The Development Process for Discovery and Clinical Advancement of

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Perspective

The development process for discovery and clinical advancement of modern antimalarials Trent D Ashton, Shane M. Devine, Joerg J. Moehrle, Benoît Laleu, Jeremy N. Burrows, Susan A. Charman, Darren John Creek, and Brad E. Sleebs J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00761 • Publication Date (Web): 06 Aug 2019 Downloaded from pubs.acs.org on August 8, 2019

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Journal of Medicinal Chemistry

The development process for discovery and clinical advancement of modern antimalarials Trent D. Ashton,†,‡,# Shane M. Devine,§,# Jörg J. Möhrle, Benoît Laleu, Jeremy N. Burrows, Susan A. Charman,§ Darren J. Creek,§,* Brad E. Sleebs.†,‡,*

†

The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia. ‡ Department

of Medical Biology, The University of Melbourne, Parkville, Victoria 3052, Australia.

§

Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria 3052, Australia.  Medicines

#These

for Malaria Venture, ICC, Route de Pré-Bois 20, 1215 Geneva, Switzerland

authors contributed equally.

*Author correspondence addressed to Brad. E. Sleebs and Darren J. Creek.

KEYWORDS: malaria, antimalarial, Plasmodium

ACS Paragon Plus Environment

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ABSTRACT

Malaria is a devastating disease caused by Plasmodium parasites resulting in approximately 435,000 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 towards 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 lifecycle 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.


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INTRODUCTION The World Health Organization (WHO) estimates that around 3.5 billion people, about half of the world’s population, live at risk of malaria.1 In 2018, there was an estimated 219 million cases of malaria and 435,000 deaths occurred globally with 92% of cases in Africa and 5% in South East Asia. Children under the age of 5 are most at risk and account for about 61% of deaths worldwide. Malaria in humans is caused by five Plasmodium species. P. falciparum (Pf) is the most prevalent species and accounts >99% of cases in Africa and 63% of cases in South East Asia, while P. vivax (Pv) is common in South East Asia and accounts for 72% of cases in the Americas.1 Pf is the most lethal of the Plasmodium species and is responsible for approximately 90% of all malaria related deaths globally. Pv is responsible for recrudescent infection via activation of dormant liverstage hypnozoites that re-establish the clinical blood-stage of infection. Although P. malariae and P. ovale may cause milder symptoms than Pf or Pv2, these parasites can be part of mixed species infections and as such, are usually not detected. Nevertheless, mixed populations of Plasmodium parasites are a significant public health threat.3 P. knowlesi is known to cause deaths in humans and is now considered the most common cause of malaria in Malaysia and is becoming increasingly widespread throughout South East Asia.4 Within the context of developing novel antimalarials to tackle elimination it is preferred for compounds to have activity against all five human-infecting Plasmodium species.5 The malaria parasite has a complex lifecycle with several morphogenesis events.6 For human infection to take place, an infected female Anopheles mosquito takes a blood meal and injects saliva filled with anticlotting agents and sporozoites. Sporozoites travel through the skin and into the blood stream, and finally reach the liver, where they invade hepatocytes and replicate over five


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to six days to produce schizonts filled with 10–30,000 merozoites. The merozoites egress from the schizonts into the bloodstream where they merozoites invade host erythrocytes. Here, the parasites self-replicate over multiple asexual stages, causing erythrocyte depletion, new merozoites are released into the blood, and sequestration of infected erythrocytes in blood vessels resulting in the symptomatic signs of malaria. On occasion, the parasite will commit to gametocytogenesis. Here the parasites sexually differentiate to produce mature male and female gametocytes. The mature gametocytes are then transmitted to the Anopheles mosquito via a blood meal and in the midgut immediately transform to male and female gametes. These forms of the parasite fuse to form a diploid zygote. These zygotes develop into motile ookinetes that invade and embed themselves in the basal lamina beneath the midgut forming an oocyst. The oocyst then divides by meiosis to produce many haploid sporozoites, which travel to the salivary glands where the parasites can be transmitted to another human host. The multifaceted lifecycle of the malaria parasite makes studying the parasite and designing new preventative measures and treatments a challenging task. There are two ways to combat malaria; prevention and treatment. Preventative measures to control the vector such as insecticide laced bed-nets have greatly reduced the incidence of infection, however this has not completely curtailed the spread of malaria.7 RTS,S/AS01 or Mosquirix is the first registered antimalarial vaccine targeting a circumsporozoite protein epitope in Pf, and although it only offers limited protection,8 it represents a significant advancement in the field. Several promising vaccines targeting different parasite protein epitopes are currently advancing through clinical trials,9 but the level of protection offered by these vaccines in a large diverse population is currently unknown. Chemo-prophylactic treatments are an important control measure, particularly for travelers to malaria endemic regions, and are also important in mitigating the spread of resistance. There are currently two classes of prophylaxis, suppressive and causal.


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Suppressive prophylactics such as chloroquine (CQ), mefloquine (MQ) (Figure 1), azithromycin and doxycycline are used as single agents and must be continually taken two to four weeks after the subject has left the endemic area, as the agents are only effective at treating the erythrocytic or asexual stage of the parasite lifecycle.10 Causal prophylactics, such as atovaquone-proguanil (Malarone), tafenoquine and primaquine (Figure 1), can stop being taken after several days of visiting an endemic area, because they target both asexual and liver stages.10 There are a number of products that are effective at curing malaria, most of which contain two or more drugs used in combination. The combination strategy was primarily implemented to deliver a cure and curb the emergence of resistance by ensuring that any resistant parasites emerging are killed by the partner drug within the combination. There are currently two types of combination therapies used, non-artemisinin combinations and artemisinin (ART) (Figure 1) combination therapies (ACTs).1 Non-ART combinations are comprised of sulfadoxinepyrimethamine in combination with either CQ, amodiaquine or MQ (Figure 1). However, in recent years the WHO stopped recommending the use of these combination therapies due to the emergence of resistance against these treatments.1


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Figure 1. Structures of currently used antimalarials and the ozonides, arterolane and artefenomel.

ACTs are now the only recommended frontline malaria treatment. The WHO currently recommends five ACTs for treatment of uncomplicated malaria and has strict compliance


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guidelines for the application of each of these therapies.1 Artemether and lumefantrine (Figure 1) (Coartem) is recommended for children but is not recommended for pregnant women due to limited safety data in this population. The combination therapy chlorproguanil-dapsone-artesunate was an encouraging ACT, but development ceased when the increased risk of hemolytic anemia in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency was discovered during the phase III program. Pyronaridine and artesunate (Figure 1) (Pyramax) is the only treatment recommended by a stringent regulatory authority for both Pv and Pf infections. The most recent data suggests ACTs have overall efficacy rates at day 28 greater than 99% outside the Greater Mekong sub-Region,11 but the current arsenal of clinically used ACTs are under threat due to the emergence of resistance. Resistance to ART, which is manifested as a decreased parasite clearance rate due to lower efficacy against resistant ring stage parasites, was first reported on the ThaiCambodian border in 200812 and as of 2017, ART resistance has been reported in four Greater Mekong sub-Region countries.1 Furthermore, resistance to all the partner antimalarials used in ACTs has also been extensively reported (reviewed in 13). To date, there have been only limited reports of resistance to ACTs in the clinic,14 but concerningly resistance to the front line therapy for malaria is progressively spreading. Compounding the issue of resistance, there has not been a new class of antimalarial registered since atovaquone at the turn of the century. Tafenoquine (Figure 1) was the most recent antimalarial to advance to registration.15 But rather than representing a new chemotype, tafenoquine is an iteration of primaquine (Figure 1); an 8-aminoquinoline developed in the 1940s.16 The recently introduced arterolane (OZ277) (Figure 1), and the structural analogue artefenomel (OZ439) (Figure 1) currently under late stage clinical assessment, possess the same endoperoxide architecture of ART.17-19 These peroxides are completely synthetic, and artefenomel


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has an extended half-life of over 2 days, compared to 1-2 h for ARTs.1, 20 Although artefenomel likely acts with a similar mode of action as ART, it still shows good activity against the same Kelch13-mutant resistance strains in vitro and in patients, and its longer half-life is an additional distinct differentiation17, 21, 22 (reviewed in 23). Common mechanism of action traits between many antimalarial clinical candidates highlights a problem with ‘me too’ chemotypes. Quinolines, phenanthrenes and antifolate chemotypes (Figure 1) are historically the mainstay of what could now be considered as ‘old world antimalarials’. The issues and limitations with current antimalarial chemo-prophylactics and treatments have been informative in constructing the current set of guidelines for new chemical entities entering the clinic.1, 5 The guidelines were created by multiple stakeholders including WHO and Medicines for Malaria Venture (MMV) to align new chemical entities with the global strategy of malaria elimination and eradication. The target product profile (TPP) encompasses the attributes of a combination therapy of two or more single agents. The TPPs are aligned with two underlying strategies: treatment and chemoprotection (Table 1) or more particularly, SERC (Single Exposure Radical Cure) and SEC (Single Exposure Chemoprotection) respectively. The Target Candidate Profiles (TCPs) for single agents, summarized in Table 1, are generally aligned with the stage of the parasite’s lifecycle. Other factors such as pharmacokinetics, parasite killing kinetics and drugdrug interactions are also considered when determining the inclusion of single agents in a combination therapy. The TPP and TCP terminologies will be referred to throughout this perspective.


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Table 1. Summary of antimalarial target product and target candidate profiles recommended by the Medicines for Malaria Venture.5

Target product profile

1

Target candidate profile

Parasite stage

Purpose

1

asexual

Treatment of acute malaria symptoms

3

liver hypnozoite (Pv)

Treatment of relapse

5

transmission (gametocytes)

Mass drug administration for treatment and transmission blocking

6

transmission (mosquito)

Chemoprevention in endemic areas

4

liver schizont

1

asexual

Treatment (SERC)

2 Chemoprotection (SEC)

Chemoprotection in endemic areas – particularly saving children’s lives in seasonal malaria chemoprevention or protecting vulnerable populations from outbreaks

Identifying new chemotypes with novel modes of action against different stages of the parasite lifecycle are now recognized as the defining traits of modern antimalarial drug discovery. Over the last twenty years there has been renewed support and funding from multiple philanthropic institutions and governments for antimalarial research and development. In particular, the public private partnership MMV has played a key role in leading, coordinating and mobilizing the development of novel antimalarial chemical entities. This has been achieved through collaborations involving MMV, industry and academia. Key early successes came from phenotypic based high throughput screening of vendor comprised and proprietary compound libraries of partners such as Novartis, GSK and St Jude Children’s Research Hospital. These


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screens were primarily conducted on the asexual stage of the parasite lifecycle,24-34 but since the advent of new parasite culturing techniques and assays, high throughput screens have also been undertaken on pre-erythrocytic and transmission stages of the lifecycle.35-40 Protein targets identified as essential to multiple stages of the malaria parasite by way of genetic studies were also screened against large compound collections (examples given in 41-47). Collectively, the initiative resulted in the discovery of thousands of unique starting points for antimalarial development. Many of these chemotypes are publicly disclosed and are now under investigation, while several compound classes developed by industry or academia or in partnership have now progressed to lead development phase or advanced to clinic trials (reviewed in 48). The most advanced of these antimalarials, KAE609, KAF156, DSM265 and MMV048 are currently in phase II clinical trials (Figure 2).

Figure 2. Structures of antimalarial clinical candidates that are the focus of this perspective.


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In this perspective, we highlight the development of the four most advanced modern antimalarials in clinical trials. The chemical starting points that ultimately led to KAE609, KAF156 and MMV048 were discovered through two independent high throughput phenotypic screens against the Pf asexual stage parasite,32, 49, 50 while the chemical starting point that led to DSM265 was identified from a target-based screen against Plasmodium dihydroorotate dehydrogenase (DHODH),51 an enzyme essential for pyrimidine biosynthesis and malaria parasite survival. The four antimalarial candidates potently inhibit the asexual stage parasite but possess differing activities against pre-erythrocytic and transmission stages of the parasites. KAF156, DSM265 and MMV048 are characterized as having plasma half-lives of several days and low clearance in keeping with the likelihood of a single dose cure or preventive therapy. Most importantly, each of these compounds have a novel mechanism of action and they are not susceptible to resistance mechanisms of existing antimalarial classes. Parasite lifecycle stage activities, parasite reduction rate (PRR), pharmacokinetic profiles, dose size, formulation and, critically, safety and tolerability are all factors that will ultimately determine the end use of these agents in a therapy. Herein, we describe in detail the identification and development of the four most promising modern antimalarials, with the goal to inform the research community of the malaria models required to characterize new chemical entities, but to also underscore the qualities desirable in a new antimalarial agent.


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KAE609 (NITD609, Cipargamin) Hit identification KAE609 was identified as part of a phenotypic high-throughput screen of ~12,000 natural products and natural product-like synthetic compounds.49 This screen identified 275 primary hits which displayed sub-micromolar growth inhibition of Pf parasites. Triaging these primary hits using a mammalian cellular cytotoxicity screening, with a cut-off of >50% viability at 10 μM, gave 17 hits. Of the 17 hits the singleton spiroazepineindole 1 displayed potent activity against wild type (NF54) and CQ resistant (K1) strains of Pf, with EC50s of 0.090 and 0.080 μM, respectively.52 The synthesis of compound 1 (Figure 3) gives a mixture of the four possible enantiomers, with the trans-diastereomers formed in favor of the cis-products in a ratio of ~9:1.52 The trans-isomers were resolved by chiral chromatography and compound 2 (the 1R,3S isomer of 1) potently inhibited NF54 (EC50 0.020 μM) and K1 (EC50 0.030 μM) parasite growth. The 1S,3R isomer 3 was inactive at 5.0 μM against both parasite lines (Figure 3). Compound 1 was also efficacious in a proof-of-concept Pb blood stage mouse model, demonstrating 96% clearance of parasitemia after a single 100 mg/kg dose.52 The spiroazepineindole 1 exhibited promising parasite efficacy, pharmacokinetic and physical properties suitable for hit-to-lead optimization.49

Figure 3. Hit compound and re-synthesized and resolved isomers.


