Title: Target identification and elucidation of the complete life cycle fingerprint of the novel Plasmodium PI4K inhibitor MMV390048

Authors: Tanya Paquet1, Claire Le Manach1, Diego Gonzalez Cabrera1, Yassir Younis1, Philipp P. Henrich2, Tara S. Abraham2, Marcus C.S. Lee2, Rajshekhar Basak2, Sonja Ghidelli-Disse3, María José Lafuente-Monasterio4, Marcus Bantscheff3, Andrea Ruecker5, Andrew M. Blagborough5, Sara E. Zakutansky5, Anne-Marie Zeeman6, Karen L. White7, David M. Shackleford7, Janne Mannila7, Julia Morizzi7, Christian Scheurer8,9, Iñigo Angulo-Barturen4, María Santos Martínez4, Santiago Ferrer4, Laura María Sanz4, Francisco Javier Gamo4, Janette Reader10, Mariette Botha10, Koen J. Dechering11, Robert W. Sauerwein11,12, Anchalee Tungtaeng13, Pattaraporn Vanachayangkul14, Chek Shik Lim15, Jeremy Burrows16, Michael J. Witty16, Kennan C. Marsh17, Christophe Bodenreider15, Rosemary Rochford18, Suresh M. Solapure19, María Belén Jiménez-Díaz4, Sergio Wittlin8,9, Susan A. Charman7, Cristina Donini16, Brice Campo16, Lyn-Marie Birkholtz10, Kirsten K. Hanson20, Gerard Drewes3, Clemens H.M. Kocken6, Michael J. Delves5, Didier Leroy16, David A. Fidock2,21, David Waterson15, Leslie J. Street1, Kelly Chibale1,22 *

Affiliations:1 Department of Chemistry, University of Cape Town, Rondebosch, 7701, South Africa2 Department of Microbiology and Immunology, Columbia University Medical Center, New York, New York 10032, United States3 Cellzome GmbH, Molecular Discovery Research, GlaxoSmithKline, Meyerhofstrasse 1, 69117 Heidelberg, Germany4 Malaria DPU, Tres Cantos Medicines Development Campus-Diseases of the Developing Word, GlaxoSmithKline, Severo Ochoa 2, 28760 Tres Cantos, Madrid, Spain5 Department of Life Sciences, Imperial College, London SW7 2AZ, United Kingdom6 Department of Parasitology, Biomedical Primate Research Centre, 2280 GH Rijswijk, The Netherlands7 Centre for Drug Candidate Optimisation, Monash University (Parkville Campus), 381 Royal Parade, Parkville, Victoria 3052, Australia8 Swiss Tropical and Public Health Institute, Socinstrasse 57, 4002 Basel, Switzerland9 University of Basel, 4003 Basel, Switzerland10 Department of Biochemistry, Centre for Sustainable Malaria Control, University of Pretoria, Pretoria, South Africa11 TropIQ Health Sciences, Geert Grooteplein 28, Huispost 268, 6525 GA, Nijmegen, The Netherlands12 Radboud University Medical Center, Department of Medical Microbiology, 6500 HB, Nijmegen, The Netherlands13 Department of Veterinary Medicine, Armed Forces Research Institute, of Medical Sciences (AFRIMS), Bangkok, Thailand 10400

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14 Department of Immunology and Medicine, Armed Forces Research Institute, of Medical Sciences (AFRIMS), Bangkok, Thailand 1040015 Novartis Institute for Tropical Diseases Pte Ltd, 10 Biopolis Road, #05-01 Chromos, Singapore 13867016 Medicines for Malaria Venture, International Center Cointrin, Route de Pré-Bois 20, 1215 Geneva, Switzerland17 Abbvie, 1 North Waukegan Road, North Chicago, Il 60064-6104, United States18 Departments of Immunology and Microbiology and Environmental and Occupational Health, University of Colorado, Denver, Aurora, CO19 Nagarjuna Gardens, 60 Feet Road, Sahakaranagar, Bangalore 560092, India20 University of Texas at San Antonio, Dept. of Biology and STCEID, 1 UTSA Circle, San Antonio, TX 78249, United States21 Division of Infectious Diseases, Department of Medicine, Columbia University Medical Center, New York, New York 10032, United States22 South African Medical Research Council Drug Discovery and Development Research Unit, and Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Rondebosch, 7701, South Africa

* Corresponding author: kelly.chibale@uct.ac.za

One Sentence Summary: MMV390048 exemplifies a novel class of Plasmodium PI4K inhibitors that is active across all stages of the parasite life cycle. Although inhibition of PvPI4K as a mechanism of action will not afford a radical cure, MMV390048 has great potential for both treatment and prophylaxis due to its target and compound characteristics.

