparum sensitivity to artemisinin overlap with the parasite's responses to malarial fever, sickle trait, and other types of oxidative stresses, suggesting conserved inducible survival responses. In this study we show that intraerythrocytic stress conditions, oxidative stress and heat shock, can significantly decrease the sensitivity of the parasite to artemisinin and lumefantrine, respectively. These results indicate that an intraerythrocytic oxidative stress microenvironment and heat-shock condition can alter antimalarial drug efficacy. Evaluating efficacy of antimalarial drugs under ideal in vitro culture conditions may not accurately predict drug efficacy in all malaria patients.

MAIP: An Open-Source Tool to Enrich High-Throughput Screening Output and Identify Novel, Druglike Molecules with Antimalarial Activity.

Efforts to tackle malaria must continue for a disease that threatens half of the global population. Parasite resistance to current therapies requires new chemotypes that are able to demonstrate effectiveness and safety. Previously, we developed a machine-learning-based approach to predict compound antimalarial activity, which was trained on the compound collections of several organizations. The resulting prediction platform, MAIP, was made freely available to the scientific community and offers a solution to prioritize molecules of interest in virtual screening and hit-to-lead optimization. Here, we experimentally validate MAIP and demonstrate how the approach was used in combination with a robust compound selection workflow and a recently introduced innovative high-throughput screening (HTS) cascade to select and purchase compounds from a public library for subsequent experimental screening. We observed a 12-fold enrichment compared with a randomly selected set of molecules, and the eight hits we ultimately selected exhibit good potency and absorption, distribution, metabolism, and excretion (ADME) profiles.

Antimalarial drug discovery: progress and approaches.

Recent antimalarial drug discovery has been a race to produce new medicines that overcome emerging drug resistance, whilst considering safety and improving dosing convenience. Discovery efforts have yielded a variety of new molecules, many with novel modes of action, and the most advanced are in late-stage clinical development. These discoveries have led to a deeper understanding of how antimalarial drugs act, the identification of a new generation of drug targets, and multiple structure-based chemistry initiatives. The limited pool of funding means it is vital to prioritize new drug candidates. They should exhibit high potency, a low propensity for resistance, a pharmacokinetic profile that favours infrequent dosing, low cost, preclinical results that demonstrate safety and tolerability in women and infants, and preferably the ability to block Plasmodium transmission to Anopheles mosquito vectors. In this Review, we describe the approaches that have been successful, progress in preclinical and clinical development, and existing challenges. We illustrate how antimalarial drug discovery can serve as a model for drug discovery in diseases of poverty.

Development and application of a PBPK modeling strategy to support antimalarial drug development.

As part of a collaboration between Medicines for Malaria Venture (MMV), Certara UK and Monash University, physiologically-based pharmacokinetic (PBPK) models were developed for 20 antimalarials, using data obtained from standardized in vitro assays and clinical studies within the literature. The models have been applied within antimalarial drug development at MMV for more than 5 years. During this time, a strategy for their impactful use has evolved. All models are described in the supplementary material and are available to researchers. Case studies are also presented, demonstrating real-world development and clinical applications, including the assessment of the drug-drug interaction liability between combination partners or with co-administered drugs. This work emphasizes the benefit of PBPK modeling for antimalarial drug development and decision making, and presents a strategy to integrate it into the research and development process. It also provides a repository of shared information to benefit the global health research community.

The Parasite Reduction Ratio (PRR) Assay Version 2: Standardized Assessment of Plasmodium falciparum Viability after Antimalarial Treatment In Vitro.