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Lead identification and optimization Investigation of the structure activity relationship (SAR) began with exploration of halogen substituents on the indolinone ring. Replacement of the 5'-bromo substituent in 1 (Figure 3) with a chlorine atom (4) gave equipotent Pf parasite activity (NF54 EC50 0.084 μM).52 Expanding the seven-membered ring of (±)-4 to an eight-membered ring was detrimental to activity whereas potency was maintained against NF54 parasites when the 3-methyl substituent was absent from (±)-4 (Figure 4). The tetrahydro-β-carboline derivative (±)-5 displayed improved potency against parasites compared with (±)-4 with an EC50 of 0.027 μM. As with the original screening hit, the stereochemistry was crucial for antimalarial activity. The (1R,3S)-enantiomer 6 exhibited enhanced activity against NF54 parasites (EC50 0.009 μM) (Figure 4), while the (1S,3R)enantiomer was considerably less active (EC50 >5.0 μM).52 Additional halogen substituents on the indolinone moiety failed to improve parasite growth inhibitory activity.

Figure 4. Structures of spiroindoles in lead identification phase including clinical candidate KAE609.


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Variations made to the C3-substituent of (±)-5 were not well tolerated and typically resulted in >5-fold decreases in efficacy; deletion the 3-methyl substituent or 3,3-dimethyl substitution were detrimental modifications. Replacing the 3-methyl substituent of 6 with a trifluoromethyl group also decreased potency against Pf NF54 (EC50 of 0.054 μM).52 Extending the 3-methyl group to a propyl chain or oxidation of the methyl substituent also led to reduced Pf parasite activity. Methylation of N9 of (±)-5 was also tolerated but was ~2-fold less potent against Pf NF54 (EC50 0.017 μM). The lactam carbonyl was found to be necessary for potency as the reduced form of the spiro-lactam exhibited micromolar potency.52 The active 1R,3S enantiomer 6 displayed moderate clearance (CL 50 mL/min/kg) in mice (dosed at 5.4 mg/kg by i.v.) and also inhibited CYP2C9 (IC50 1.5 μM).52 The metabolic stability of the active 1R,3S enantiomer was addressed while investigating SAR around the indole motif of (±)-5 (Figure 4). Specifically, halogenation led to potency gains and improved pharmacokinetics, presumably by blocking metabolically susceptible sites. Inclusion of a 6-fluoro substituent (7) did not impact the inhibition of CYP2C9 (EC50 1.7 μM) but it did result in a modest improvement in potency against NF54 parasites (EC50 0.003 μM) and in vivo clearance in mice was markedly improved (CL 24 mL/min/kg). The addition of the 7-chloro moiety in 8 impacted potency in a similar manner (NF54, EC50 0.004 μM) and decreased CYP2C9 inhibition (IC50 4.1 μM). However, in isolation, the 7-chloro substituent of 8 proved unfavorable with respect to clearance in mice (CL 60 mL/min/kg). In combination (KAE609), these two additions resulted in slightly decreased CYP2C9 inhibition (IC50 5.4 μM), improved clearance in mice (CL 9.8 mL/min/kg) and sub-nanomolar potency against NF54 parasites (EC50 0.0009 μM). Similarly, the 6,7-difluoro analogue was also highly efficacious against NF54 parasites (EC50 0.0002 μM), displayed low


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clearance in mice (CL 8.5 mL/min/kg) and reduced inhibition of CYP2C9 (IC50 7.4 μM). Interestingly, the gains in potency achieved through the metabolic blocking strategy was replicated for the enantiomers. For example, the enantiomer of KAE609 displayed reasonable potency against the NF54 line (EC50 0.077 μM), low clearance in mice (CL 2.6 mL/min/kg) and no CYP2C9 inhibition at 10 μM.52 The changes incorporated in the spiroindolone scaffold culminated in the preclinical candidate, KAE609.49 After i.v. dosing to mice (5.4 mg/kg), KAE609 displayed moderate steady state volume of distribution (Vss 2.11 L/kg), good exposure (AUCinf 23.88 μM·h), moderate clearance (CL 9.75 mL/min/kg) and a half-life of 3.44 h. Upon oral administration at 24.6 mg/kg to mice, KAE609 was rapidly absorbed (Tmax 1 h), displayed good exposure (AUCinf 138.65 μM·h), a moderate halflife of 10.0 h and excellent oral bioavailability (F 100%). After an i.v. dose of 5.0 mg/kg and oral dose of 23.7 mg/kg to rats, exposure levels were high (AUCinf 61.32 and 524.15 μM·h, respectively), with a long half-life (T1/2 10.69 and 27.73 h for i.v. and p.o., respectively), low clearance (CL 3.48 mL/min/kg) and excellent oral bioavailability (F 100%).49 The PK profile of KAE609 suggested that once a day dosing was a suitable dosing regimen for malaria efficacy models.

Mechanism of action KAE609 displays moderate to slow-killing activity in vitro against asexual blood stages of Pf as measured in the PRR assay.49 The compound does not exhibit cross-resistance with any of the clinically-used antimalarials, suggesting that the spiroindolones act by a novel mechanism of


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action.49 Current evidence indicates that the primary target is the ATP-dependent Na+ channel, PfATP4 (Figure 5).49 The mechanisms of action and resistance were initially determined by generation of resistant parasites in vitro, which required 3–4 months of continuous exposure to sub-lethal concentrations of KAE609 to provide low levels of resistance, indicated by EC50 values 7- to 24-fold greater than the parental strain.49 Genomic analysis of six drug-resistant clones identified eleven nonsynonymous mutations in the Pfatp4 gene, and confirmation of the role of Pfatp4 in KAE609 susceptibility was provided by transgenic expression of two mutant Pfatp4 alleles (D1247Y or double-mutant I398F/P990R) (Figure 5) in a wild-type parasite, resulting in elevated EC50 values.49 In addition to providing a mechanism for resistance, it is proposed that PfATP4 is also the molecular target of KAE609. The Pfatp4 gene was annotated as a cation-transporting P-type ATPase and was previously thought to play a role in Ca2+ transport.53 However, more recent studies have confirmed a primary role in the maintenance of intra-parasitic Na+ levels, consistent with a function as an ATPdependent Na+/H+ antiporter.54 This transporter plays a critical role in parasite survival by pumping Na+ out of the intracellular parasite, in order to maintain a low intra-parasitic Na+ concentration in the context of high Na+ levels in the infected red blood cell (RBC) cytosol. Direct analysis of the impact of spiroindolones (KAE246 and KAE139) on Na+ homeostasis revealed a rapid and extensive increase in intracellular Na+ concentration immediately after drug addition, consistent with inhibition of PfATP4 function.54 Furthermore, baseline intracellular Na+ concentrations were moderately higher in Pfatp4-mutant KAE609-resistant parasites (11.9 and 14.8 M) compared to their parental wild-type strains (8.4 and 7.2 M), suggesting that resistance-associated mutations modify the Na+-transporting function of PfATP4. Importantly, the inhibition of Na+ homeostasis


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by the spiroindolone (KAE246) was significantly impaired in Pfatp4-mutant strains, consistent with a key role for PfATP4 in the mechanism of action of the spiroindolones.54 Whilst the spiroindolones were the first class of novel antimalarials shown to target PfATP4, a wide range of antimalarial chemotypes have subsequently been identified with the same resistance marker and phenotype. Sequencing of resistant strains generated by prolonged in vitro drug pressure has identified mutations in Pfatp4 associated with antimalarial dihydroisoquinolones (including the preclinical candidate SJ-733),55 aminopyrazoles (including GNF-Pf4492),56 pyrazolamides (including PA21A092),57 MMV77258 and two compounds from the MMV Malaria box.59 Furthermore, analysis of Na+ homeostasis revealed 28 chemically diverse compounds from the MMV Malaria Box,59 and 11 from the MMV Pathogen Box,60 that appear to inhibit PfATP4. The findings that so many structurally diverse compounds target PfATP4 raises the question of whether the mode of action involves direct inhibition of PfATP4, or whether PfATP4 is a secondary target downstream of a range of primary targets that regulate Na+ homeostasis. Unfortunately, to date it has not been possible to heterologously express PfATP4 in order to directly demonstrate drug binding. However, analyses of ATPase activity in parasite plasma membrane preparations have confirmed that KAE609 and a range of other PfATP4 inhibitors directly inhibit the Na+-dependent ATPase activity in membrane fractions,54, 61 thereby ruling out a potential role of cellular targets in other compartments that might modulate PfATP4 activity and confirming that these compounds act directly on PfATP4 itself, or on a membrane-associated interacting partner. In addition, the inhibition of PfATP4-associated ATPase activity in membrane fractions by KAE609 was attenuated in Pfatp4-mutant strains that demonstrated KAE609 resistance.61 These findings, and the extensive cross-resistance observed between different PfATP4-targeting molecules associated with various mutations in the Pfatp4 gene, strongly


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suggest that KAE609, and a range of other novel antiparasitic compounds, act by direct inhibition of PfATP4.49, 54, 55, 57, 58, 61

Figure 5. A homology model of PfATP4. Cartoon representation of PfATP4 showing mutations (magenta) that confer resistance to KAE609,49 and mutations orange that confer resistance to KAE678, a structurally related analogue of KAE609. Spiroindolones are cross resistant to parasites that possess these mutations.54 The previously described homology model of PfATP4 was recreated with SWISS-MODEL based on the crystal structure of the rabbit SERCA pump (PDB accession code: 2C8862).49 The predicted transmembrane region of PfATP4 is shown by the dotted line.


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Unfortunately, the inability to obtain purified PfATP4 protein (to date) has prevented direct measurement of drug-target interactions and structural analysis of the ligand binding site. A majority of the resistance-associated PfATP4 mutations occur in the transmembrane domain (Figure 5), suggesting that the binding site of KAE609 is likely within this transmembrane domain.49 A comparative genomics study investigating the mechanisms of action and resistance of KAE609 in modified Saccharomyces cerevisiae identified the primary target as ScPMA1, a Ptype ATPase proton pump with homology to Pfatp4.63 In silico docking studies for KAE609 in the yeast protein structure revealed a putative binding site containing resistance-associated mutations in a region with high homology to Pfatp4, thereby providing a hypothetical binding site for KAE609 (and dihydroisoquinolones) in PfATP4, although further work is required to determine the structure of PfATP4 in order to better understand drug binding. The mechanism of cell death that follows from inhibition of PfATP4 is not entirely clear. The elevated intraparasitic Na+ concentration that results from inhibition of Na+ extrusion could have direct effects on metabolic functions within the parasite, or could inhibit processes that rely on the plasma membrane electrochemical gradient (such as inorganic phosphate uptake), or could lead to osmotic imbalance and subsequent cell swelling or bursting.58 Metabolomic profiling of KAE609 and other PfATP4 inhibitors demonstrated significant inhibition of metabolic pathways involved in nucleotide, hemoglobin and central carbon metabolism.64,

65

Compound KAE609 has been

shown to dramatically inhibit protein translation within 1 h of treatment,49 and to induce accumulation of cholesterol in the parasite plasma membrane within 2 h.66 Morphological changes similar to merozoite formation were observed following KAE609 exposure,66 while other studies observed swelling, increased rigidity and membrane blebbing that may indicate eryptosis.55, 60, 67


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There are conflicting reports about whether or not PfATP4 inhibitors induce phosphatidylserine exposure, which is indicative of eryptosis.55, 67 However, multiple studies have demonstrated cell swelling and increased rigidity,55, 60, 67 which has been attributed to Na+-induced osmotic stress.60 These properties likely enhance the clearance of infected RBCs by the spleen,67 which may provide an explanation for the higher rate of parasite clearance observed in vivo compared to the rate of parasite killing measured in vitro.55

Antimalarial activity KAE609 has potent asexual Pf activity with EC50 values in the range of 0.0005–0.0014 μM for multiple strains.49 KAE609 also has similar potency against variety of multidrug resistant parasite lines, implying KAE609 is not susceptible to the same drug resistance mechanism as known drugs, such as CQ, MQ and ART (Figure 1). These results are consistent with PfATP4 inhibition as the mechanism of action for KAE609. KAE609 displays single digit nanomolar activity against Pv,49 however, P. knowlesi is less sensitive to KAE609 (Table 6).68 This phenomenon was also observed for other PfATP4 inhibitors.68 KAE609 possesses potent activity in asexual ring, trophozoite and schizont stages at low nanomolar (EC50 30 days and 100% cure rate (Table 6). Under the same dosage regimen, the reference drugs (ART, CQ and MQ) failed to cure infection. A three day 10, 30 or 50 mg/kg q.d. dosing regimen of KAE609, resulted in 50%, 90% and 100% cure rate of Pb infected mice.49 Collectively,


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the pharmacokinetic, parasite kinetic and stage specificity data suggested that KAE609 could be used as a fast acting partner agent in a SERC therapy (Table 1).