Abstract: Stemming from phenotypic whole-cell screening, the novel 2-aminopyridine antimalarial, MMV390048, lacked cross-resistance with marketed and clinical compounds and was efficacious against all Plasmodium life cycle stages, apart from hypnozoites, in vivo. Efficacy in both rodent (Plasmodium berghei) and humanized (Plasmodium falciparum) mouse models of malaria was obtained, with results supporting the inclusion of MMV390048 in single dose treatments. Modest reductions in population-to-population transmission were achieved in rodent models, as well as causal prophylaxis and full chemoprotection in monkeys. Albeit delaying the onset of relapse in a monkey model, MMV390048 did not deliver a radical cure. Both genomic and chemoproteomic studies identified Plasmodium PI4K as the target.  The complete life cycle profile of MMV390048 thus elucidates the full implication of targeting Plasmodium PI4K in the context of malaria eradication.

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Introduction

Malaria, the parasitic disease caused by Plasmodium falciparum and Plasmodium vivax infections, still causes an estimated 236,000 – 635,000 deaths per annum in spite of the positive impact of interventions around access to medication, indoor spraying, and insecticide-treated bed nets (1). Resistance to treatment regimes, especially the emergence of resistance to artemisinins in currently recommended artemisinin-based combination therapies (2,3), still poses a threat and

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highlights the importance of developing treatments containing new chemical classes that function through different modes of action to marketed anti-malarials. The global aim to eradicate malaria brings about additional requirements of chemoprevention and transmission blocking, and the ability to eliminate dormant liver parasites that are responsible for relapse in the case of P. vivax infection. To this effect, new chemical entities should exhibit potency across the different life cycle stages of the parasite in both the human host and mosquito vector (4).

A number of new chemical classes (5) with activity across different life cycle stages of the malaria parasite have emerged and novel mechanisms of action have been identified, including classes of Plasmodium PI4K inhibitors (6-8). We herein describe the 2-aminopyridine, MMV390048 (Fig. 1a) (9), as representative of a new chemical class of Plasmodium PI4K inhibitors, whose properties provide confirmation of the positioning of a PI4K inhibitor as a chemoprotective and transmission-blocking agent, and discernment about its anti-relapse potential.

MMV390048 was developed based on a series of hits identified from a phenotypic high-throughput screen of a commercial BioFocus library (9). Although kinetic solubility of MMV390048 was not optimal at pH 6.5, it was well absorbed and gave high exposure in rats. In vitro potency and good pharmacokinetics translated to efficacy in a Plasmodium berghei mouse model for malaria (9, and Supplementary Section 1), which spurred further investigations into the potential of MMV390048 as an anti-malarial agent.

Results In vitro and in vivo antimalarial blood stage activity The in vitro activity of MMV390048 against intra-erythrocytic stages of P. falciparum (NF54 sensitive strain) showed a steep inhibition curve with IC50 and IC90 values of 28 and 40 nM, respectively. Against a panel of resistant isolates, the ratio of the Max/Min IC50 values for MMV390048 was 1.5-fold, suggesting that cross-resistance is a low risk for MMV390048 (Supplementary Section 2, Table S1) (10).

Parasite killing rates were also determined using limiting dilution experiments (11). The in vitro log parasite reduction ratio, PRR (log10 number of parasites that are killed in a single 48 h life cycle) for MMV390048 was 2.7 at 10x IC50 with a lag phase of 24 h (Fig. 1b). In the recently developed erythrocytic IC50 speed and stage-specificity assays (12), the IC50 values of pyrimethamine and MMV390048 were 7.8 and 4.8-fold higher at the 24 h time point compared to data generated at the 72 h time point (Fig. 1c). These results indicate that MMV390048 is a moderately “slow” killer in vitro. The stage-specificity profile of MMV390048 was also similar to pyrimethamine, with highest activity against young schizonts (Supplementary Section 2, Fig. S2).

Against P. falciparum 3D7, in a humanised SCID mouse efficacy model (13), efficacy was assessed following once daily oral administration of MMV390048 for four consecutive days with blood parasitemia measured by flow cytometry (Supplementary Section 3, Table S4). MMV390048 achieved an ED90 at day 7 of 0.57 mg/kg in this model. The rate of in vivo parasite

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clearance (Fig. 1d) was intermediate, comparable to the reference drug mefloquine (14). From this efficacy study, the minimum blood concentration of MMV390048 necessary to inhibit growth (minimum inhibitory concentration or MIC) was calculated to be 71 ng/mL (Supplementary Section 4).

