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

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

Here we perform a detailed in vitro and in vivo characterisation of the antimalarial and pharmacokinetic properties of DDD01034957 using three different Plasmodium species, test for development of resistance in vitro and perform structure-function studies of analogues which share the same chemical scaffold.

Results
DDD01034957 shows no cross-resistance to a range of drug resistant P. falciparum strains. The use of partner drugs in combination therapies requires two molecules with distinct modes of action to protect against the emergence of resistance to the monotherapy. To determine whether DDD01034957 has a unique mode of action or acts through an already recognised mechanism, its efficacy was tested in asexual growth assays against a range of selection-derived drug resistant P. falciparum parasites and their parental non-resistant lines ( Table 1). In all assays, the resistant strains were sensitive to DDD01034957, giving no greater than a threefold increase in IC 50 when compared to their parental lines. This result suggests that DDD01034957 has a distinct mode of resistance and is active against parasites harbouring resistance mutations linked to a loss of efficacy against many new antimalarials currently under clinical development.
DDD01034957 is a fast-acting antimalarial against P. falciparum asexual stages in vitro. Fast-acting antimalarials are crucial to rapidly reduce the parasite burden and relieve the patient from malarial symptoms as fast as possible 11 . To determine the speed of action of DDD01034957, it was evaluated in an established parasite viability assay 12 and compared to artesunate (fast kill), chloroquine (fast kill), pyrimethamine (medium kill) and atovaquone (slow kill) 12 . At 3.2 µM (10xIC 50 ), DDD01034957 rapidly reduced parasite viability to baseline within 24 h at a similar rate to 10xIC 50 artesunate and chloroquine (Fig. 2), thus demonstrating its fastacting antimalarial activity. In contrast, the slower acting antimalarials pyrimethamine and atovaquone showed only partial reduction in parasite viability at 24 h.   Table 1). Whole-genome sequencing highlighted two genes in common that possessed a variety of single point mutations in all eleven clones-pfabci3 (PF3D7_0319700) and pflsa1 (PF3D7_10364000).
Although each clone possessed up to two different mutations in pflsa1 from a combination of six mutations observed, it was discounted as a potential mode of action/mechanism of resistance due to the reported exclusive expression of its gene product, liver stage antigen 1, at only the liver stages of the parasite life cycle 13 . The remaining gene pfabci3 encodes an essential ATP-binding cassette transport protein that is known to mediate resistance to other experimental antimalarial compounds. These compounds encompass several distinct chemical scaffolds 14,15 unrelated to DDD01034957. Eight out of eleven clones harboured the mutation F2010L, two possessed a H2181D mutation and the remaining clone acquired a L79F mutation. The F2010L and H2181D mutations map to a region of the ABCI3 protein that is predicted to harbour multiple transmembrane domains which may form part of the transporter channel (Fig. 3C). To determine whether DDD01034957 resistance is mediated by PfABCI3, this compound was tested against a previously reported line harbouring a R2180P mutation (the amino acid adjacent to the H2181D mutation observed in this study) and a pfabci3 copy number variant (CNV) (Fig. 3B) 15 . The R2180P mutant was 29.8-fold less susceptible to DDD01034957 than the parental strain, while the CNV line displayed 5.3-fold decreased susceptibility. Together, these data strongly suggest that PfABCI3 does indeed mediate resistance to DDD01034957. A combined dataset of 6,230 sequenced P. falciparum isolates from 22 countries [16][17][18] was searched and none of the identified mutations in pfabci3 conferring DDD01034957 resistance were found. Similarly, evidence of pfabci3 CNV was only found in 0.89% of isolates, which possessed duplications of different lengths implying independent events rather than selection pressure at this locus. This implies that an optimised scaffold of DDD01034957 would not encounter existing PfABCI3-mediated resistance in the clinic.