Synthesis During medicinal chemistry optimization, led by Thierry Diagana and Bryan Yeung at the Novartis Institute for Tropical Disease, the synthesis of KAE609 and analogues was conducted using a Pictet-Spengler reaction between a tryptamine derivative and an isatin building block (Scheme 2).52, 70 The diastereoselective nature of the Pictet-Spengler reaction allows for the synthesis of the active enantiomer of KAE609 from the enantiopure tryptamine 15. The synthesis of 15 began from accessing the ketone 14 by two pathways (Scheme 1). The first started with the addition of acetone to 5-chloro-6-fluoroisatin (9) using a catalytic Et2NH and K2CO3 to give 10 in high yield.71 Acetal protection of the ketone 10 afforded 11 which is then converted to 14 by Red-Al reduction and subsequent treatment with HCl. Alternatively, aldehyde 12 can be converted to the nitroalkene 13 by condensation with EtNO2 in the presence of NH4OAc.52 Hydrogenolysis of 13 using Pt/C and H2 afforded an intermediate oxime which is converted to 14 upon treatment with NaHSO3.71


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Scheme 1. Reagents and conditions: (a) Et2NH (10 mol%), K2CO3 (10 mol%), acetone, 56 °C, 2 h, 84%; (b) (EtO)3CH, p-TsOH·H2O, ethylene glycol, 40–50 °C, 2 h, 95%; (c) (i) Red-Al, THF, 3 h; (ii) 20% HCl, 20 min, 75% (over two steps); (d) EtNO2, NH4OAc, 100 °C, 4 h, 97%; (e) (i) 5% Pt/C, H2, EtOAc, 21 °C; (ii) NaHSO3, H2O, EtOH, 80 °C, 60% (over two steps).52, 71

Treating indole ketone 14 with an engineered transaminase ATA25672 and i-PrNH2·HCl as an amine source in the presence of P5P in triethanolamine buffer, adjusted to pH 7 using aqueous NaOH, afforded the chiral amine which was isolated as the (+)-CSA salt 15 (Scheme 2).71 Spiroindoline formation is then achieved by treating the amine salt 15 with 5-chloroisatin and Et3N. Addition of (+)-CSA was followed by solvent exchange to EtOH/EtOAc (1:14) to yield the spiroindole (+)-CSA salt 16 in high yield. Neutralization of 16 was affected using Na2CO3 in EtOH/H2O (3:1), followed by particle filtration and recrystallization affording KAE609 as a hemihydrate.


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Scheme 2. Reagents and conditions: (a) (i) i-PrNH2·HCl, ATA256, P5P, PEG-200, triethanolamine buffer pH 7, NaOH(aq), 50 °C, 24 h; (ii) (+)-CSA, i-PrOAc, 89 °C then cooled to 0 °C, 84% (over two steps); (b) (i) Et3N, 5-chloroisatin, i-PrOH, 83 °C, 24 h; (ii) (+)-CSA; (iii) solvent exchange to EtOH/EtOAc (1:14), 77 °C then cooled to 0 °C for 24 h, 86%; (c) (i) Na2CO3, EtOH/H2O (3/1), 58 °C; (ii) recrystallization, 91%.71

Preclinical assessment ADME profiling of 14C-labelled KAE609 in rats and dogs was undertaken as part of preclinical assessment.73 In the ADME study, KAE609 was the major component found in plasma and excreta after intravenous and oral administration. On intravenous dosing (5 mg/kg for rats; 1 mg/kg for dogs), KAE609 displayed low clearance (0.252 L/h/kg in rats and 0.201 L/h/kg in dogs), moderate volume distribution (3.18 L/kg in rats and 2.17 L/kg in dogs) and a moderate half-life (8.2 in rats and 10.9 h in dogs). On oral dosing, KAE609 (10 mg/kg for rats; 3 mg/kg for dogs) was slowly absorbed in rats (Cmax 1210 ng/mL observed at Tmax 5.3 h), but efficiently absorbed in dogs (Cmax 868 ng/mL observed at Tmax 1.7 h). The estimated oral bioavailability was 85% in rats and 68% in


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dogs. Combined with human in vitro metabolism data, it was predicted that KAE609 had a pharmacokinetic profile suitable for once a day dosing in humans. In ADME and safety studies in rats and dogs, KAE609 and several major metabolic products were primarily excreted in the feces. Of the metabolites identified, M18 was the major metabolite identified in plasma of rats and dogs (Figure 6). M17 and M19 were the major metabolites detected in rat and dog feces, respectively. The M17 metabolite was also the major metabolite formed during incubation of KAE609 in presence of human hepatocytes. Characterization of the metabolites revealed the structure of M17 was a result of a novel ring expansion biotransformation mediated by CYP3A4 (Figure 6).74 The CYP3A4 oxidative biotransformation is proposed to proceed via a C-C bond cleavage by a single electron transfer to produce an isocyanate intermediate. The isocyanate intermediate then undergoes nucleophilic attack of the tetrahydro carboline nitrogen affording the ring closure product M17. Subsequent oxidation by CYP3A4 and CYP1A2 produced metabolites M18 and M19, respectively. M19 was further hydroxylated to produce M20, which was also detected as a trace metabolite. The major metabolites were synthesized and were inactive against Pf in vitro, and importantly were non-toxic in human cellular assays.73


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Figure 6. The proposed mechanism for the formation of metabolite M17 and the metabolic pathway of major metabolites detected in humans and preclinical species after oral dosing of 14Clabelled KAE609.74

In vitro safety profiling established that KAE609 was non-toxic (CC50 >10 M) to a variety of human neural, renal, hepatic or monocytic cell lines.49 KAE609 was screened against a panel of high-risk human receptors and ion channels in a biochemical format and revealed KAE609 did not inhibit any of these targets at a therapeutically relevant concentration (IC50s all >30 M). Importantly, negligible inhibition of the hERG ion channel (IC50 ≥30 M) was observed by patch clamp and binding assay methods, indicating a low risk of cardiotoxicity. KAE609 was also nonmutagenic in an Ames assay suggesting a low risk of genotoxicity.49


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In exploratory toxicology studies, rats were dosed with KAE609 daily for 14 days and showed plasma exposure levels (AUC0-24h) after this period that were 10 to 20 times higher than the dose required to clear parasitemia in a Pb mouse model (ED99 5.3 mg/kg).49 Additionally, KAE609 was well tolerated with no adverse events recorded and normal histopathology. Further safety data in other preclinical species have not been reported. Collectively, the preclinical assessment established that KAE609 was well tolerated and has a safety profile suitable for dosing in humans.

Clinical trial progress. A phase I trial was undertaken to assess the pharmacokinetics, safety and tolerability of KAE609 in healthy individuals (Table 2).75 In the first component of the trial, a single dose of KAE609 was administered to cohorts at escalating doses from 1 mg to 300 mg. In part two of the phase I trial, three once-daily doses of KAE609 ranging from 10 to 150 mg were administered over three consecutive days to different study cohorts. In part one and part two trial arms, bioanalysis sampling occurred at multiple time-points to assess the pharmacokinetic profile over 96 and 144 h post-dosing. In the single dose study, the systemic exposure of KAE609 increased in a doseproportional manner. At a 300 mg single oral dose of KAE609, the Tmax was 8 h and with an average Cmax of approximately 2000 ng/mL. The systemic exposure of KAE609 (AUC0–24 h) was 36 μg·h/mL, the elimination half-life was approximately 24 h and the apparent clearance was 5.5 L/h. In part 2 of the trial, KAE609 dosed at 150 mg over 3 days, showed increased plasma exposure from day 1 (Cmax 1170 ng/mL; AUC0–24 h 15 μg·h/mL) to day 3 (Cmax 1770 ng/mL; AUC0–24 h 29.4 μg·h/mL). The extent of exposure was not affected by food intake. In both arms of the KAE609 trial, no serious adverse events were observed. Mild adverse events were observed in 99.7%. Approximately 85% of the dose (based on total radioactivity) was recovered in feces with less than 5% of the total dose in urine. The two major circulating metabolites were M18 and M19 (shown in Figure 6) comprising 8 and 12%, respectively of plasma radioactivity. Other trace metabolites (75 mg. Phase IIa – Uncomplicated Single dose determinat Pf infected range of 30 to 10 ion of MIC adults from mg. Vietnam

Primary measure: MIC up to day 8.

NCT0183 80 Secondary measures: parasite clearance up to 6458, 72 h and cure rate by qPCR at days 28, 25 and 42. Outcomes: Predicted median MIC: 0.126 ng/mL.


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Cure rate: 30 mg, 57% (n = 7); 20 mg, 15 mg 14% (n = 7); 50% (n = 4); 10 mg, 29% (n = 7). Phase IIa - Healthy adults 30 mg daily dose efficacy infected with for 3 days. either Pv or Pf asexual parasites

Primary measure: Parasite clearance up to day NCT0152 5. 4341, 81 Secondary measures: Tolerability and PK. Outcomes: 100% clearance for Pv and Pf. 12 h median Pf and Pv parasite clearance. No serious adverse events. Nausea in 67% of subjects. Half-life of KAE609 was 20.8 h.

Phase IIa – tolerability and efficacy

Uncomplicated Pf infected adults from multiple sites in Africa

Single dose and Primary measure: AST and ALT for liver NCT0333 multiple daily function. 4747 doses over 1 to 3 Secondary measures: Parasite clearance (12, days. 24 and 48 h post dose) and recrudescence (day 15 and 28). Outcome expected Sep 2019.

KAF156 (GNF156, Ganaplacide) Hit identification The Genomics Institute of the Novartis Research Foundation, via a HTS of 1.7 million compounds led by Prof. Elizabeth Winzeler, identified 6,000 molecules from >530 different scaffolds active against Pf 3D7 in a cell-based proliferation assay.32 The team led by Thierry Diagana and Arnab Chatterjee interrogated the data to identify a novel scaffold that had attractive features for further development including: a) potency (EC50 20-fold, toxicity panel against six cell lines) and c) synthetic tractability. One such scaffold was the imidazolopiperazine class, whereby GNF-Pf-5069, GNF-Pf-5179 and GNF-Pf-5466 (2123) were identified (Figure 7).84 The imidazolopiperazine class (2123) demonstrated good activity against Pf wildtype (3D7) and drug resistant (W2) strains and >20 fold selectivity window over the Huh7 cell line. Additionally, 21 was highly aqueous soluble (>175 M at pH 6.8) and did not


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have cytochrome P450-mediated effects associated with other hit compounds. The imidazolopiperazine chemotype is distinct from known antimalarial scaffolds and appeared an ideal class for optimization.

Figure 7. Hit compounds in the imidazolopiperazine class.

Lead identification and optimization The imidazolopiperazine scaffold, although considered an ideal starting point for medicinal chemistry, it was not without risk, including potential metabolic soft spots on the aryl groups and the presence of a toxicophoric methylenedioxy aryl motif. Compound 21 demonstrated low hERG susceptibility (IC50 19 M),84 low oral plasma exposure with Cmax 320 nM and AUC(0–5h) 972 h·nM,85 and low bioavailability when dosed orally in rodents.84 Optimization of the imidazolopiperazine class first aimed to enhance asexual parasite potency while addressing metabolic liabilities. This was achieved by investigating the amino acid moiety (A), the 2-phenyl moiety (B) and the aniline substituent (C) (Figure 8).


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Figure 8. Modifications to optimize the imidazolopiperazine core.

The first generation compounds designed by Wu et al84 demonstrated that substitution on the secondary amine (A-position, Figure 8) was necessary for antiparasitic activity. For example, the glycine derived 24 and dimethyl analogue 25 were the most potent analogues with EC50 values of 20 nM against 3D7 Pf when p-fluorophenyl groups were attached at the other key positions. Modifying the B-position (see Figure 8) to include the 3-fluorophenyl (26) group led to a 2-fold improvement in Pf activity (EC50 10 nM) (Figure 9). However, electron donating groups on the aryl ring-B (e.g. 4-methyl and 4-methoxy) were less advantageous demonstrating EC50 values >2000 nM. Many substituents were tolerated on the aryl ring-C, for example, the 3,4difluorophenyl (27) analogue inhibited parasite growth with an EC50 of 60 nM (Figure 9). But the most potent derivative was the 3-fluoro-4-chloro substituents (28) in combination with the Nglycine and 4-fluorophenyl groups at positions A and B, respectively, exhibiting an EC50 of 3 nM (Figure 9).


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Figure 9. Key SAR for the first generation imidazolopiperazine series with activity against NF54 parasites.

The SAR from this first series was fine-tuned to enhance the physicochemical properties when coupled with the incorporation of changes to the 5-, 6- and 8-positions of the piperazine ring. This was required, as without further modifications at these positions, potent molecules such as 26 resulted in reduced oral exposure and inferior in vivo efficacy. Another factor that was a prerequisite for piperazine modification was the identification of inactive metabolites (M29 and M30) after oxidation of the unsubstituted piperazine ring of a 3,4-difluoroaniline derivative (27) and relative chemical instability (Figure 10).86


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Figure 10. Major metabolites resulted from chemical instability and C8-oxidation of M29 leading to inactive compounds.86

Although activity gains were achieved with 5- and/or 6-position modification to the piperazine ring, the greatest benefit was seen when 8-substitution was examined. The addition of a methyl substituent at either the 5-position (31) or 6-position (32) of the piperazine ring resulted in reduced activity with EC50 values of 80 and 82 nM, respectively (Figure 11).86 Incorporation of a substituent at the 8-position resulted in abrogation of the unwanted oxidation profile by blocking the benzylic position and the chirality at this position was unimportant for activity. The gemdimethyl substituent was the most synthetically attractive as it avoided the incorporation of a stereogenic carbon and gave the most potent analogues. The combination of 8-dimethyl substituent on the piperazine ring, N-glycine substitution, 4-fluorophenyl group and 4-fluoroaniline produced the additive effect of KAF156 with an EC50 value of 10 nM against Pf 3D7 parasites. In general, the imidazolopiperazines were Lipinski rule-of-five compliant and highly soluble in aqueous media.