In vitro and in vivo potential to reduce transmissionIn vitro assays were conducted to analyze the potential of MMV390048 to reduce transmission (Fig. 2a). MMV390048 inhibited the viability of stage IV/V gametocytes in culture with a potency of 285 nM as demonstrated by a decrease in pLDH activity (Fig. 2b). Gametocyte viability assays were also performed on early and late gametocytes expressing luciferase, with MMV390048 achieving potencies of 214 nM and 140 nM respectively (Fig. 2c). Kinetic studies were also performed on these transgenic parasites. As shown in Figure 2d, late stage gametocytes were killed 2.5 times faster than early stage gametocytes, with a 24 h rate of onset of action (correlating to the asexual rate of onset of action). In addition, MMV390048 showed similar efficacy against late gametocytes (>90% stage IV/V) produced from five clinical isolates of P. falciparum compared to laboratory adapted strains, providing evidence of its activity against gametocytes from current clinical populations (Supplementary Section 5, Table S13). When transmitted to the mosquito midgut after a blood meal, stage V gametocytes differentiate rapidly into male and female gametes. This step has been reconstituted in vitro to assess the impact of a compound on the gametocyte functional viability through the formation of gametes. Stage V male gametocytes typically differentiate into eight flagellated motile male gametes by exflagellation, an event that can be observed and quantified under the microscope. After incubating stage V gametocytes with MMV390048 for 24 h and inducing gametogenesis, exflagellation was inhibited with a potency of 90 nM (Fig. 2e).

The transmission-blocking assay displaying the highest biological content is the standard membrane-feeding assay (SMFA). It is performed by incubating the test molecule with stage V gametocytes for either 24 h prior to mosquito feeding (indirect SMFA) or directly at the time of the blood meal (direct SMFA). When tested in the indirect SMFA, MMV390048 inhibited the formation of oocysts (oocyst density) in the mosquito midgut at 111 nM (Fig. 2f). When added directly to the blood meal, MMV390048 inhibited the formation of oocysts by less than 25% at 1 µM (Fig. 2f inset). The difference in potency observed for the indirect feeding mode supports a transmission-reducing role that targets stage V gametocytes in the host blood more efficiently than the forms subsequently developing in the mosquito midgut.

To test the transmission-blocking efficacy of MMV390048 in vivo, a model comprising mouse-to-mouse transmission of Plasmodium berghei infection was employed (15, 16). Briefly, the effect of a transmission-blocking drug can be expressed in terms of impact on the mosquito population, and subsequent vertebrate populations following mosquito bites at variable transmission intensities. Within the course of this study, MMV390048 (administered orally at 2 mg/kg) significantly inhibited transmission to the mosquito host, with a 69.3% and a 30.3% reduction in oocyst intensity and prevalence respectively observed over two replicate experiments (Table 1, Fig. 2g). This resulted in a 37.2% and 46.5% reduction in sporozoite intensity and prevalence. Mosquitoes previously fed on infected, drug-treated mice were allowed to feed on uninfected mice (16). Over multiple mosquito biting rates, a 10.1% reduction in the number of mice that developed blood stage infection was observed in comparison with mice

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bitten by mosquitoes that fed on non-drug-treated infected mice. The overall effectiveness of an intervention over a round of transmission (from mouse to mosquito to mouse) can be quantified by estimating its ability to reduce the basic reproductive number (R0). This has been termed the “effect size” of an intervention. By fitting data from the mouse-to-mouse assay to a chain binomial model, we can estimate the effect size of the intervention, assessing the ability of MMV390048 usage to reduce the basic reproductive number R0 (assuming 100% coverage). Our results estimated an effect size of 28.5% (95% Cls 22.8-33.7), suggesting that MMV390048 is capable of acting as a transmission-blocking agent at lower transmission settings within a field context (15). Positive and negative controls (atovaquone at 0.3 mg/kg and sulfadiazine at 8.4 mg/kg, respectively) performed as expected, and as in previous studies (16).

In vitro and in vivo liver stage prophylactic activity The prophylactic activity of MMV390048 on liver stages that precede symptomatic blood stage infections was determined in vitro against Plasmodium cynomolgi, a simian parasite species closely related to P. vivax (17). This parasite produces in vitro and in vivo both large developing and small non-dividing exo-erythrocytic forms, recently validated as being schizonts and hypnozoites respectively (18). Hypnozoites are capable of later reinitiating growth, causing subsequent disease relapses that are a key clinical feature of P. vivax infection. In the cell-based assay, MMV390048, administered to the primary hepatocyte cell culture 2 h post-inoculum (allowing sporozoites to invade the hepatocytes), showed potent inhibition of liver-stage development of both schizonts and hypnozoites, with IC50 values of 64 nM and 61 nM respectively (Supplementary Section 6, Fig. S6).