With artemisinin-resistant Plasmodium falciparum parasites emerging in Africa, the need for new antimalarial chemotypes is persistently high. The ideal pharmacodynamic parameters of a candidate drug are a rapid onset of action and a fast rate of parasite killing or clearance. To determine these parameters, it is essential to discriminate viable from nonviable parasites, which is complicated by the fact that viable parasites can be metabolically inactive, whilst dying parasites can still be metabolically active and morphologically unaffected. Standard growth inhibition assays, read out via microscopy or [3H] hypoxanthine incorporation, cannot reliably discriminate between viable and nonviable parasites. Conversely, the in vitro parasite reduction ratio (PRR) assay is able to measure viable parasites with high sensitivity. It provides valuable pharmacodynamic parameters, such as PRR, 99.9% parasite clearance time (PCT99.9%) and lag phase. Here we report the development of the PRR assay version 2 (V2), which comes with a shorter assay duration, optimized quality controls and an objective, automated analysis pipeline that systematically estimates PRR, PCT99.9% and lag time and returns meaningful secondary parameters such as the maximal killing rate of a drug (Emax) at the assayed concentration. These parameters can be fed directly into pharmacokinetic/pharmacodynamic models, hence aiding and standardizing lead selection, optimization, and dose prediction.

Discovery and Characterization of Potent, Efficacious, Orally Available Antimalarial Plasmepsin X Inhibitors and Preclinical Safety Assessment of UCB7362.

Plasmepsin X (PMX) is an essential aspartyl protease controlling malaria parasite egress and invasion of erythrocytes, development of functional liver merozoites (prophylactic activity), and blocking transmission to mosquitoes, making it a potential multistage drug target. We report the optimization of an aspartyl protease binding scaffold and the discovery of potent, orally active PMX inhibitors with in vivo antimalarial efficacy. Incorporation of safety evaluation early in the characterization of PMX inhibitors precluded compounds with a long human half-life (t1/2) to be developed. Optimization focused on improving the off-target safety profile led to the identification of UCB7362 that had an improved in vitro and in vivo safety profile but a shorter predicted human t1/2. UCB7362 is estimated to achieve 9 log 10 unit reduction in asexual blood-stage parasites with once-daily dosing of 50 mg for 7 days. This work demonstrates the potential to deliver PMX inhibitors with in vivo efficacy to treat malaria.

Optimized Pyridazinone Nutrient Channel Inhibitors Are Potent and Specific Antimalarial Leads.

Human and animal malaria parasites increase their host erythrocyte permeability to a broad range of solutes as mediated by parasite-associated ion channels. Molecular and pharmacological studies have implicated an essential role in parasite nutrient acquisition, but inhibitors suitable for development of antimalarial drugs are missing. Here, we generated a potent and specific drug lead using Plasmodium falciparum, a virulent human pathogen, and derivatives of MBX-2366, a nanomolar affinity pyridazinone inhibitor from a high-throughput screen. As this screening hit lacks the bioavailability and stability needed for in vivo efficacy, we synthesized 315 derivatives to optimize drug-like properties, establish target specificity, and retain potent activity against the parasite-induced permeability. Using a robust, iterative pipeline, we generated MBX-4055, a derivative active against divergent human parasite strains. MBX-4055 has improved oral absorption with acceptable in vivo tolerability and pharmacokinetics. It also has no activity against a battery of 35 human channels and receptors and is refractory to acquired resistance during extended in vitro selection. Single-molecule and single-cell patch-clamp indicate direct action on the plasmodial surface anion channel, a channel linked to parasite-encoded RhopH proteins. These studies identify pyridazinones as novel and tractable antimalarial scaffolds with a defined mechanism of action. SIGNIFICANCE STATEMENT: Because antimalarial drugs are prone to evolving resistance in the virulent human P. falciparum pathogen, new therapies are needed. This study has now developed a novel drug-like series of pyridazinones that target an unexploited parasite anion channel on the host cell surface, display excellent in vitro and in vivo ADME properties, are refractory to acquired resistance, and demonstrate a well defined mechanism of action.

The Antifungal Itraconazole Is a Potent Inhibitor of Chikungunya Virus Replication.