Determining the in vivo efficacy and pharmacokinetic properties of DDD01034957.
Having established in vitro antimalarial efficacy, the in vivo efficacy of DDD01034957 was investigated using the standard 4-day suppression test in the Plasmodium berghei rodent model of infection 19 . A 50 mg/kg oral dose of DDD01034957 administered on four consecutive days to infected mice reduced parasitaemia by 59.7-79.8% compared to the vehicle control (Fig. 4A). However, at this level of suppression DDD01034957 did not appear to protect mice from the symptoms of malaria and there was no statistically significant difference in time taken for mice to reach their humane endpoint (unpaired t-test). In contrast, the 10 mg/kg chloroquine treatment completely supressed parasitaemia and all mice survived to the end of the experiment. Whole blood levels of DDD01034957 in naïve mice treated with a 50 mg/kg dose by intraperitoneal (IP) injection rapidly peaked at 1092 ng/ml but reduced with an elimination half-life of ~ 106 min suggesting a rapid clearance mechanism (Fig. 4B).
In vitro antimalarial structure-activity relationship. It was hypothesised that the methoxy group on the phenyl ring of DDD01034957 could be a potential metabolic liability and partly account for its fast in vivo clearance following metabolic cleavage of the methyl group. Therefore, commercially available analogues Removing the methoxy group altogether also greatly reduced antimalarial activity (MolPort-008-120-629 and MolPort-006-386-365) suggesting that the methoxy group of DDD01034957 may be important for target binding. Conversely, replacement of the fivemembered amine ring with a six-membered ring did not appreciably modify activity suggesting that at least small changes of this part of the molecule are tolerated, and further alterations will need to be investigated.
DDD01034957 is more efficacious against P. falciparum than P. knowlesi. It has been established that some antimalarials show different efficacy against different species of Plasmodium 20 . Therefore, the antimalarial activity of DDD01034957 was tested on culture adapted Plasmodium knowlesi-a species causing malaria in humans and primates common in Southeast Asia. Over one asexual cycle (48 h P. falciparum vs. 27 h P. knowlesi), DDD01034957 showed greatly reduced potency against P. knowlesi with inhibition not reaching an upper plateau by the maximum concentration tested (40 µM) (Fig. 6).

Discussion
Herein, we report the in vitro and in vivo efficacy of DDD01034957 together with its mechanism of resistance mediated through the transporter PfABCI3. DDD01034957 demonstrates promising fast-acting killing of P. falciparum asexual blood stage parasites in vitro, and shows efficacy against parasite lines harbouring resistance to many diverse antimalarials. These data support the continued development of DDD01034957 as a new chemotype with antimalarial potential. However, DDD01034957 had limited in vivo efficacy against P. berghei. Whole blood concentrations peaked at 25-times the in vitro IC 50 and maintained levels of DDD01034957 above the IC 50 for at least 480 min (the last time point sampled), indicating that lack of efficacy may not be due to bioavailability, but in part may be related to fast clearance. Interestingly however, the pbabci3 gene harbours the equivalent histidine to aspartate point mutation that gave the ~ 12-fold increase in IC 50 in the P. falciparum H2181D resistant line, suggesting that the P. berghei line tested may be naturally resistant to DDD01034957. Consequently, future work should prioritise studying in vivo efficacy of DDD01034957 in the humanised P. www.nature.com/scientificreports/ falciparum mouse model or alternatively in a P. berghei line modified transgenetically to contain the D2181H reverting mutation. DDD01034957 lacked efficacy against P. knowlesi which does not contain any of the point mutations in pkabci3 linked to resistance in P. falciparum. It has previously been reported that P. knowlesi shows modulated sensitivity to a range of different antimalarials when compared to P. falciparum 20 . Currently it is unclear as to why this phenomenon occurs. However P. falciparum possesses a number of species-specific influx/ efflux transporters that might play an important role in modulating the uptake of DDD0103957 from the red blood cell 21 . Alternatively, DDD010349757 efficacy could be linked to different binding affinities to its target, which might differ between Plasmodium species. A preliminary structure-activity relationship (SAR) study using commercially available analogues indicates that modification of the methoxy group of DDD01034957 substantially impacted its antimalarial activity. Replacement with a fluorine or chlorine atom results in a reduced activity by 10-to 14-fold respectively. Deeper medicinal chemistry studies are required to determine whether metabolism of the methoxy group to a phenol is important for in vivo clearance. Key to such studies could be the synthesis and testing of phenol analogues of our series and/or methoxy surrogates. Additional future medicinal chemistry could include exploration of whether modification of the pyrido-pyrimidinone core can increase efficacy, while improving pharmacokinetics.
In conclusion, a detailed study of the antimalarial efficacy of new chemotypes is highly valuable for the progression of molecules into the lead optimisation stage. DDD01034957 shows promising in vitro activity and demonstrated, albeit reduced, oral in vivo efficacy (which is unsurprising for a non-optimised primary hit at this preliminary stage). Its mode of action is potentially distinct from other antimalarials under development and its mechanism of resistance is now defined, implicating the transporter PfABCI3 in drug influx/efflux. Future work will focus on reducing in vivo metabolic clearance, further improving efficacy, and mitigating PfABCI3mediated resistance.