The prophylactic effect of MMV390048 was also evaluated in vivo in a P. cynomolgi infected Macaca mulatta model (19). Two cohorts of malaria-naïve monkeys were used during this experiment. In cohort 1, 20 mg/kg of MMV390048 was administered orally to three study animals on day 1 before sporozoite inoculum, while in cohort 2, two control monkeys were administered orally with the same volume of vehicle. On day 0, the monkeys were infected with 1 x 106 sporozoites intravenously and blood smears were taken daily up to day 100 to detect malarial parasites that progressed through the liver stage into blood stage forms. As shown in Figure 3a, parasite patency occurred on day 8 post-inoculum in the two control monkeys. Parasitemia continued to develop until day 11 post-inoculum, at which time both control monkeys received a treatment consisting of a 7-day course of chloroquine (CQ), 10 mg base/kg orally. Parasite clearance occurred in all animals treated with CQ. As CQ cannot eliminate hypnozoites, relapse was expected for the control group due to reactivation of the dormant forms (20). Figure 3a shows that relapse occurred at day 26 for monkey 1 and day 29 for monkey 2 of the control group. These monkeys were then radically cured using a standard 7-day regimen of oral CQ and primaquine (10 and 1.78 mg base/kg, respectively). Once parasitemia was below the limit of detection for at least 5 consecutive days, monkeys were considered as radically cured and taken out of the study (colour arrows in Figure 3a). In contrast to the control group, animals from cohort 1 did not present any parasitemia when observed up to day 100 post-inoculum, revealing the causal prophylactic efficacy and full protection conveyed by MMV390048 (Fig. 3a, monkey 3, 4 and 5).

Blood samples were taken at different time points in order to study MMV390048 plasma-concentration time curves and to relate the pharmacokinetic profile to the efficacy of the

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compound. A peak concentration (Cmax) was obtained shortly after oral administration, reaching 1.98 µg/mL within 24 h. The concentration then decreased below the limit of quantification at around 320 h post administration (Fig. 3c). The terminal half-life of the compound was long (~30 h), allowing coverage above the liver stage IC99 (determined to be 90 nM in the in vitro Pc liver stage assay), for about 6.5 days (arrow in Fig. 3c).

In vitro and in vivo liver stage radical cure activityThe potential for MMV390048 as a new radical cure agent that could lead to the killing of the hypnozoites in the liver was also investigated using the same P. cynomolgi in vitro and in vivo models used to investigate its chemoprotective effect. In the cell-based assay, MMV390048 was incubated with P. cynomolgi infected hepatocytes at different time points of development of the parasites, such as day 1, 3, 4 and 5 post-inoculum, while maintaining a 6-day treatment regimen. The cidality of MMV390048 reduces when the compound is administered to later stage (>24 h old) hypnozoites, with a complete loss of activity when applied at day 5 post-infection (IC 50 shift from 61 nM at day 0 to >10 µM at day 5). Interestingly, the compound remained active when applied to later stage (>24 h old) schizonts despite reduced potency (IC50 shift from 64 nM at day 0 to 1.5 μM at day 5) suggesting that this compound is still active against late stage schizonts (Supplementary Section 6, Table S21).

The lack of radical cure effect of MMV390048 was also confirmed in vivo in the P. cynomolgi infected Macaca mulatta model (19). Similar to the prophylactic experiment, two cohorts of malaria-naïve monkeys were used. On day 0, the monkeys were injected intravenously with with 1 x 106 sporozoites and blood smears were taken daily up to day 100 to detect parasites. The two control monkeys in cohort 1 were administered orally with vehicle (HPMCT). In cohort 2, three monkeys were administered orally with MMV390048 (20 mg/kg oral daily dose for 5 days) at the same time as a 7-day course of CQ, 10 mg base/kg that was given to treat the primary parasitemia. Parasitemia was then monitored on a daily basis until relapse occurred, which happened at day 24 for control monkey 1 and day 29 for control monkey 2. On average, relapses occurred 16.7 days after primary parasitemia vs 9.5 days in the control group (p-value = 0.0397, considered significant - unpaired t test). Blood samples were also taken during this study in order to relate effect to exposure. As shown in Figure 3d, a 5-day administration of MMV390048 led to a high Cmax, reaching up to 5697 ng/mL without any toxicity observed, and a very long coverage of the liver stage IC99 for about 15.8 days. Interestingly, this pharmacokinetic profile demonstrated that the delay in relapse relative to the control group was likely due to suppressive blood stage activity. Indeed, blood stage parasitemia due to reactivation of liver hypnozoites appeared only once exposure of MMV390048 dropped below its minimum inhibitory concentration (determined to be 11.5 ng/mL based on the in vivo PfSCID mice model and correcting for protein binding and blood to plasma partitioning).