Chikungunya virus (CHIKV) is the causative agent of chikungunya fever, a disabling disease that can cause long-term severe arthritis. Since the last large CHIKV outbreak in 2015, the reemergence of the virus represents a serious public health concern. The morbidity associated with viral infection emphasizes the need for the development of specific anti-CHIKV drugs. Herein, we describe the development and characterization of a CHIKV reporter replicon cell line and its use in replicon-based screenings. We tested 960 compounds from MMV/DNDi Open Box libraries and identified four candidates with interesting antiviral activities, which were confirmed in viral infection assays employing CHIKV-nanoluc and BHK-21 cells. The most noteworthy compound identified was itraconazole (ITZ), an orally available, safe, and cheap antifungal, that showed high selectivity indexes of >312 and >294 in both replicon-based and viral infection assays, respectively. The antiviral activity of this molecule has been described against positive-sense single stranded RNA viruses (+ssRNA) and was related to cholesterol metabolism that could affect the formation of the replication organelles. Although its precise mechanism of action against CHIKV still needs to be elucidated, our results demonstrate that ITZ is a potent inhibitor of the viral replication that could be repurposed as a broad-spectrum antiviral.

Assessing risks of Plasmodium falciparum resistance to select next-generation antimalarials.

Strategies to counteract or prevent emerging drug resistance are crucial for the design of next-generation antimalarials. In the past, resistant parasites were generally identified following treatment failures in patients, and compounds would have to be abandoned late in development. An early understanding of how candidate therapeutics lose efficacy as parasites evolve resistance is important to facilitate drug design and improve resistance detection and monitoring up to the postregistration phase. We describe a new strategy to assess resistance to antimalarial compounds as early as possible in preclinical development by leveraging tools to define the Plasmodium falciparum resistome, predict potential resistance risks of clinical failure for candidate therapeutics, and inform decisions to guide antimalarial drug development.

Potent Antimalarials with Development Potential Identified by Structure-Guided Computational Optimization of a Pyrrole-Based Dihydroorotate Dehydrogenase Inhibitor Series.

Dihydroorotate dehydrogenase (DHODH) has been clinically validated as a target for the development of new antimalarials. Experience with clinical candidate triazolopyrimidine DSM265 (1) suggested that DHODH inhibitors have great potential for use in prophylaxis, which represents an unmet need in the malaria drug discovery portfolio for endemic countries, particularly in areas of high transmission in Africa. We describe a structure-based computationally driven lead optimization program of a pyrrole-based series of DHODH inhibitors, leading to the discovery of two candidates for potential advancement to preclinical development. These compounds have improved physicochemical properties over prior series frontrunners and they show no time-dependent CYP inhibition, characteristic of earlier compounds. Frontrunners have potent antimalarial activity in vitro against blood and liver schizont stages and show good efficacy in Plasmodium falciparum SCID mouse models. They are equally active against P. falciparum and Plasmodium vivax field isolates and are selective for Plasmodium DHODHs versus mammalian enzymes.

Actives from MMV Open Access Boxes? A suggested way forward.

It is estimated that more than 1 billion people across the world are affected by a neglected tropical disease (NTD) that requires medical intervention. These diseases tend to afflict people in areas with high rates of poverty and cost economies billions of dollars every year. Collaborative drug discovery efforts are required to reduce the burden of these diseases in endemic regions. The release of "Open Access Boxes" is an initiative launched by Medicines for Malaria Venture (MMV) in collaboration with its partners to catalyze new drug discovery in neglected diseases. These boxes are mainly requested by biology researchers across the globe who may not otherwise have access to compounds to screen nor knowledge of the workflow that needs to be followed after identification of actives from their screening campaigns. Here, we present guidelines on how to move such actives beyond the hit identification stage, to help in capacity strengthening and enable a greater impact of the initiative.

Targeting Plasmepsins-An Achilles' Heel of the Malaria Parasite.

Recently, two Plasmodium aspartyl proteases were identified as druggable targets impacting parasite survival. In this issue of Cell Host & Microbe, Favuzza et al. describe the optimization of a compound series acting on both targets, heralding the prospect of a new class of antimalarials for clinical studies.