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Figure 11. Effect of methyl substitution on the imidazolopiperazine series.86

Key compounds in the series were evaluated for permeability in the Caco-2 and PAMPA assays.86 A range of permeability values exist across the series, with no apparent correlation between Caco-2, PAMPA and cLogP values. Compounds with cLogP values higher than 4.5 were required for high percentage absorbed values in the PAMPA assay. In these assays, KAF156 exhibited a 52% absorption by the PAMPA assay, and permeability coefficients of 1.26 (apical to basolateral) and 2.99 (basolateral to apical) × 10-6 cm/s in the Caco-2 cell assay. The introduction of the carbonyl group adjacent to the piperazine nitrogen thereby eliminating the basicity of the nitrogen resulted in improved microsomal stability in mouse and human liver microsomes. The in vitro hepatic extraction ratio for KAF156 was intermediate (0.48, 0.66 and 0.49 in mouse, rat and human liver microsomes, respectively). The PK profile of KAF156 following i.v. administration at 5 mg/kg to male Balb/c mice showed a high volume of distribution (Vd 10.2 L/kg), moderate clearance (49.2 mL/min/kg), short half-life (T1/2 2.0 h) and AUC0–24 h of 4119 h·nM. After oral administration at a dose of 20 mg/kg, the maximal concentration (Cmax) was 1538 nM at 1.0 h, the half-life was modest (T1/2 4.35 h), and there was good exposure (AUC0–24 h of 12,155 h·nM) and good oral bioavailability (F 74%).

Mechanism of action


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The mechanism of action of KAF156 has not been clearly defined. In vitro directed evolution studies using KAF156 produced resistant parasites with a range of mutations in the Pfcarl gene that demonstrated up to 700-fold resistance to KAF156.87, 88 The role of five Pfcarl mutations in drug resistance was confirmed using CRISPR-Cas9 gene editing to introduce the mutations into wild-type parasites.89 The PfCARL protein contains 7 transmembrane domains and is localised to the cis-Golgi region,89 and whilst the biological function of PfCARL has not been characterized, a homologous protein in yeast plays a role in protein folding.38 The critical role of Pfcarl mutations in KAF156 resistance raises the likelihood that PfCARL is the direct target of KAF156. However, other structurally unrelated compounds have also been shown to induce Pfcarl mutations following drug pressure, raising the possibility that PfCARL represents a non-specific resistance protein.89, 90

An alternative resistance selection strategy, aiming to allow selection of mutations that confer a fitness cost, identified mutations in two other resistance-associated genes, Pfact and Pfugt.91 These genes encode an acetyl-CoA transporter (Pfact) and a UDP-galactose transporter (Pfugt) that reside on the ER membrane of the parasite, and their role in drug resistance was confirmed by CRISPR-Cas9 gene editing. Mutations in the acetyl-CoA transporter were also identified in KAF156 resistant Pb selected by directed evolution in the murine in vivo model.91 Interestingly, cross-resistance was observed with the chemically unrelated compounds that were associated with Pfcarl-based cross-resistance, suggesting that all three proteins, PfCARL, PfACT and PfUGT, can mediate resistance to a selection of antimalarial compounds.91 It has been proposed that these drugs may exert their action in the ER/Golgi apparatus and that mutations in these three proteins may limit access of the drugs to this organelle, in a manner similar to the impact of Pfcrt mutations on accumulation of CQ in the digestive vacuole. However, direct evidence for this proposed


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mechanism of resistance is lacking, and further work is necessary to elucidate the mechanism of action of KAF156.

Antimalarial activity KAF156 displays potent Pf asexual activity against 3D7 (EC50 10 nM) and W2 (EC50 6 nM) strains (Table 6).86 The Pv asexual antimalarial activity was determined by an ex vivo schizont maturation assay utilizing field isolates from the Thai-Myanmar border and Indonesia (Papua). In this assay KAF156 displayed activity against Pv and Pf, with EC50 values of 5.5 and 12.6 nM respectively (Table 6).87 In addition to asexual activity, KAF156 demonstrates potent multistage activity with single digit nanomolar EC50 values against liver and gametocyte stages (Table 6).90 The 4-chloroanilino variant of KAF156 arrested P. yoelii exo-erythrocytic forms (EEFs).88 Early stage gametocytes treated with KAF156 demonstrated a substantial dose-dependent reduction in total numbers of mature gametocytes (stage V) after the induction of gametocytogenesis.87 When 5 nM of KAF156 was fed to mosquitoes in a SMFA, no oocysts were detected in the mosquito midgut. This evidence demonstrates KAF156 blocks pre-erythrocytic development and transmission to the mosquito and implies KAF156 is suitable for the TCP 1, 4 and 5 (Table 1). KAF156 was evaluated in a Pb mouse survival model treated orally one day post infection.86 At doses of 1 × 30, 1 × 100 or 3 × 30 mg/kg, the average parasitemia reduction was 99.5% at 16.3 d, 99.4% at 14.0 d and 99.8% at 17.7 d survival (untreated control mice survive 6–7 days), respectively (Table 6). The effective dose giving 99% reduction in parasitemia (ED99) was 2.2 mg/kg. These results were more favorable than for the known antimalarials; CQ and artesunate.


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After i.v. dosing to rats, KAF156, showed a high volume of distribution (Vd 13.7 L/kg), high clearance (67.5 mL/min/kg), moderate half-life (T1/2 4.6 h) and reduced AUC0–24 h of 1831 h·nM. The maximal concentration (Cmax) was 91 nM at 1.5 h, half-life (T1/2 4.7 h), AUC0–24 h of 974 h·nM and oral bioavailability (F 20%) at 10 mg/kg, whereas these values increased to Cmax 580 nM at 4.3 h, half-life (T1/2 8.4 h), AUC0–24 h of 34,885 h·nM and oral bioavailability (F 57%) at a dose of 100 mg/kg.

Synthesis The synthesis of KAF156 begins with the base-mediated alkylation of Cbz-protected 2-amino2-methylpropionic acid 33 with 4-fluorophenacyl bromide (34) to give ester 35 (Scheme 3).86 Condensation of 35 with NH4OAc affords the imidazole 36 in good yield. The imidazole 36 was then alkylated using ethyl 2-bromoacetate under basic conditions, followed by hydrogenolysis of the benzyl carbamate to give lactam 37 in high yield. Reduction of the lactam and HATU-mediated coupling with N-Boc-glycine furnished imidazolopiperazine 38. Treatment of 38 with Br2 gives quantitative conversion to bromide 39 which can then be converted to KAF156 using a BuchwaldHartwig amination with 4-fluoroaniline and Boc-deprotection.


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Scheme 3. Reagents and conditions: (a) K2CO3, DMF, 25 °C, 4 h, 84%; (b) NH4OAc, toluene, 111 °C, 3 h, 88%; (c) (i) ethyl 2-bromoacetate, K2CO3, DMF, 25 °C, 2.5 h, 83%; (ii) H2, Pd/C, EtOH, 25 °C, 3 d, 91%; (d) (i) BH3–THF, THF, 66 °C, 16 h, 95%; (ii) 2-(tertbutoxycarbonylamino)acetic acid, HATU, DIPEA, CH2Cl2, 25 °C, 8 h, 70%; (e) Br2, CH2Cl2, 25 °C, 30 min, 100%; (f) (i) 4-fluoroaniline, Pd2(dba)3, xantphos, Cs2CO3, 1,4-dioxane, 25 °C, 15 min, then 120 °C, 8 h, 89%; (ii) TFA, CH2Cl2, 25 °C, 2 h, 52%.86

Preclinical assessment KAF156 was assessed for in vitro toxicology as a component of preclinical evaluation.86 KAF156 was non-cytotoxic in a panel of hematopoietic, epithelial, laryngeal, hepatoma human and mammalian cell lines at physiologically relevant concentrations (CC50 >12 M). KAF156 had IC50 values >6 M against a panel of cytochrome P450s and panel screening against high risk adverse


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targets revealed KAF156 inhibited hERG with an IC50 value of 13.4 M, 170-fold above the efficacious plasma concentration (Cmax 75 nM) of KAF156 dosed (2 mg/kg) in a mouse model of malaria, suggesting a low risk of cardiotoxicity. In an Ames and a micronucleus assay KAF156 was negative suggesting a low risk of genotoxicity in humans. ADME profiling of

14C-

and

13C-labelled

KAF156 in rats was undertaken as part of the

preclinical assessment studies.92 The primary goal was to pharmacokinetically characterize KAF156 and identify metabolites that may display toxicity. In this study, KAF156 was dosed orally and intravenously to rats at 10 and 3 mg/kg, respectively. After i.v. dosing, KAF156 exhibited a half-life of 6.6 h, high plasma clearance (CL 5.4 L/h/kg) and a very high volume of distribution (Vss 29.5 L/kg). After oral dosing, KAF156 was absorbed efficiently with a Cmax of 139 ng/mL at 2 h and displayed a half-life of 5.7 h with an estimated oral bioavailability of 48%. After both i.v. and oral dosing to rats, KAF156 was extensively metabolized and metabolites were excreted in urine and feces. Three major polar metabolites of KAF156, (including M41) (Figure 12), and several minor metabolites (including M40) were detected in rat urine, accounting for 24– 37% of the intravenous or oral dose. While five major metabolites, (including M40) were detected in feces. The characterized metabolites revealed KAF156 was oxidatively metabolized, underwent oxidative defluorination, oxidative deamination, hydroxylation and ring opening, and acetylation. O-Sulfonation by sulfotransferases gave further byproducts of these metabolites. The M40 metabolite, present in both urine and feces, was also a major product formed upon incubation of KAF156 with rat or human hepatocytes. The ketoamide metabolite M40 was not a result of an oxidative transformation with any of the 19 CYP450 isoforms, therefore it was proposed that KAF156 is metabolized to the ring opened product, M40, by way of two hydrolytic events. These metabolism events were proposed to proceed via hydrolytic cleavage of the imidazole ring


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followed by amide hydrolysis to form M41 and 4-fluoroaniline which is subsequently hydroxylated, O-sulfonated, acetylated and oxidatively defluorinated, producing several metabolites (M42–M45) (Figure 12). Overall, the in vitro toxicology data and dosing in rodent models suggested that KAF156 was well tolerated. Limited toxicology data in preclinical species was published at the time of writing this review, however KAF156 was advanced to human trials.


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Figure 12. The proposed mechanism for the formation of metabolite M40 and the metabolic pathway of major metabolites detected in rats after oral and intravenous dosing of

14C-labelled

KAF156.92

Clinical trial progress. A phase I trial was undertaken to determine the safety, tolerability and pharmacokinetics of KAF156 (Table 3).93 In this trial, KAF156 was studied in two arms, the first was a single-ascending dose from 10 to 1200 mg; the second arm included multiple ascending doses of 60 to 600 mg once daily for 3 days. KAF156 was well tolerated in both single and multiple dose cohorts, with no serious adverse events observed. The most common adverse events reported were headache, nausea and dizziness. Gastrointestinal disorders appeared to be more frequent with increasing dose of KAF156. No liver-related issues nor significant cardiac QTc prolongations were detected. A single dose of KAF156 was quickly absorbed with a Tmax between 1 and 6 h. The systemic circulation of KAF156 was dose-dependent, with Cmax values ranging from 7.6 to 2720 ng/L, and AUC0–24h ranging from 83 to 31,000 mg·h/mL for 10 to 1200 mg doses respectively. The apparent clearance decreased from 35 to 16 L/h and the volume of distribution decreased from 2,410 to 1,610 L with an increase in dose from 10 to 1200 mg, suggesting possible saturation of metabolism enzymes. The half-life of the KAF156 ranged from 47 to 70 h for 10 to 1200 mg doses. The absorption profile of KAF156 was not significantly affected when administered with food compared to fasted, although the Tmax was extended to 6 h with food, compared to 3 h fasted. After multiple doses of KAF156, the systemic exposure on day 3 increased more than the increase in dose.93 In summary, the study demonstrated that KAF156 well tolerated and suitable for single or


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multiple dose administration, although a pharmacodynamic study was required to determine the effective dose and dosing regimen necessary to treat humans infected with Plasmodium. PPQ (Figure 1) was considered as a potential antimalarial combination partner of KAF156, and therefore a phase I clinical trial was performed to determine drug-drug interactions between KAF156 and PPQ in healthy individuals (Table 3). Both PPQ and KAF156 are substrates and inhibitors of CYP3A4. Leong et al. report that both KAF156 and PPQ inhibit CYP3A4 with a Ki of 156 and 90 nM, respectively94 (although this is inconsistent with previous reports that KAF156 does not inhibit CYP3A486). Thus, it was important to compare the pharmacokinetic and safety profile in healthy individuals of the two antimalarials dosed as single agents and in combination.94 The pharmacokinetic profile for KAF156 (800 mg dose) as a single agent was consistent with the previous clinic data75 and this was relatively unchanged when it was co-administered with PPQ (800 mg and 1280 mg dose for KAF156 and PPQ, respectively). However, the Cmax of both KAF156 and PPQ increased 1.2- and 1.7-fold respectively, compared to the Cmax when either was dosed alone. The reason for the change in the absorption is unknown. KAF156 and PPQ either alone or in combination were well tolerated and no serious adverse events were recorded. The frequency of adverse events was not significantly different between the PPQ alone and KAF156 and PPQ combination cohorts. Consistent with the previous clinical trial for KAF156, the most common side effects reported were nausea and headache. Importantly, there was no evidence of a synergistic increase in QT interval compared to the QT interval when PPQ was dosed alone. In summary, the trial demonstrated there were no safety issues or drug-drug indications that would prevent the use of KAF156 and PPQ in a combination therapy for treatment of malaria. To determine the antimalarial efficacy of KAF156 in humans a phase II trial was undertaken across sites in South East Asia (NCT01753323) (Table 3).95 The trial was conducted in two parts.