Phenotypic correlates of liver stage prophylaxis A P. berghei – HepG2 in vitro model was used to search for phenotypic correlates of the liver stage prophylactic activity seen against P. cynomolgi. Treatment with 460 nM MMV390048 from 2-8 hours post-infection led to clear mislocalization of the parasitophorous vacuole membrane (PVM) protein UIS4 to the parasite interior (Fig. 4). This phenotype was previously associated with efficient liver stage parasite clearance in vitro and causal prophylaxis in vivo by

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the compound Torin2, but is not provoked by known liver stage active compounds in current clinical use (21).

In vitro P. falciparum resistance generation and genome sequencing To identify the mechanism of action of MMV390048, resistant mutants were generated and genetic mutations identified by whole-genome sequencing (22). Initial drug pressure at 46 nM (3x IC50, IC50 determined to be 16 nM in this instance) over a range of inocula (106 to 109) indicated that mutants were obtained with a frequency of about one per 3x106 infected red blood cells in vitro (Supplementary Section 7, Table S22). To generate more resistant parasites, three parasite cultures (109 Dd2 parasites per flask) were subjected to a higher selective pressure of 76 nM of MMV390048 (5x IC50). The cultures recrudesced between days 28 to 35, and were cloned by limiting dilution. Both the bulk culture and clones showed shifts in IC50 and IC90 values, with clones giving an approximately 4- to 5-fold IC50 shift (70 - 90 nM) relative to the parental line (Supplementary Section 7, Fig. S7). Three clones (E6, A2, and B9) were selected for genome analysis.

Whole-genome sequencing of resistant parasites identified a phosphatidylinositol 4-kinase (PfPI4K, Pf3D7_0509800) as a potential resistance determinant. Two clones from the same culture, (flask 2 clones A2 and E6) harbored an A1319V mutation, whereas a third clone (flask 3 clone B9) yielded a S743T mutation in PfPI4K (Supplementary Section 7, Table S23). The read count at both positions exceeded 120-fold coverage with no contaminating wild-type reads. Pfpi4k was the only gene mutated in all three drug-pressured clones, providing evidence in favor of PfPI4K being the target of MMV390048. The program BIC-Seq (23) was used to search for copy-number variations (CNV) in resistant mutants A2, E6, and B9, by varying input parameters empirically. No CNV was detectable in any iteration, in particular none in the pi4k gene (Supplementary Section 7, Fig. S8).

Cross-resistance of MMV390048 was tested against three transgenic parasite lines possessing mutations in the PfPI4K kinase domain that confer resistance to the chemically distinct imidazopyrazine (PI4K-S1320L and -H1484Y) and quinoxaline (PI4K-Y1356F) scaffolds (6). In comparison to the Dd2 parental line (IC50 = 22.7 nM), parasites bearing PfPI4K mutations at H1484Y (IC50 = 62.8 nM) and S1320L (IC50 = 57.7 nM), which is immediately adjacent to the A1319V mutation identified in this study, were cross-resistant to MMV390048, further supporting PfPI4K as the target of this compound.

Chemoproteomics identifies PfPI4K as the target While resistant mutants were being generated, a novel chemoproteomic approach to determine the mechanism of action of MMV390048 was also explored. Covalent immobilization on Sepharose beads of MMV666845, an active analogue containing a primary amine functionality (NF54 EC50 = 0.019 µM, Supplementary Section 8, Table S24), was used to affinity capture potential target proteins from a P. falciparum blood stage extract. Pull-down experiments were performed in the absence or presence of excess “free” MMV390048 to delineate target proteins for which capture is competitively inhibited. In a second experiment, an analogue devoid of antimalarial activity, MMV034137 (NF54 EC50 = 9.2 µM, Supplementary Section 8, Table S24), was added. All proteins captured by the beads were quantified by isotope tagging of tryptic

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peptides followed by LC-MS/MS analysis (24). Notably, MMV390048 competitively inhibited the binding of only a single protein, PfPI4K (PFE0485W, Fig. 5a), to the beads.

A second experiment was performed in which we added “free” MMV390048 over a range of concentrations in order to establish a competition-binding curve and determine a half-maximal inhibition (IC50) value (Fig. 5b and 5c). The IC50 values obtained in these experiments represent a measure of target affinity, but are also affected by the affinity of the target for the bead-immobilized ligand. The latter effect can be deduced by determining the depletion of the target proteins by the beads, such that apparent dissociation constants (Kd

app) can be calculated that are largely independent from the bead ligand (25). The apparent Kd value was determined to be 0.30 µM for PfPI4K (Fig. 5c).