Lead Optimization of a Pyrrole-Based Dihydroorotate Dehydrogenase Inhibitor Series for the Treatment of Malaria.

Malaria puts at risk nearly half the world's population and causes high mortality in sub-Saharan Africa, while drug resistance threatens current therapies. The pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase (DHODH) is a validated target for malaria treatment based on our finding that triazolopyrimidine DSM265 (1) showed efficacy in clinical studies. Herein, we describe optimization of a pyrrole-based series identified using a target-based DHODH screen. Compounds with nanomolar potency versus Plasmodium DHODH and Plasmodium parasites were identified with good pharmacological properties. X-ray studies showed that the pyrroles bind an alternative enzyme conformation from 1 leading to improved species selectivity versus mammalian enzymes and equivalent activity on Plasmodium falciparum and Plasmodium vivax DHODH. The best lead DSM502 (37) showed in vivo efficacy at similar levels of blood exposure to 1, although metabolic stability was reduced. Overall, the pyrrole-based DHODH inhibitors provide an attractive alternative scaffold for the development of new antimalarial compounds.

An in vitro toolbox to accelerate anti-malarial drug discovery and development.

BACKGROUND: Modelling and simulation are being increasingly utilized to support the discovery and development of new anti-malarial drugs. These approaches require reliable in vitro data for physicochemical properties, permeability, binding, intrinsic clearance and cytochrome P450 inhibition. This work was conducted to generate an in vitro data toolbox using standardized methods for a set of 45 anti-malarial drugs and to assess changes in physicochemical properties in relation to changing target product and candidate profiles. METHODS: Ionization constants were determined by potentiometric titration and partition coefficients were measured using a shake-flask method. Solubility was assessed in biorelevant media and permeability coefficients and efflux ratios were determined using Caco-2 cell monolayers. Binding to plasma and media proteins was measured using either ultracentrifugation or rapid equilibrium dialysis. Metabolic stability and cytochrome P450 inhibition were assessed using human liver microsomes. Sample analysis was conducted by LC-MS/MS. RESULTS: Both solubility and fraction unbound decreased, and permeability and unbound intrinsic clearance increased, with increasing Log D7.4. In general, development compounds were somewhat more lipophilic than legacy drugs. For many compounds, permeability and protein binding were challenging to assess and both required the use of experimental conditions that minimized the impact of non-specific binding. Intrinsic clearance in human liver microsomes was varied across the data set and several compounds exhibited no measurable substrate loss under the conditions used. Inhibition of cytochrome P450 enzymes was minimal for most compounds. CONCLUSIONS: This is the first data set to describe in vitro properties for 45 legacy and development anti-malarial drugs. The studies identified several practical methodological issues common to many of the more lipophilic compounds and highlighted areas which require more work to customize experimental conditions for compounds being designed to meet the new target product profiles. The dataset will be a valuable tool for malaria researchers aiming to develop PBPK models for the prediction of human PK properties and/or drug-drug interactions. Furthermore, generation of this comprehensive data set within a single laboratory allows direct comparison of properties across a large dataset and evaluation of changing property trends that have occurred over time with changing target product and candidate profiles.

The Development Process for Discovery and Clinical Advancement of Modern Antimalarials.

Malaria is a devastating disease caused by Plasmodium parasites, resulting in approximately 435000 deaths in 2018. The impact of malaria is compounded by the emergence of widespread resistance to current antimalarial therapies. Recently, a new strategy was initiated to screen small molecule collections against the Plasmodium parasite enabling the identification of new antimalarial chemotypes with novel modes of action. This initiative ushered in the modern era of antimalarial drug development, and as a result, numerous lead candidates are advancing toward or are currently in human clinical trials. In this Perspective, we describe the development pathway of four of the most clinically advanced modern antimalarials, KAE609, KAF156, DSM265, and MMV048. Additionally, the mechanism of action and life-cycle stage specificity of the four antimalarials is discussed in relation to aligning with global strategies to treat and eliminate malaria. This perspective serves as a guide to the expectations of modern antimalarial drug development.