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The first treated adults infected with either Pf or Pv with multiple doses of KAF156 (400 mg once daily for 3 days), and in the second cohort, adults infected with Pf were treated with a single 800 mg dose of KAF156. The half-life of KAF156 in malaria infected patients in this trial was approximately 44 h, consistent with the long half-life in earlier clinical trials.93, 94 KAF156 was generally well tolerated and there were no serious adverse events. Approximately 23% of all patients experienced a drug-related increase in aspartate aminotransferase levels – a key indicator of liver dysfunction. Other adverse events reported were likely related to malaria infection and were not directly attributed to KAF156. In this phase II trial, KAF156 cleared Pf infection efficiently, with median parasite clearance times of 45 h for a multiple dose regime and 49 h for treatment with a single dose of. Clearance of Pv with a multiple dose regime of KAF156 was significantly quicker than for Pf, with a median parasite clearance time of 24 h. The median parasite clearance half-life (calculated using the Worldwide Antimalarial Resistance Network parasite clearance estimator82) was between 1.9 h for Pv and approximately 3.5 h for Pf. In comparison, this is a slightly slower clearance rate than seen with ART derivatives, but significantly slower than for KAE609.81 Of the patients treated with a single dose of KAF156, 67% were cured at day 28 (blood smear), while 33% had recrudescent infections. Gametocytes were detected in independent Pf and Pv infections of 4 patients. Gametocytes were cleared by 16 h for Pv and between 54- and 74-h post-treatment for Pf consistent with in vitro gametocytocidal activity of KAF15687 and suggesting a possible use for KAF156 in a transmission blocking therapy (Table 1). Of the patients infected with Pf in the phase II trial, 14 patients had parasites with singlenucleotide polymorphisms (SNPs) in the K13 gene, 10 of which possessed the C580Y variant, strongly associated with ART resistance.96,

97

In infections with or without this mutation the


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parasite clearance rates were the same, suggesting KAF156 is not susceptible to the same resistance mechanism as ART. Patients infected with Pf parasites harboring mutations in the PfMDR and PfCRT genes associated with aminoquinoline resistance,98,

99

also responded to

treatment with KAF156 equally as well compared to patients infected with Pf without these genetic perturbations. Of the 31 patients infected with Pf in the trial, 28 isolates had either one of eight distinct SNPs or one of three insertions or deletions in the Pfcarl gene. None of the mutations detected were the same as the previously identified mutations associated with acquired resistance to KAF156 in the laboratory.87 There was no discernible difference between the clearance rates of infections or recrudescent parasites with the different Pfcarl genotypes, nor compared to isolates with the wild-type Pfcarl gene. Overall, KAF156 dosed three times in three consecutive days was effective in clearing both Pf and Pv in a small cohort of adults. Presently, a phase IIb clinical trial (NCT03167242) of KAF156 has commenced100 across multiple sites in Africa and South East Asia, to determine the efficacy KAF156 in combination with a solid-dispersion formulation of lumefantrine to treat adults and children infected with uncomplicated Pf malaria (Table 3). According to a 2018 report by the trial partner organization, MMV, “Interim analysis on 261 patients is promising and shows no safety signal; more than 90% of subjects show no recrudescence at 45 days”.101 The complete outcome of the trial is expected around April 2020.


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Table 3. Overview of KAF156 clinical trials completed or in progress. Phase/ Study

Study Dose population

Endpoint and outcome

Reference

Phase Ia - Healthy PK and adults tolerability

Arm 1: single Primary measure: PK dose from 10 Secondary measure: Tolerability to 1200 mg; Outcomes: half-life: 43–72 h. AUC0–24h: 83–31,000 Arm 2: 60 to mg·h/mL; T : 1–6 h; C : 7.6–2720 ng/L. max max 600 mg daily dose for 3 No serious adverse events, few mild adverse symptoms. days.

93

Phase Ia - Healthy tolerability adults with partner agent

single dose for Arm 1: 800 mg KAF156 and 1280 mg PPQ;

94

Primary measures: PK and tolerability up to 61 days. Secondary measure: Cardiotoxicity up to 48 h. Outcomes: KAF156/PPQ combination did not alter AUC.

Arm 2: 800 KAF156 did not affect the QT parameters of PPQ. mg KAF156; Frequency of mild adverse events: 87.5, 79.2 and Arm 3: 1280 58.3% for KAF156 + PPQ, PPQ and KAF156. mg PPQ. Phase IIa

Phase IIb Efficacy and tolerability with partner agent

Pv or Pf Arm 1: Daily monodoses of 400 infected mg for 3 days. adults Arm 2: Single from dose of 800 Vietnam mg. and Thailand

Primary measures: Arm 1: Pf and Pv parasite NCT01753 clearance at day 5; Arm 2: Pf parasitemia at day 28. 323, 95

Infected patients

Primary measure: Parasite clearance at day 28

Part A: >12 yrs age; Part B: >2 to 5,000-fold selectivity compared to the human form of the enzyme (HsDHODH, IC50 > 200 μM).51 It was noted that 46 was 5-fold less active against PbDHODH (IC50 0.23 μM) compared to PfDHODH. The decreased activity against PbDHODH was a trend throughout the development of DSM265 and would impact the evaluation of the series in Pb mouse models. Nevertheless, the PfDHODH activity of 46 robustly correlated with Pf 3D7 parasite activity (EC50 0.079 μM) 51 and therefore throughout the development of the DSM series, promising candidates were evaluated in P. falciparum humanized mouse models.


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Figure 13. Hit identified from HT screen against PfDHODH.

Lead identification and optimization Early structure activity studies focused on variation of the N7-(naphth-2-yl) substituent of 46 and preference for larger non-polar aromatic rings, such as an anthracen-2-yl substituent (47, Figure 14) was established (PfDHODH IC50 0.056 μM, 3D7 EC50 0.19 μM).51 Activity against PfDHODH was decreased when an endocyclic nitrogen was substituted at any position of the naphthyl ring, and indicated that the naphthyl ring was sensitive to modification.51 The 5,6,7,8-tetrahydronaphth2-yl analogue 48 had promising activity against Pf and PbDHODH (IC50 0.035 and 0.19 μM, respectively) and inhibited Pf parasite growth with an EC50 of 0.16 μM.103 On the other hand, the 1,2,3,4-tetrahydronaphth-2-yl analogue 49 had markedly reduced PfDHODH inhibition (IC50 0.54 μM) and micromolar potency against the Pb orthologue and in parasites.103 Interestingly, the aniline derived 50 (Figure 15) showed no activity against the Pf enzyme or parasites (>100 μM).


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Figure 14. Structures of PfDHODH inhibitors in lead identification phase.

Early in the program it was established that additional substitution of the exo-cyclic amine51 or C6 were not favorable for PfDHODH inhibition and efficacy. In general, replacing the 5-methyl substituent was detrimental for either PfDHODH or Pf3D7 activity, with only a single example of a 5-chloro group exhibiting favorable PfDHODH inhibition.103 Despite the promising inhibition of PbDHODH by the original hit compound 46 and 47, these compounds demonstrated a lack of efficacy in the Pb mouse model.104 This was likely a result of poor exposure of both compounds based on data in parallel studies in healthy mice. Even though 47 displayed higher plasma concentrations that were maintained longer than for 46, exposure was insufficient given the poor potency of 47 against PbDHODH (3.7 μM) (Figure 14). After multiple days of dosing, the observed Cmax values for 46 and 47 were lower than after a single day treatment which indicates that these compounds were likely metabolic inducers. In addition, 46 exhibited poor pharmacokinetics with high intrinsic clearance (CLint 96 μL/min/mg) in human liver microsome preparations and the estimated hepatic extraction ratio was high (Eh 0.84). Given the perceived liabilities of the naphthyl moiety,104 phenyl substituted analogues were prepared with the aim of investigating electronic and steric requirements which could improve the activity of 50.


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The structure activity relationship of mono-substituted N7-phenyl rings showed a clear pattern of activity associated with the substituent position. Substitution at the ortho-position (relative to the nitrogen) was not tolerated and in most cases resulted in no enzyme activity (IC50 > 100 μM).104 In isolation, meta-substitution gave PfDHODH IC50s in the range of 1.4–14 μM, bis-metasubstitution was also largely inactive.104 Analogues with para-substitution displayed good activity against Pf and Pb DHODH. Substituents such as bromine104 and linear aliphatic groups up to 5 carbons in length (≤ n-pentyl) produced sub micromolar inhibitors of PfDHODH.103 The 4-tert-butylphenyl derivative 51 (Figure 15) was the most active in the aliphatic series with Pf and Pb DHODH inhibition of 0.08 and 0.40 μM, respectively and modest efficacy against 3D7 parasites (EC50 0.32 μM).103 Inclusion of polar functional groups such as ether or amine led to profound decreases in activity.103


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Figure 15. Structures of PfDHODH inhibitors in lead identification phase. * denotes the use of albumax supplemented media.

The 4-(trifluoromethyl)aniline analogue 52 (Figure 15) was identified in this series with activity against PfDHODH (IC50 of 0.28 μM) and similar potency in the 3D7 growth inhibition assay with an EC50 of 0.34 μM.103 Importantly, 52 inhibition of PbDHODH (IC50 0.38 μM) was comparable to that for Pf, and studies in the Pb mouse model provided the first in vivo evidence that inhibition of plasmodial DHODH led to a reduction in parasitemia.104 The para-trifluoromethyl compound 52 exhibited improved metabolic stability in the presence of human liver microsomes (CLint 7.5 μL/min/mg) compared with 46.104 Additionally, 52 had a LogD7.4 of 2.5, modest aqueous solubility (21–43 μM) while human plasma protein binding (hPPB) was relatively low 86.9%.103 In general, combinations of 3,4-disubstitution on the aryl ring were less active against PfDHODH than the parent derivatives with an exception being the presence of 3-fluoro substituents adjacent to an activity-inducing para-substituent.26, 104, 105 For example, the 3-fluoro4-(trifluoromethyl)aniline derived analogue 53 (Figure 15) demonstrated improved inhibition of PfDHODH (IC50 0.077 μM) compared to 52. However, 53 had suboptimal 3D7 parasite activity (EC50 of 1.3 μM).104 With two fluorine atoms adjacent to the trifluoromethyl moiety in 54 (Figure 15), Pf and PbDHODH inhibition (IC50 0.19 and 0.28 μM, respectively) and Pf parasite activity (EC50 1.1 μM) was modest.103 The human protein binding of 54 was high (~97%) and it was proposed that high serum binding in the in vitro assay negatively impacted parasite activity. When the whole cell parasite assay was conducted using albumax supplemented media (lower protein


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concentration), the potency of 54 against Pf 3D7 (EC50 0.22 μM) was reflective of PfDHODH inhibition. Species selectivity was adversely impacted by the inclusion of additional fluorine substituents which was a potential obstacle for future preclinical toxicological studies.105 In the case of 54, rat and mouse DHODH were inhibited with IC50 values of 37 and >30 μM, respectively.105 In contrast, 52, was inactive against rat and mouse DHODH (IC50 >100 μM). A systematic investigation illustrated a trend between increasing fluorine substitution and off-species DHODH inhibition. In some instances low micromolar inhibition of human DHODH was demonstrated and could be ascribed, in part, to close contacts between the fluorine atoms and hydrophobic side chains of HsDHODH.105 The preference for hydrophobic moieties was further exploited with the inclusion of a 4pentafluorosulfanyl group (55) (Figure 15).103 This analogue was the most active of the monosubstituted N7-phenyl analogues with good activity against Pf and Pb DHODH (IC50 0.13 and 0.28 μM, respectively). 55 also had high protein binding and therefore the 3D7 growth inhibition (EC50 1.3 μM) did not correlate with enzyme inhibition unless serum was replaced with albumax supplemented media (EC50 0.18 μM).103 The aqueous solubility at pH 6.5 of 55 was also improved (71–142 μM) in comparison to trifluoromethyl anilino-derived analogues. In mice, a single oral dose of 20 mg/kg of 55 resulted in high plasma exposure (Cmax 12 μM) with plasma levels maintained above 1 μM for 19 h. This was similar in rats where a single oral dose (20 mg/kg) resulted in high plasma exposure (Cmax 31 μM), a long half-life of 33 h and excellent bioavailability (F 100%). In a Pb mouse model using ascending doses from 3 to 100 mg/kg once daily, 55 was efficacious with an ED50 of 17 mg/kg.


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The C2 position on the triazolopyrimidine scaffolds of 52 and 55 (Figure 15) were further explored with the aim to exploit further binding interactions with PfDHODH.26 Aliphatic substituents which were less than three carbons (or atoms) in length were tolerated in the C2 position of the scaffold. Short alkoxy groups and methylsulfanyl substitution were well tolerated in the 2-position resulting in potent inhibitors of PfDHODH (IC50 24 h and good oral bioavailability (F 61%).26


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Mechanism of action The mechanism of action of DSM265 is based on direct inhibition of the Plasmodium DHODH enzyme, and the resulting inhibition of de novo synthesis of essential pyrimidine nucleotides. Binding of DSM265 to PfDHODH has been directly demonstrated by X-ray crystallography (Figure 16),106 and direct inhibition of PfDHODH enzyme activity has been demonstrated in vitro for a large number of DSM265 analogues, with a high correlation observed between the in vitro potency against the enzyme and the antiparasitic activity against Pf.26, 102 Genetic confirmation of the role of DHODH in the mechanism of action of DSM265 was achieved by the use of a parasite line that over-expressed a yeast isoform of DHODH that was highly resistant to DSM265.26 Unlike the plasmodium enzyme, the yeast DHODH (yDHODH) is not dependent on mitochondriallyderived ubiquinone for regeneration of the FMN cofactor, and therefore can also induce resistance to mitochondrial inhibitors such as atovaquone (Figure 1).107 However, parasites expressing yDHODH become sensitive to mitochondrial inhibitors (e.g. atovaquone) when tested in combination with proguanil (Figure 1), whereas these parasites remained resistant to DSM265 even in the presence of proguanil, indicating that DSM265 targets the DHODH enzyme itself, rather than indirectly inhibiting the function of DHODH via a mitochondrial target.26


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Figure 16. Crystal structure of Pf dihydroorotate dehydrogenase bound to DSM265 (green), orotate and FMN (magenta) (PDB accession code: 4RX0).106 A. Cartoon representation of PfDHODH showing mutations (orange) that confer resistance to DSM265 (G181C only)106 and structurally related analogues of DSM265.108 The alpha-helix 181–193 (center-front) is transparent for clarity. B. Amino acids (blue) in the binding site of DSM265. Yellow dotted lines show hydrogen bonds between DSM265 and PfDHODH.