Immobilization of a drug via a linker may not be compatible with binding to all of its targets. Therefore, a similar capturing experiment with “Kinobeads” was performed, which represent a combination of immobilized promiscuous ATP-competitive kinase inhibitors (26,27). As in the previous experiments, PfPI4K was the only P. falciparum protein that exhibited a dose-dependent reduction of bead binding upon the addition of MMV390048 to the extract. These studies yielded an apparent dissociation constant (Kd

app) of 0.1 µM, which is concordant with the previous Kd

app value derived with the bead-immobilized analogue (Fig. 5c). The apparent Kd

values obtained from the chemoproteomics experiments were about 10-fold lower compared to the potency of MMV390048 in parasite growth assays. There are a number of possible reasons for this deviation. Many kinase inhibitors exhibit higher affinity for their target kinases in activated conformations (28,29), whereas the bulk of the PfPI4K protein present in the parasite extracts might adopt an inactive, lower affinity conformation. Alternatively, the compound might accumulate at the site of action in the parasite. We also assessed a set of MMV390048 analogues with antiparasitic activity ranging from low nanomolar to mid micromolar. The chemoproteomics competitive-binding data showed a good correlation between the degree of binding to PfPI4K and antiparasitic activity (Fig. 5d and Supplementaty Section 8, Table S24).

For the identification of possible human host cell targets and off-targets, we adopted the same chemoproteomics strategy and used the same bead-immobilized ligands for pull-down experiments in human K562 erythroleukemia cells. Given the fact that MMV390048 is an ATP-competitive kinase inhibitor, the compound exhibited a remarkably clean selectivity profile. We did not observe any binding to the human PfPI4K orthologues PI4KB (Q9UBF8) and PI4KA (P42356) (Supplementary Section 8, Fig S9, and Supplementary Proteomics Data). Only three human kinases showed any binding affinity to MMV390048. Human PIP4K2C (Q8TBX8) was the major human target and had similar affinity to PfPI4K, whereas the other two targets, ATM (Q13315) and TNIK (Q9UKE5), showed low binding affinity (Fig. 5b and Supplementary Section 8, Fig. S9). The consequences of PIP4K2C inhibition in the host are unknown, but it is interesting to note that this kinase represents an off-target of the marketed chronic myeloid leukemia drug imatinib (gleevec) (26).

Functional evaluation against recombinant P. vivax PI4KBecause phosphatidylinositol 4-kinases are highly homologous across P. falciparum and P. vivax species (30), MMV390048 was evaluated for inhibition of recombinant PvPI4K (6). MMV390048 inhibited the activity of PvPI4K with an IC50 of 0.0034 µM. Similarly, analogue

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MMV034137 that was devoid of in vitro whole cell activity (EC50 = 2.8 µM), had decreased activity against PvPI4K (IC50 = 0.24 µM) compared to MMV390048, and MMV666845, the analogue that was used for affinity capturing, was equipotent (IC50 = 0.0015 µM) to MMV390048. Across a set of analogues evaluated in the PvPI4K functional assay, a close correlation between PvPI4K inhibition and activity against the drug sensitive PfNF54 parasite strain was observed (Supplementary Section 8, Table S24, and Section 9, Fig. S10). This result further supports the genomic and chemoproteomic findings and confirms the mode of action of MMV390048 to be through inhibition of Plasmodium PI4K.

Discussion MMV390048 is a new chemical entity derived from high-throughput screening and phenotypic based optimization. Its lack of cross-resistance with marketed and clinical compounds suggests a potentially novel mechanism of action that strongly affects young schizonts in the erythrocytic stage of the parasite life cycle. Potent in vivo efficacy was shown in both P. berghei (Supplementary Section 1) and P. falciparum mouse models with ED90 values of 1.1 mg/kg and 0.57 mg/kg respectively. Cures were also achieved with a single dose as low as 30 mg/kg, driven by the excellent potency and pharmacokinetic behaviour of the compound (9). Across mouse, rat, dog, and monkey species, MMV390048 maintained a low clearance and a long half-life. Good oral bioavailability was also evident across all species, supporting the correlation between the in vitro potency and the in vivo efficacy. The consistent pharmacokinetic profiles led to a human half-life prediction of around 90 h, and a low dose of 80-100 mg to maintain a therapeutic concentration for 8 days (9, and Supplementary Section 10). MMV390048 therefore has the potential to be used as a component of a single-dose combination therapy.