Materials and methods
Comparison of resistant strains. The asexual activity of published drug resistant strains (Table 1) were tested in the P. falciparum lactate dehydrogenase assay (pLDH) 3 which is a surrogate for asexual parasite growth. Each strain was tested in comparison to its parental (non-resistant) line. Parasites were prepared at 0.25% parasitaemia/2% haematocrit in RPMI-1640 supplemented with 5% Albumax and 150 µM hypoxanthine (except the parasite strain adapted to low folate conditions which was cultured in culture medium depleted of paraaminobenzoic acid and with low (100 ng/ml) folic acid concentrations) before being dispensed into wells of a 384 well plate (25 µl per well) containing test compounds in DMSO. After incubation for 72 h at 37 °C under a 5% CO 2 /5% O 2 /90% N 2 atmosphere, the plates were frozen at − 70 °C to lyse cells. After thawing, 70 µl of reaction Speed of kill assay. This assay was performed as described in Linares et al. 22 . Briefly, P. falciparum 3D7 strain parasites were incubated at 2% haematocrit, 0.5% parasitaemia in 96 well plates in the presence of test compounds at 10xIC 50 . At 24 and 48 h post drug treatment parasite samples were transferred and diluted into Selection of compound-resistant lines was performed using a high-pressure intermittent method 6 . 1 × 10 9 asexual parasites were cultured in the presence of 0.2 µM or 2 µM DDD01034957 and monitored regularly until parasitaemia recrudesced (11-15 days). Then, compound pressure was removed to allow cultures to expand up to ~ 2% parasitaemia, DDD01034957 was added again and sensitivity was compared to the parental line. Resistant parasites were then cloned by limiting dilution to obtain up to six clones per selection experiment (n = 3). The sensitivity of a PfABCI3 copy number variant (CNV) and PfABCI3 R2180P 14,15 mutant to DDD01034957 was tested in asexual growth assays. Parasites were maintained at 3% haematocrit with human O + red blood cells in RPMI medium supplemented with 50 μM hypoxanthine, 2 g/l sodium bicarbonate, 2 mM l-glutamine, 25 mM HEPES, 0.5% AlbuMAXII (Invitrogen) and 10 μg/ml gentamycin in 5% O 2 , 5% CO 2 and 90% N 2 at 37 °C. Parasites were diluted to 0.3% parasitaemia/1% haematocrit in 96 well plates containing serial dilutions of DDD01034957 and incubated for 72 h at 37 °C. Parasite survival in each well was assessed by SYBR Green and MitoTracker Deep Red FM staining (Life Technologies) and subsequent flow-cytometric analysis (Accuri C6, BD Biosciences).
Whole genome sequencing. Resistant parasite cultures were pelleted by centrifugation and then frozen at − 80 °C to lyse the cells. Genomic DNA was then extracted using a Blood & Cell Culture DNA Mini Kit (Qiagen) and sequenced together on one lane of an Illumina HiSeqX machine with a 150 bp paired end protocol. The resulting sequences were mapped to the 3D7 reference genome (v3) using bwa-mem software, and genomic variants called using GATK and samtools software tools within existing bioinformatic pipelines 23 . A collection of genomes from 6230 P. falciparum isolates from 22 countries [16][17][18] , was searched for identified PfABCI3 mutations. The same dataset was analysed for evidence of CNV using Delly 24 with a < 200kbp cut-off.

P. berghei 4-day suppression test.
All procedures were performed in accordance with the UK Animals (Scientific Procedures) Act (PPL 70/8788) and approved by the Imperial College and University of Cambridge AWERB. The Office of Laboratory Animal Welfare Assurance for Imperial College covers all Public Health Service supported activities involving live vertebrates in the US (no. A5634-01). This study was carried out in compliance with the ARRIVE guidelines (https ://arriv eguid eline s.org/). Naïve T0 mice (n = 5 per treatment) were infected by IP injection of 2 × 10 7 P. berghei parasites obtained from the blood of a donor mouse. DDD01034957 and chloroquine (positive control) were dissolved in 7% Tween 80/3% Ethanol in dH 2 0 and dosed orally to infected mice to result in a 50 mg/kg and 10 mg/kg exposure respectively. Five additional infected mice received a vehicle-only dose as a negative control. Mouse in vivo PK analysis. Pharmacokinetic analysis was outsourced commercially to Dundee Drug Discovery Unit (DDU). Briefly, DDD01034957 was dissolved in 7% Tween 80/3% Ethanol in dH 2 0 and 50 mg/kg and administered by IP injection to three BALB/c mice. Whole blood was sampled at intervals over 8 h and the concentration of DDD01034957 determined by mass spectroscopy.
Analogue asexual growth assays. P. falciparum 3D7 strain parasites were synchronised by sorbitol lysis to obtain a pure ring stage population and maintained in RPMI 1640 supplemented with 25 mM HEPES, 25 mM Figure 6. A comparison of the in vitro asexual efficacy of DDD01034957 against P. falciparum (Pf) and P. knowlesi (Pk) over one asexual cycle. Dose response data shows the mean of eight independent replicates with error bars denoting the SEM.