In vitro exposure of Pf to DSM265 readily generated resistant parasites that displayed gene amplification and/or point mutations in Pfdhodh. Phillips et al. reported copy number amplification along with a non-synonymous polymorphism at G181C (Figure 16), which lies within the binding pocket for DSM265.106 A more recent study generated resistant clones associated with 5 different Pfdhodh mutations. Four of these mutations involved amino acids within the DSM265 binding pocket (Figure 16), whereas one resistance-conferring mutation, C276F, was located in the adjacent FMN-binding site.109 CRISPR-Cas9 gene editing confirmed that the C276F mutation was sufficient to induce resistance to DSM265, and X-ray structural


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analysis of this mutant enzyme revealed conformational changes that affected the DSM265binding pocket. It is proposed that inhibition of DHODH would lead to depletion of pyrimidine nucleotides, and subsequently prevent the formation of RNA and DNA that are necessary for cellular transcription and replication. Indeed, the activity of DSM265 within the asexual blood-stage parasite is greatest against early trophozoite and schizont stages when these processes are most active.106 Functional confirmation of the role of pyrimidine synthesis in the mechanism of DSM265 action has been demonstrated by metabolic profiling of drug-treated Pf, whereby levels of the DHODH substrate, dihydroorotate, and its upstream precursor, N-carbamoyl L-aspartate, are significantly elevated, along with decreases in the levels of downstream pyrimidine nucleotides.64 Stable isotope labelling of parasites treated with DSM265 analogues confirmed the inhibition of metabolic flux through the de novo pyrimidine synthesis pathway at the point of DHODH, and untargeted metabolomics revealed that the impact on the cellular metabolome was highly specific to this pathway.65 The species-specific potency of DSM265 against Pb and Pv is not fully understood. It has been hypothesized that the weak activity against Pv and Pb may be due to the tropism of these parasites for immature red blood cells (reticulocytes) that may be able to supply the necessary pyrimidine nucleotides to the parasite in the absence of de novo pyrimidine synthesis.110 However, whilst reticulocytes have been shown to be rich in nutrients, including pyrimidines, genetic studies have shown that the enzymes of de novo pyrimidine synthesis appear to be essential in blood-stage Pb,110 suggesting that these parasites would not be able to survive potent inhibition of DHODH. It has therefore been proposed that species-specific differences in drug-enzyme interactions contribute to the different antiparasitic potencies observed for different Plasmodium species.


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Indeed, the DSM265 IC50 for enzyme inhibition of PbDHODH is about 300-fold higher than for the Pf enzyme, whereas inhibition of the Pv enzyme is only about 3-fold weaker than Pf.26, 111 The 3-fold difference in enzyme IC50 for DSM265 does not completely explain the 5-fold higher EC50 for Pv compared to Pf in the whole parasite maturation assay.111 This raises the possibility that the physicochemical properties of the drug may play an additional role in determining potency in different parasite species.111

Structural biology X-ray crystal structures of PfDHODH bound to DSM265 (Figure 16, PDB accession code: 4RX0) reveal important information of the interactions present upon binding, justification for the observed SAR and trends in species specificity.106 All DHODH structures with DSM ligands bound contain the FMN cofactor bound in the oxidized form with an adjacent molecule of orotate. An additional binding pocket, known as the species-selective inhibitor site, extends from the bound FMN cofactor to the N-terminal α-helix and is near or overlapping with the binding site for CoQ which is required for the regeneration of FMN. Sequence homology across species is well conserved for the FMN and dihydroorotate binding sites, but sequence variability within the inhibitor binding site is responsible for species selective inhibition. In the bound state DSM265 exists as a tautomeric form, resulting in a temporary dipole and electrostatic interactions with the protein. The Arg265 forms a hydrogen bonding interaction with N4 of the pyrimidine ring while the exo-cyclic N-H projects towards His185 making a hydrogen bond. The proximity of the histidine side chain to DSM265 occludes space for any further substitution of N7 and the replacement of N1, which is reflected in the SAR of early DSM


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analogues where the addition of N7-methyl abolished PfDHODH inhibition.51 Similarly, there is limited space in the binding pocket to accommodate substituents larger than a chloro or methyl substituent in the 5-position.103 Inclusion of substituents at C6 was also detrimental to activity.103 The remainder of the DHODH inhibitor site is hydrophobic which is exploited by the inclusion of the C2-, N7-substituents. A small tunnel extends from the species selective pocket and runs towards the FMN cofactor and is composed of Ile263, Ile272 the hydrophobic portion of the Arg265 side chain and of the hydroxyl and ring of Tyr528 (Figure 16).106 Small halogenated aliphatic moieties in the C2-position, such as the CF2CH3 of DSM265, were found to be optimal in terms of enzyme inhibition and whole cell parasite activity.26 Small aliphatic and sulfanyl moieties were also accommodated by this tunnel, although extending the carbon chain beyond 2– 3 carbons led to reduced potency.26 In PbDHODH, Ser181 is present in the tunnel (Gly181 for PfDHODH) and restricts the size of the substituent that is tolerated. This structural difference in PbDHODH resulted in C2-substituted DSM analogues displaying weak activity against the murine parasite strain.26 The pocket which accommodates the N7-is also completely hydrophobic in nature which precludes polar or basic substituents (Figure 16).106 The limited space near N7 and N1 also accounts for the poor activity observed for the ortho-substituted phenyl derivatives.103 The structure also demonstrates that para-substituents extend outward from the pocket which is consistent with the presence of 4-CF3 and 4-SF5 in the highest potency analogues. There is space for meta-fluorine substituents in addition to para-moieties and this pattern often resulted in greater potency at PfDHODH.103-105 However, this can also lead to a loss of species-selectivity as increasing the number of hydrophobic interactions restores activity in HsDHODH.105


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Antimalarial activity DSM265 has potent activity against asexual Pf strains (EC50s 7–16 nM) (Table 6) that are resistant to known drugs, such as CQ and MQ (Figure 1).106 This indicates that DSM265 is unlikely to be susceptible to drug resistance mechanisms of known antimalarials. Several experiments show the killing kinetics of DSM265 to be similar to that for atovaquone (Figure 1) which is consistent with their inherently related mechanism of action. The phenotypic and developmental response to treating blood-stage parasites with DSM265 at 10 × EC50 was similar to atovaquone. Microscopy evaluation of parasites 48 h post-treatment with DSM265 showed that parasites were killed in the young trophozoite stage.106 Determination of parasite kill rate at the EC50 was shown to have a 96 h lag phase, while at ≥3 × EC50 the lag phase was 24–48 h before parasite killing (Table 6) which is similar to that for atovaquone (Figure 1).106 This delayed phenotype was further exemplified with parasite clearance times of 85 and 82 h with treatment of 10 and 100-fold EC50 concentrations of DSM265. DSM265 did not inhibit growth of the early and late gametocyte (NF54) forms (Table 6).106 This is consistent with the observation that gametocyte levels were not affected by administration of DSM265 in humans.112 Additionally, DSM265 displayed no activity in the dual gamete formation assay, suggesting DSM265 would not be effective in a therapy aimed at transmission blocking.106 Liver cell invasion by sporozoites was not blocked by DSM265,106 however EEF growth to schizonts was arrested with DSM265 with an EC50 of 0.0057 μg/mL (Table 6) which is 30-fold more potent than primaquine (Figure 1). Against P. cynomolgi in vitro, DSM265 was active against


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multinucleated large EEFs (EC50 0.13 μg/mL), but not against hypnozoites. The pre-erythrocytic activity combined with the long plasma half-life suggests DSM265 has potential as a single exposure chemo-protectant. DSM265 displays potent activity against both asexual blood and liver schizont parasites and therefore would be suitable for use in TCP 1 and 4 (Table 1). As the program progressed, the humanized Pf SCID mouse became the gold standard preclinical efficacy model virtually replacing the earlier Pb mouse model.113 DSM265 was tested for efficacy in this model to enable calculation of the minimum parasiticidal concentration (MPC). DSM265 (as a tosylate salt) was dosed once or twice daily in the SCID mouse model where the 90% effective dose (ED90) was 3 mg/kg (q.d.) and 1.5 mg/kg (b.i.d.).106 The MPC was estimated to be 1–2 µg/mL (dose of 13 mg/kg per day or 6.4 mg/kg b.i.d.) (Table 6). The detection of late trophozoites and early schizonts in the blood further supports a mechanism which interferes with parasite metabolism.106

Synthesis The GMP synthesis of DSM265 is a scale-up of the method developed for analogue generation in the medicinal chemistry campaign and the pathway is shown in Scheme 4. The 2,3-diamino-6methyl-4(3H)-pyrimidinone (56) portion of the core was formed through the condensation of ethyl acetoacetate and aminoguanidine hydrochloride under basic conditions.106 Treatment of 56 with ethyl 2,2-difluoropropanoate in the presence of base affords the C2-substituted-7-hydroxy pyrazolopyrimidine in high yield. Chlorination was achieved using POCl3 in MeCN at elevated temperature to afford 58. Displacement of the chloride by pentafluorosulfanyl aniline gave DSM265 in high yield.


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During GMP scale-up the 3-fluoro phenyl by-product 59 (Scheme 4) was present at ~0.6% in the GMP scale-up material which was due to a contaminant formed during the synthesis of the pentafluorosulfanyl aniline. The formation of 59 was synthetically unavoidable and could not be removed on purification of DSM265.106 Compound 59 exhibited the same activity (IC50 0.010 μM) against PfDHODH as DSM265 but was 10-fold more potent against mammalian DHODH, including the human ortholog (IC50 3.7 M). In contrast to the biochemical activity, 59 was 10fold more potent against Pf in vitro (EC50 2 nM) than DSM265. Dosing DSM265, with 0.6% 59 present in the formulation, was well tolerated in preclinical species but needs to be monitored closely in the human clinical trials because of potential human DHODH activity.

Scheme 4. Reagents and conditions: (a) CH3CF2COOEt, NaOEt, EtOH, 78 °C, 3 h, 84%; (b) POCl3, MeCN, 80 °C, 5 h, 82%; (c) Pentafluorosulfanyl aniline, EtOH, 60 °C, 3.5 h, 86%, 99.5% purity. Inset: byproduct 59 resulting from the synthesis of DSM265.106


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Preclinical assessment Evaluation of DSM265 in preclinical species were undertaken by Phillips et al.106 to ensure safety and to predict an effective dose for humans in clinical trials. The pharmacokinetic profile of DSM265 in dogs and monkeys was improved compared to that seen in rodents (discussed in an earlier section).106 In monkeys and dogs, DSM265 (both 1 mg/kg i.v.) displayed a long half-life ranging from 10 to 45 h, low plasma clearance (0.054 to 0.022 L/h/kg) and good exposure (AUC 16.7 to 46.9 μg·h/mL). The oral bioavailability of DSM265 was lower in dogs than in rats and monkeys, however assessment of bioavailability in dogs was complicated by the presence of enterohepatic recirculation. A significant increase in plasma exposure of DSM265 was observed in the fed state compared to the fasted state in dogs, most likely resulting from poor aqueous dissolution of the freebase form of DSM265 in fasted versus fed state intestinal fluids. To improve solubility, a tosylate salt of DSM265 was produced, improving aqueous solubility somewhat and increasing exposure in mice compared to the free base form of DSM265, but a similar trend was not observed in rats or dogs. To improve the dissolution and absorption profile of DSM265, an amorphous spray dried dispersion (SDD) formulation was prepared. This formulation enhanced the simulated intestinal solubility approximately 10-fold (to 100 g/mL) and increased plasma exposure levels approximately 8-fold under both fasted and fed conditions in dogs. Furthermore, the SDD formulation was stable for at least 9 months under temperate conditions. The pharmacokinetic studies undertaken in preclinical species enabled the estimation of human pharmacokinetic parameters using a physiologically based pharmacokinetic (PBPK) model.114 Allometric scaling of the unbound clearance in preclinical species gave an estimated human clearance value of 0.25 L/h, and the PBPK model predicted a half-life of approximately 130 h, leading to an estimated human dose between 200 and 400 mg to achieve plasma concentrations above the plasma MPC (1–2 µg/mL) for at least 8 days.


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Metabolite profiling was performed to identify metabolites of DSM265 and their potential toxicity.106 One major metabolite and 4 minor metabolites were identified in in vivo samples from pharmacokinetic studies in animals. The structure of the major metabolite, M60, was confirmed by synthesis and mass spectra comparison. M60 was produced from mono-oxidation of the 5methyl group of the triazolo[1,5-a]pyrimidine core of DSM265. Other minor metabolites were formed by dehydrogenation (M61) and glucuronidation (M62) (Figure 17). In preclinical species, M60 was estimated to represent approximately 4–27% of the parent AUC. In vitro studies with each CYP450 isoform showed CYP2C8 and CYP2C19 were primarily associated with the oxidative biotransformation of DSM265. The metabolite M60 is ~3-fold less active against PfDHODH and 5-fold less active against PvDHODH in vitro compared to DSM265. Compound M60 is inactive against human DHODH and was well tolerated in dose ranging experiments in preclinical species. Collectively the data indicated that this metabolite was unlikely to pose a safety risk for DSM265 in the clinic.