Cyclization-blocked proguanil as a strategy to improve the antimalarial activity of atovaquone.

Atovaquone-proguanil (Malarone®) is used for malaria prophylaxis and treatment. While the cytochrome bc1-inhibitor atovaquone has potent activity, proguanil's action is attributed to its cyclization-metabolite, cycloguanil. Evidence suggests that proguanil has limited intrinsic activity, associated with mitochondrial-function. Here we demonstrate that proguanil, and cyclization-blocked analogue tBuPG, have potent, but slow-acting, in vitro anti-plasmodial activity. Activity is folate-metabolism and isoprenoid biosynthesis-independent. In yeast dihydroorotate dehydrogenase-expressing parasites, proguanil and tBuPG slow-action remains, while bc1-inhibitor activity switches from comparatively fast to slow-acting. Like proguanil, tBuPG has activity against P. berghei liver-stage parasites. Both analogues act synergistically with bc1-inhibitors against blood-stages in vitro, however cycloguanil antagonizes activity. Together, these data suggest that proguanil is a potent slow-acting anti-plasmodial agent, that bc1 is essential to parasite survival independent of dihydroorotate dehydrogenase-activity, that Malarone® is a triple-drug combination that includes antagonistic partners and that a cyclization-blocked proguanil may be a superior combination partner for bc1-inhibitors in vivo.

Partnering to fight malaria in India: Past, present and future.

The global fight against malaria requires continual development of new tools. Collaborations in India have played a key role in MMV's partnerships to discover, develop and deliver new medicines. Over the last decade, India has become a focal point of global medicinal chemistry, and combined with investments in basic science, this has led to the discovery of new potential drugs. India also brings significant experience to drug development, in clinical trials, but also in formulation and manufacturing. Finally, innovative new approaches in case management have streamlined impact at the level of communities and the patients.

Injectable anti-malarials revisited: discovery and development of new agents to protect against malaria.

Over the last 15 years, the majority of malaria drug discovery and development efforts have focused on new molecules and regimens to treat patients with uncomplicated or severe disease. In addition, a number of new molecular scaffolds have been discovered which block the replication of the parasite in the liver, offering the possibility of new tools for oral prophylaxis or chemoprotection, potentially with once-weekly dosing. However, an intervention which requires less frequent administration than this would be a key tool for the control and elimination of malaria. Recent progress in HIV drug discovery has shown that small molecules can be formulated for injections as native molecules or pro-drugs which provide protection for at least 2 months. Advances in antibody engineering offer an alternative approach whereby a single injection could potentially provide protection for several months. Building on earlier profiles for uncomplicated and severe malaria, a target product profile is proposed here for an injectable medicine providing long-term protection from this disease. As with all of such profiles, factors such as efficacy, cost, safety and tolerability are key, but with the changing disease landscape in Africa, new clinical and regulatory approaches are required to develop prophylactic/chemoprotective medicines. An overall framework for these approaches is suggested here.

Target Validation and Identification of Novel Boronate Inhibitors of the Plasmodium falciparum Proteasome.

The Plasmodium proteasome represents a potential antimalarial drug target for compounds with activity against multiple life cycle stages. We screened a library of human proteasome inhibitors (peptidyl boronic acids) and compared activities against purified P. falciparum and human 20S proteasomes. We chose four hits that potently inhibit parasite growth and show a range of selectivities for inhibition of the growth of P. falciparum compared with human cell lines. P. falciparum was selected for resistance in vitro to the clinically used proteasome inhibitor, bortezomib, and whole genome sequencing was applied to identify mutations in the proteasome ß5 subunit. Active site profiling revealed inhibitor features that enable retention of potent activity against the bortezomib-resistant line. Substrate profiling reveals P. falciparum 20S proteasome active site preferences that will inform attempts to design more selective inhibitors. This work provides a starting point for the identification of antimalarial drug leads that selectively target the P. falciparum proteasome.