In addition to its activity on asexual blood-stage parasites, MMV390048 also has potential for interrupting transmission. Its potency against gametocytes was submicromolar and well aligned with the efficacy observed against male gametes and oocysts in mosquitoes. When assessed in the population-to-population transmission model with P. berghei, a moderate reduction in the number of mice that developed blood-stage infection was observed. Further clinical studies will be necessary to assess the compound’s effects on gametocyte viability and fully evaluate its potential for transmission blocking in patients. Nevertheless, the data demonstrating inhibition of transmission from vertebrates to vertebrates is promising for the role that such a molecule could play toward malaria eradication.

MMV390048 also holds promise as a prophylactic and chemoprotective agent as demonstrated by its impact on parasite liver-stage development. Potent in vitro prophylactic activity against the P. vivax-related simian parasite species, P. cynomolgi, was evident in preventing both hypnozoite and schizont development. This prophylactic effect translated in vivo to monkeys infected with P. cynomolgi, with MMV390048 providing causal prophylaxis and full protection. In contrast, liver-stage activity was not evident against late stage hypnozoites (>24 h old and refered to as late stage) in vitro and in vivo as MMV390048 did not deliver a radical cure. This was also recently observed for the PI4K inhibitor KDU691 (31). While MMV390048 remains active on late-stage schizonts, there is a drop in activity when the compound is added at day 5, which could imply that eventually a higher dose will be needed to kill developed liver schizonts in vivo than is needed for blood stage infection. This requires more understanding and will be the subject of future studies. The phenotype in early developing P. berghei liver stages was shown to be

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mislocalization of the parasitophorous vacuole membrane protein UIS4 to the parasite interior, similar to a prior observation with Torin2 (21). Further investigations into the RNA and protein expression levels for the PI4K target will be necessary to evaluate the phenotype in later stage schizonts and hypnozoites.

Chemoproteomic pull-down experiments based on different types of immobilized ligands provided compelling evidence that Plasmodium PI4K is the target of MMV390048. Single nucleotide mutations in the PfPI4K-encoding gene of MMV390048 resistant strains further confirmed PI4K as the target. Although the frequency of resistance to MMV390048 appears high, combination treatment partners will be identified to prevent or delay the emergence of resistance in field isolates. The correlation between phenotypic effect and inhibition of PfPI4K was evident for the 2-aminopyridine series during competitive binding experiments, and also with inhibition studies using recombinant PvPI4K.

PI4K was recently revealed as a novel malaria target (6), and the full implication of this target in the context of malaria eradication has now been elucidated through the complete life cycle profile of MMV390048. Inhibitors of Plasmodium PI4K are important as treatments and as chemoprotective and transmission-blocking agents, but are unlikely to radically cure P. vivax infection based on findings with P. cynomolgi. As a chemoprotective, based on the in vivo P. cynomolgi data, MMV390048 has the potential to be a prophylactic for all human malaria infections without any known risk of hemolysis in G6PD-deficient patients (Supplementary Section 11). As a kinase inhibitor, MMV390048 is selective with respect to almost all P. falciparum and human kinases, thus alleviating potential kinase-mediated safety concerns. MMV390048 has shown an acceptable preclinical safety profile and is currently undergoing clinical assessment. This compound holds the potential to clinically validate Plasmodium PI4K as a target in the treatment of malaria, as well as to establish the role of a PI4K inhibitor in combination therapy.

Notwithstanding the promise shown by MMV390048 based on the data presented, there are limitations to the studies that will need to be validated in humans. Parasite viability and inhibition studies are subject to in vitro analysis and, in the case of liver stage investigations, the use of simian and rodent parasite species. Likewise, in vivo models for transmission, prophylaxis, and radical cure are conducted in animals using the appropriate parasite species, namely P. berghei for mouse to mouse transmission (related to P. falciparum) and P. cynomolgi for liver stage infections in monkeys (related to P.vivax). Pharmacokinetic/pharmacodynamic estimations were also based on preclinical species. Lastly, although MMV390048 is a generally highly selective PfPI4 kinase inhibitor, the full implication of PIP4K2C inhibition in the host, including when used in combination with other antimalarial drugs, is yet to be elucidated.

Materials and MethodsStudy designThe objective of the work was to fully characterize the treatment, prophylactic, and radical cure potential of antimalarial, MMV390048, and elucidate the molecular target of the compound. Efficacy was assessed across blood, liver and transmission stages of the Plasmodium life cycle using both in vitro and in vivo models of infection. The pharmacokinetic/pharmacodynamic relationship was modeled based on compound exposure during efficacy studies and

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pharmacokinetic profiles in pre-clinical species. Target identification was conducted through genetic and chemoproteamic means. In vitro mutant generation indicated a possible drug resistance mechanism linked to a target. The protein target was identified through chemoproteomic pull-down, “Kinobead”, and competitive binding experiments, and was further confirmed by functional enzyme assays.