Figure 17. The proposed biotransformation pathway for the formation of metabolite M60 and minor metabolites M61 and M62 from incubation of DSM265 in liver microsomes from preclinical species.106


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In vitro toxicology was evaluated to identify potential safety issues with DSM265.106 DSM265 did not inhibit or induce any human CYP450 isoform indicating a low likelihood of it impacting the metabolism of a co-administered partner agent. DSM265 displayed minimal inhibition (IC50 >4.2 mg/mL) of a panel of high-risk human receptors, ion channels and a panel of kinases. DSM265 was shown to inhibit the hERG cardiac ion channel at elevated concentrations (IC50 values of 2.9 and 0.66 μg/mL) using two patch clamp assays. However, DSM265 is unlikely to attain these concentrations in humans because of its low free fraction in plasma (unbound Cmax 250 parasites/mL in the placebo group was between 8 and 9 days compared to 15.3 for the -7-day cohort to 20.9 days for the -3-day treated cohort. Between individual treated cohorts there was no significant difference in the median rate to achieve >250 parasites/mL. Of the 18 patients in the trial, two patients were protected, 1 each from -3- and -7day treatment cohorts. In summary, although two patients were protected from infection, DSM265 overall displayed partial protection, suggesting it will require a fixed dose combination regimen for chemoprotection.

Table 4. Overview of DSM265 clinical trials completed or in progress. Phase/ Study


Phase Ia – Healthy PK and adults tolerability


Reference

Single dose of Primary measures: MTD; PK. ACTRN 5–1200 mg. Outcomes: Half-life: 86–118 h; AUC0–480: 12613000 104,000–4,720,000 ng·h/mL; Tmax: 1.5–4 h, Cmax: 522718, 116 1,310–34,800 ng/mL. No serious adverse events.

Phase Ib - Healthy Single 150 mg efficacy adults dose. infected with Pf asexual parasites

Primary measure: Parasite clearance kinetics.

Phase Ib - Healthy Single 400 mg efficacy adults dose. infected with Pf asexual parasites

Primary measures: Tolerability, PK.

ACTRN12 61300052 7763, 116

Outcome: Parasite clearance half-life: 9.4 h. MIC: 1040 ng/mL. Predicted single efficacious dose: 340 mg.

112

Secondary measure: Asexual parasite and gametocyte clearance. PK and tolerability consistent with Phase 1a. 100% asexual parasite clearance at day 12 (n = 7). No recrudescence at day 28.


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All patients became gametocytemic post treatment. Gametocytes not cleared with second 400 mg dose of DSM265. Phase IIa - Pv or Pf efficacy monoinfected adults from Peru

Phase Ib – efficacy with partner agent

Pf infected Primary measures: Parasitemia at day 14; PK. NCT0212 118 arm: Secondary measures: Parasite clearance kinetics; 3290, 250 or 400 mg tolerability; gametocytemia up to day 28; single dose. parasitemia at day 28; PK/PD. Pv infected arm: 400, 600 or 800 mg single dose.

Healthy Arm 1: single adults dose of 200 infected mg of OZ439 with Pf and 100 mg of asexual DSM265. parasites Arm 2: single dose of 400 mg for DSM265 and 500 mg for OZ439.

Outcomes: % of patients cured from Pf infection at day 25: 400 mg, 11%; 250 mg, 38%. Percentage of patients cured from Pv infection at day 14: 400 mg, 0%; 600 mg, 50%; 800 mg, 25%. Primary measure: PK/PD.

NCT0238 Secondary measures: Tolerability and safety; PK 9348 parameters. Outcome expected: unknown.

Phase Ib – Healthy prophylactic adults efficacy infected with Pf sporozoites

400 mg at 3 time points pre and post Pf sporozoite infection.

Primary measure: Time from sporozoite infection NCT0245 to asexual parasitemia (blood smear). 0578, 123

Phase Ib – Healthy prophylactic adults efficacy infected with Pf sporozoites via mosquito bite

400 mg at 3 time points pre and post Pf infection.

Primary measures: percentage with parasitemia NCT0256 (qRT-PCR) at day 28 and time from infection to 2872, 124 asexual parasitemia.

Secondary measure: Tolerability, PK and PK/PD. Outcomes: DSM265 dosed 1 day pre-infection: 100% protection (n = 5); DSM265 dosed 7 days pre-infection: 50% protection (n = 6) at day 24.

Outcomes: DSM265 dosed at 3 or 7 days preinfection: 33% protection (n = 6).


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MMV048 (MMV390048) Hit identification A HTS campaign performed by Prof. Vicky Avery at the Griffith Research Institute for Drug Discovery (GRIDD), as part of a collaborative team led by Prof. Kelly Chibale at the University of Cape Town, used an image based screen27 of the BioFocus DPI SoftFocus kinase library125 to identify 3,5-diarylaminopyridines as in vitro antimalarial hit compounds.50 The HTS was performed on 3D7 and Dd2 parasite strains with 36,608 compounds tested. Of these, 442 demonstrated 50% inhibition at 1.82 µM against these strains and structural variation was seen between the 3- and 5-postitions of the pyridine ring. Representative examples (63–65) of the initial hits are shown in Figure 18.

Figure 18. Initial hits of the 3,5-diarylaminopyridine class.

Lead identification and optimization Exploration of SAR started with the 5-(p-methylsulfonylphenyl)aminopyridine core and investigating the importance of the 3-position.50 These compounds featured various electrondonating and electron-withdrawing groups attached to either benzene or heteroaromatics such as pyridine and pyrazine. These compounds were examined for antimalarial activity against asexual NF54 Pf and they had equivalent or greater activity than CQ. The most notable changes amongst this series were compounds featuring pyridine, and/or electron-withdrawing groups, all of which


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exhibited EC50 values 28,000 nM for 76). Only the pyrazine compound (77) showed good potency with an EC50 value of 48 nM against Pf NF54.

Figure 21. Changes to the scaffold core and associated NF54 asexual parasite activity.

The pyrazine scaffold modification was further examined in combination with the otrifluoromethylpyridine at the 3-position.126 This was further extended to improve aqueous solubility and potency by replacement of the p-methylsulfonylphenyl group with a number of varied substituents; with the piperazine amide (79) demonstrating the most attractive features.127


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The key lead compounds across this series are shown in Figure 22. Ultimately, 77 was not further pursued due to limited aqueous solubility at physiological pH and 79, also known as UCT943128 is a potential clinical substitute for MMV048 exhibiting greater aqueous solubility.

Figure 22. The frontrunner compounds in the 3,5-diarylaminopyridine (pyrazine) series.50, 126, 127

MMV048 displays a LogD7.4 of 2.6 and solubility of 6.3–12.5 µg/mL at pH 6.5.50 Metabolism in human liver microsomes (HLM) demonstrated low clearance (CLint 10 µM at day 5. This inability to eliminate mature hypnozoites was reconfirmed in vivo in P. cynomolgus infected macaques suggesting that MMV048 will not have anti-relapse (TCP3) potential (Table 1).


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MMV048 inhibited both early stage (I, II, III) and late stage (IV, V) gametocytes in a luciferase reporter assay with EC50s of 214 nM and 140 nM, respectively.130 MMV048 also impacted gametogenesis exflagellation with an EC50 of 90 nM (Table 6). MMV048 was examined by direct SMFA (at the time of blood-meal) or indirect SMFA (before mosquito feeding) by incubation with stage V Pf gametocytes at 24 h. In the SMFA MMV048 was able to inhibit the formation of oocysts in the midgut with activity of 111 nM. However, in the direct SMFA, MMV048 inhibited >25% oocyst formation at 1 M. This difference infers a greater transmission-blocking ability against the stage V gametocytes in host blood rather than subsequent forms matured in the midgut of the mosquito. In summary, the multi-stage activity of MMV048 suggests it would be suitable for TCPs 1, 4 and 5 (Table 1). MMV048 administered to Pb-infected mice at five dose levels resulted in inhibition of between 98.0–99.5% parasitemia. When MMV048 was given as a single dose of 30 mg/kg or 100 mg/kg all mice were cured. In this mouse model, CQ, ART and MQ (Figure 1) do not result in a single dose cure. Efficacy in a Pf humanized SCID mouse model after a once daily dose of MMV048 over four consecutive days achieved an ED90 of 0.57 mg/kg at day 7 (Table 6).130 The mouse efficacy models support the use of MMV048 as a possible partner agent in a SERC therapy (Table 1).

Synthesis The process used to generate analogues during the discovery phase was also used to synthesize MMV048.50 The process was a relatively straightforward 3-step route beginning with iodination of 3-bromopyridin-2-amine (81) to give 82 in high yield. The aryl substituents were then successively installed in the 5- and then 3-positions using sequential Suzuki-Miyaura crosscoupling reactions in modest yields to generate MMV048 (Scheme 5).


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Scheme 5. Reagents and conditions: (a) I2, DMSO, 100 °C, 4 h, 79%; (b) p-MeSO2PhB(OH)2, 1,4-dioxane, K2CO3, Pd(PPh3)2Cl2 (5 mol%), 110 °C, 16 h, 58%; (c) 6-(trifluoromethyl)pyridin3-yl)boronic acid, 1,4-dioxane, K2CO3, Pd(PPh3)2Cl2 (7 mol%), 110 °C, 16 h, 65%.50

Preclinical assessment. Paquet et al. performed pharmacokinetic studies using preclinical species to assist in prediction of human pharmacokinetics and dose for MMV048.130 On intravenous administration of MMV048 to mice, rats, dogs and monkeys there were subtle differences in the pharmacokinetic profile between species. MMV048 possessed a low plasma clearance in dogs (CL 0.036 L/h/kg) and clearance was elevated in mice and rats (0.18 and 0.39 L/h/kg, respectively). The volume of distribution was moderate in all preclinical species (1.3–3.0 L/kg), while the half-life in dogs and monkeys (58 and 56 h) was longer than the half-life in mice and rats (5.6 and 9.2 h). On oral administration, the maximum concentrations of MMV048 attained in all species were dosedependent. MMV048 on oral dosing was characterized by a long half-life in dogs and monkeys and an oral bioavailability between 45 and 74%. Using allometric scaling of data from preclinical species, it was predicted that in humans MMV048 would have a low clearance (0.02 L/h), a modest volume of distribution (2.8 L/kg) and long half-life of 90 h. At a dose of 80–100 mg in humans,


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MMV048 was predicted to maintain the desired plasma concentrations for 8 days, suggesting MMV048 has potential in a one dose curative therapy. The long half-life of MMV048 combined with its liver stage activity imply MMV048 is also a suitable single agent for a chemoprotective or prophylaxis therapy (Table 1). In vitro safety data indicated that MMV048 is non-toxic to a panel of human cells at a therapeutically relevant concentration (CC50 >10 μM).128, 130 MMV048 does not inhibit hERG or ion channels Nav1.5 and Cav1.2 at concentrations >10 μM, suggesting MMV048 represents a low cardiac risk in the clinic.128 MMV048 was not active in the Ames or micronucleus test suggesting it represents a low genotoxicity risk in humans.128 MMV048 did not decrease red blood cell levels or affect spleen weight in G6PD-deficient immunodeficient mice at 30 mg/kg daily for 4 days, indicating MMV048 is suitable for administration in patients with G6PD deficiency. At the time of writing this review, exploratory toxicology or MTD data in preclinical species has not been published and therefore further safety aspects cannot be reported on, nonetheless, MMV048 has advanced into human clinical trials.

Clinical trial progress. MMV048 is currently in a phase II clinical trial. At time of writing this review, there were no literature reports regarding the efficacy outcome of MMV048 in human clinical trials (Table 5). The phase I trial assessing the pharmacokinetic profile and tolerability of MMV048 (NCT02230579)133 was the only clinical trial with results available at the NIH Clinical Trial Registry. In this trial, healthy fasted individuals were administered a single oral dose of MMV048 ranging from 5 to 120 mg. One cohort was fed and dosed 40 mg/kg orally to compare the effect of food on the pharmacokinetics. In the fasted arm, MMV048 was characterized by high plasma


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exposure (AUC0–∞) levels ranging between 2,136–156,036 h.ng/mL and a long half-life of between 163–252 h, correlating well with the half-life of MMV048 observed in preclinical species.130 The plasma exposure levels of the fed versus fasted cohort (29,004 and 21,050 h·ng/mL for fed and fasted respectively) indicated higher plasma exposure in the fasted group, while the half-life of MMV048 was not significantly affected (210 and 193 h for fed and fasted respectively). A secondary outcome of the phase I trial (NCT02230579)133 was to determine differences between the ex vivo EC50 value of a serum sample taken 144 h post dosing 40 mg of MMV048 and a reference serum sample spiked with a known amount of MMV048 titrated into the Pf asexual assay. The sample taken from MMV048 treated volunteers at 144 h gave an EC50 value of 24 nM which correlated well with the reported EC50 value of 28 nM.130 During the trial, a generalized myoclonic seizure was reported, but it is not known if this was a pre-existing condition or related to treatment with MMV048. Increased blood creatine kinase levels were noted at 40–80 mg doses of MMV048 but not at the lower doses of 5 and 30 mg. The cause of increased creatine kinase levels is not clear, as no other adverse drug-related events or cardiac aberrations were noted with increasing dose of MMV048. This trial gave a preliminary indication of the human pharmacokinetic profile of MMV048 and demonstrated that it was well tolerated. Additional phase I trials (NCT02554799 and NCT02783820) investigated the effect of new formulations of MMV048 on the pharmacokinetic profile and safety in healthy individuals (Table 5). The trials have been completed, but to date the results have not been reported. Further phase Ib trials to investigate the efficacy of MMV048 in healthy individuals infected with Pf (NCT02281344 or NCT02783833) have also been completed (Table 5). The malaria research community awaits the results of these trials as phase II assessment of MMV048 continues.101


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Table 5. Overview of MMV048 clinical trials completed or in progress. Phase/ Study




Reference

Single dose of Primary measures: Tolerability, PK 5–120 mg. Secondary measure: ex vivo efficacy up to 144 h.

NCT0223 0579

Outcomes: Half-life: 163–210 h; AUC0-24: 2137– 156,036 h·ng/mL. No serious adverse events. Mild adverse events reported in 67–100% subjects. ex vivo efficacy for 40 mg dose (IC50): 9.48 ng/mL.


40 mg in 2 Primary measure: PK formulations. Secondary measure: Tolerability

NCT0255 4799


Single dose of Primary measure: Tolerability up to 28 days. 40, 80 and 120 Secondary measure: PK. mg. Outcome expected: unknown.

Phase Ib - Healthy efficacy adults infected with Pf asexual parasites

Arm 1: single Primary measure: Parasitemia (qPCR) over 21 days. NCT0228 20 mg; 1344 Secondary measure: PK. Arm 2: single Outcome expected: unknown. TBD;

Phase Ib - Healthy efficacy adults infected with Pf asexual parasites

Arm 1: single Primary measure: Parasitemia (qPCR) over 28 days. NCT0278 40 mg; 3833 Secondary measure: PK; tolerability. Arm 2: single Outcome expected: unknown. TBD.

Outcome expected: unknown. NCT0278 3820

Arm 3: single TBD.


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Table 6. Summary of antimalarial activity for each clinical candidates. Compound Protein target activity IC50 (nM) Asexual blood stage (72 h) EC50 (nM) Pf ABS PRR

KAE609 12 (PfATP4 Na+ homeostasis assay)

KAF156

DSM265

MMV048

n/a

33 (PfDHODH)

3.4 (PvPI4K)

0.9 (NF54)

10 (3D7)

7.8 (3D7)

28 (NF54)

Kills early and late trophozoites and schizonts. Moderate acting in vitro. Very fast acting in vivo.

Fast acting.

Kills at early trophozoite stage. Slow acting.

Moderate rate of kill.

EC99 50 nM

not active

not active

EC50 214 nM

EC99 500 nM

EC50 4 nM

not active

EC50 140 nM

not active

no data

not active

90

not active

no data

not active

not active

EC99 500 nM

EC99 500 nM

not active

EC50 111 nM

not active

10 (Pb)

5.7 (Pf)

64 (Pc)

not active

not active

not active

not active

Gametocytes – early Gametocytes – late Dual gamete formation assay – male EC50 (nM) Dual gamete formation assay – female EC50 (nM) SMFA Liver – schizont EC50 (nM) Liver – hypnozoite EC50 (nM) Pb mouse efficacy

ED99 5.3 mg/kg

ED99 2.2 mg/kg

no data

cure at a single 100 mg/kg oral dose

Pf mouse SCID efficacy

no data

no data

ED90 3 mg/kg (q.d.) and 1.5 mg/kg (b.i.d)

ED90 1.1 mg/kg

38, 49, 61, 69

84, 87, 90

26, 106

128, 130

References


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Table 7. Overview of each antimalarial clinical candidate. KAE609 Chemotype Identification

spiroindolone

KAF156

DSM265

imidazolopiperazine triazolopyrimidine

MMV048 amino pyridine

phenotypic

phenotypic

target based

phenotypic

PfATP4

PfCARL a

PfDHODH

PfPI4K

Parasite stage

asexual,

asexual, liver,

activity

gametocyte

gametocyte

not active

not active

not active

not active

Pf, Pv, Pk

Pf, Pv, Pk

Pf, Pv, Pk

Pf, Pv, Pk

24 h

2–3 days

3–4 days

6–8 days

TCP

1 and 5

1, 4 and 5

1 and 4

1, 4 and 5

Potential TPP

1

1 or 2

1 or 2

1 or 2

strategy Target or resistance marker

Pv mature hypnozoite Plasmodium species b Human half life

a

asexual, liver

asexual, liver, gametocyte

Mechanism of resistance. b no data on P. ovale or P. malariae.

CONCLUSIONS In the past l5 years, mass screening of academic and industry libraries against the Plasmodium parasite has produced a plethora of different chemotypes as starting points for development of antimalarials. The development of these scaffolds has been driven by collaborative efforts between multiple organizations from academia and industry with different infrastructure and skill sets. The highly collaborative programs, principally managed through the work of MMV and its partners, are underpinned by significant advancements in specialized in vitro and in vivo platforms to assess


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the antimalarial activity of compounds. These platforms have enabled thorough characterization of each antimalarial, informing decisions on developmental workflow, clinical trial design and ultimately its suitability as a partner agent in an antimalarial therapy. Four of the most advanced of the antimalarials to emerge from the multi-disciplinary collaborative initiatives, KAE609, KAF156, DSM265 and MMV048 (Figure 2), are currently under assessment in phase II clinical trials and are the focus of this perspective. A cornerstone of modern antimalarials is their activity across multiple stages of the parasite lifecycle and alignment with treatment and elimination strategies outlined by leading malaria organizations; nevertheless, antimalarials with rapid action on asexual blood stages only and low propensity to generate resistance are also critically needed. All four focal antimalarials differentially target multiple stages of the malaria parasite lifecycle (Tables 6 and 7). In addition to targeting the asexual stage, KAF156, DSM265 and MMV048 kill liver stage schizonts. Combined with a long human plasma half-life, these three compounds could serve as chemoprotectants according to TPP2 (Table 1). KAF156 and MMV048 also show transmission blocking activity and combined with their long half-life could find potential use, ultimately, in campaigns focused on the control and elimination of malaria in endemic areas provided that the safety and tolerability is sufficient. KAE609 is very fast acting in vivo, displays transmission blocking activity and has a moderate half-life. KAE609 will most likely find use in a combination therapy for treatment of uncomplicated malaria and could also be considered for severe malaria. Antimalarials KAF156, DSM265 and MMV048 could also serve as partner agents in a combination treatment therapy, but DSM265 is likely to need to be partnered with a fast-acting partner antimalarial because of its slower rate of parasite killing.


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Laboratory models assist in determining the potential utility of an antimalarial agent, but ultimately clinical evidence is required. The clinical trial data currently available on the four focal antimalarials appears positive (Tables 2–5). In clinical trials, all four antimalarials were well tolerated as single agents. In preliminary efficacy studies; KAE609, KAF156 and DSM265 as single agents were able to significantly reduce or clear Pf and Pv infections in either healthy individuals or patients. DSM265 did not clear Pv as efficiently, and therefore its use for treating Pv infections may be limited unless combined with a suitable partner agent. The results for the efficacy of MMV048 in human clinical trials are yet to be reported. Of the four focal antimalarials, DSM265 as a single agent has been assessed for prophylactic inhibition of liver stage infection development under controlled sporozoite or mosquito fed challenge conditions. The liver schizont activity in the two trials suggest that the compound has potential for once weekly chemoprotection. Overall clinical trials using KAE609, KAF156 and DSM265 as single agents for treating Plasmodium parasite infection in humans are positive, but further trials are necessary to determine their potential as partner agents for implementation in a SERC and/or SEC therapy (Table 1). Furthermore, the trials to date are on small demographic cohorts, and therefore more data are required to determine if the modern antimalarials demonstrate sufficient safety and tolerability and will be effective when implemented in a larger population with greater genetic diversity, and more particularly for treating high risk groups such as infants and pregnant women.


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AUTHOR INFORMATION Corresponding Author * Dr Brad E. Sleebs. Phone: +61 3 9345 2718. Email: [email protected]; Dr Darren J. Creek. Phone: +61 3 9903 9249. Email: [email protected]. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Dr Trent D. Ashton and Dr Shane M. Devine contributed equally.

ACKNOWLEDGMENTS This work was supported by the National Health and Medical Research Council of Australia (Development Grant 1113712 to B.E.S.; Fellowship 1148700 to D.J.C.; Project Grant 1163235 to D.J.C. and S.M.D.), Australian Cancer Research Foundation, the Victorian State Government Operational Infrastructure Support and Australian Government NHMRC IRIISS. B.E.S. is a Corin Centenary Fellow.

ABBREVIATIONS ACT, artemisinin combination therapy; ACPR, adequate clinical and parasitological response; ART, artemisinin; AUC, area under the curve; CARL, cyclic amine resistance locus; Cmax, maximum

concentration;

CRT,

CQ

resistance

transporter;

DHODH,

dihydroorotate

dehydrogenase; EEF, exoerythrocytic form; fu, fraction unbound in plasma; G6PD, glucose-6phosphate dehydrogenase; GMP, good manufacturing practice; hERG, ether-à-go-go-related gene;


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Hs, Homo sapiens; HTS, high throughput screening; MDR, multi-drug resistant; MQ, mefloquine; MIC, minimum inhibitory concentration; MMV, Medicines for Malaria Venture; MPC, minimal parasiticidal concentration; MTD, maximum tolerated dose; PAMPA, parallel artificial membrane permeability assay; Pb, P. berghei; Pf, P. falciparum; Pv, P. vivax; PPQ, piperaquine, PRR, parasite reduction ratio; QT, interval between Q and T wave of cardiac rhythm; RBC, red blood cell; SCID, severe combined immunodeficiency; SERC, single exposure radical cure; SEC, single exposure chemoprotection; SMFA, standard membrane feeding assay; SNP, single-nucleotide polymorphism; TCP, target candidate profile; Tmax, time to reach the maximum concentration; TPP, target product profile; WHO, World Health Organization.

BIOGRAPHIES Trent Ashton obtained his PhD from the Monash Institute of Pharmaceutical Sciences in 2008. During post-doctoral positions at Boston University and Deakin University he worked on multiple programs developing anti-infective agents and epigenetic modulators for the treatment of metabolic disorders. In 2018, he joined the Sleebs Laboratory at the Walter and Eliza Hall Institute where his current research focuses on the development of antimalarial compounds with novel modes of action.

Shane Devine obtained his PhD from La Trobe University in 2006 researching the synthesis of novel heterocyclic systems. He then joined Monash Institute of Pharmaceutical Sciences, where his research has included using fragment-based and structure guided drug design approaches in the design of G protein-coupled receptor ligands, novel anticancer agents and more recently antimalarial compounds with novel mechanisms of action.


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Jörg J. Möhrle obtained his PhD from Basel University researching protein kinases of Plasmodium falciparum. He then held several positions in clinical development in the pharmaceutical and biotech industry researching oncology, inflammatory diseases and neurology. He joined MMV in 2005 and was subsequently appointed Vice President, Head of Translational Medicine, Research & Development for Medicines for Malaria Venture. Since 2017, he is also an Associate Professor for Infection Biology and Epidemiology at Basel University.

Benoît Laleu obtained a PhD in Chemistry from the University of Geneva, Switzerland, in 2006. After completing postdoctoral studies at the University of Toronto, Canada, in 2007 he joined GenKyoTex −a Swiss biotechnology company− where he spent 7 years as a medicinal chemist. In 2015, Benoît joined Medicines for Malaria Venture where he is currently Associate Director of Drug Discovery and has responsibility for a portfolio of antimalarial drug discovery projects working with pharmaceutical companies and academic groups.

Jeremy Burrows obtained a PhD in organic chemistry at Oxford University in 1996. He then joined AstraZeneca in the UK and focused on Infection, Cardiovascular and Inflammation research. In 2005, he moved to AstraZeneca in Sweden to focus on Alzheimer’s disease. In 2010, he was appointed Head of Drug Discovery at the Medicines for Malaria Venture and manages a portfolio of enabling technology, screening, hit-to-lead and lead optimization projects in malaria and neglected diseases.


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Susan Charman obtained a PhD in pharmaceutical sciences from the University of Florida in 1987. She spent 2 years in the pharmaceutical industry in the US before assuming an academic position at the Monash Institute of Pharmaceutical Sciences. She is currently Professor and Director of the Centre for Drug Candidate Optimisation. She leads a team of 25 post-doctoral fellows and research assistants that provide expertise in physicochemical, ADME and pharmacokinetic properties to enhance the discovery and development of novel drug candidates to treat tropical infectious diseases, cancer, metabolic and CNS disorders.

Darren Creek obtained a PhD from the Monash Institute of Pharmaceutical Sciences in 2008. He has post-doctoral experience in antimalarial pharmacology, biochemical parasitology and metabolomics from Makerere University-UCSF, Monash University, University of Glasgow and University of Melbourne. Since 2014 he has led a laboratory focused on understanding mechanisms of antiparasitic drug action using metabolomics and proteomics. He is also the metabolomics director of the Monash Proteomics and Metabolomics Facility.

Brad Sleebs obtained his PhD from La Trobe University in 2005. He then joined The Walter and Eliza Hall Institute as a Research Officer and focused on the development of novel anxiolytics and agents that target the BH3 family of proteins for treating blood cancers. He was recently appointed Laboratory Head in the Chemical Biology Division at the Walter and Eliza Hall Institute. His current research focuses on designing small molecule probes to better understand biological processes that are critical to the survival of the malaria parasite and the progression of cancer malignancies, and in collaboration with industry partners the development of novel antimalarial and oncology therapies.


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GRAPHICAL ABSTRACT

123

The Development Process for Discovery and Clinical Advancement of

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