Animal welfareIn vivo studies were conducted in accordance with the applicable legislation and respective institutional policies on animal use and welfare.

In vitro antimalarial blood stage activity In vitro antimalarial activity was measured using the [3H]-hypoxanthine incorporation assay, which measures dose-dependent drug inhibition of P. falciparum growth (32), as described in Supplementary Section 2.

In vivo antimalarial efficacy studies MMV390048 were tested in the murine P. falciparum SCID model (13), as described in Supplementary Section 3.

P. falciparum gametocyte viability assaysGametocyte viability was determined essentially as described (33,34). Details are provided in Supplementary Section 5.

Male gamete formation assayThe “functional viability” of mature stage V gametocytes was evaluated based on their ability to undergo onward development and form gametes. The compound was incubated with mature stage V gametocytes for 24 h in 96 well plates, after which gamete formation was triggered by a drop in temperature and the addition of xanthurenic acid. 25 minutes post-triggering, male gamete exflagellation was recorded and quantified by automated microscopy.

Standard membrane feeding assayGametocyte infectiousness was determined in the Standard Membrane Feeding Assay as previously described (35). Details are provided in Supplementary Section 5.

In vivo  transmission blockage assessed by a mouse-to-mouse population modelMouse-to-mouse transmission was assessed as described in Supplementary Section 5.

In vitro P. cynomolgi liver stage assayThe liver-stage activity of the test compound was assessed using a validated P. cynomolgi in vitro assay as previously described (36). Furter details are given in Supplementary Section 6.

In vivo prophylactic and radical cure efficacy studies in the P. cynomolgi infected monkey model The prophylactic and radical cure activity of the test compound was investigated using a P. cynomolgi-infected Rhesus monkey model (37), further described in Supplementary Section 6.

P. berghei liver stage phenotyping

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HepG2 human hepatoma cells (ATCC) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 2 mM Glutamax, and penicillin-streptimycin mix (100U/mL) (all Gibco/ThermoFisher). 15,000 P. berghei sporozoites were added to 100,000 HepG2 human hepatoma cells seeded one day prior to infection on glass coverslips, and allowed to invade. Extracellular sporozoites were washed off at 2 hours post-invasion (hpi), and the cells incubated with 460 nM MMV390048 or an equivalent amount of the DMSO vehicle in the control. Cells were fixed 8 hpi, and immunolabelled as in Hanson et al. (21). Images were acquired on a Leica SP8 confocal.

In vitro P. falciparum resistance generation and genome sequencingIn vitro resistance generation and whole-genome sequencing were conducted as described in Supplementary Section 7, including Selecting for resistance in P. falciparum in vitro, IC50

determination for MMV390048-selected and transgenic PI4K mutant P. falciparum, and Bioinformatic analysis of Illumnia MiSeq samples.

Chemoproteomics target identificationChemoproteomics were conducted as described in Supplementary Section 8, including Synthesis of MMV032185, Plasmodium falciparum growth conditions and protein extraction for chemoproteomics, and Chemoproteomics.

PvPI4K inhibition assayThe functional enzyme assay to determine PvPI4K inhibition was performed as described in Supplementary Section 9.

Supplementary Materials:

Section 1: In vivo antimalarial blood stage activity in P. berghei modelMaterials and MethodFig. S1. Onset and recrudescence experiments in the P. berghei mouse model using a single dose of 100 mg/kg compound.

Section 2: In vitro antiplasmodial blood stage activityMaterials and MethodFig. S2. Lack of growth of schizonts compared to rings after 24 h incubation with MMV390048 at different concentrations, indicating blood stage specificity towards schizonts.Table S1. Activity against a panel of resistant strains to determine potential for cross-resistance.Table S2. In vitro parasite reduction data in support of Figure 1B.Table S3. Speed assay IC50 results in support of Figure 1C.

Section 3: In vivo antimalarial blood stage activity in PfSCID mice modelMaterials and MethodFig. S3. Whole-blood levels of MMV390048 after the first dose of treatment during the PfSCID mouse efficacy study.

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Table S4. Efficacy data in the PfSCID mouse model in support of Figure 1D.Table S5. Summary of exposure data in the Pf SCID mouse model following oral administration of MMV390048 once a day for 4 consecutive days.

Section 4: MIC and PRR determinationMaterials and MethodFig. S4. (A) Observed (symbols) and predicted (lines) blood PK profiles from PfSCID mouse efficacy study. (B) Predicted blood PK profile over 4 days after once-daily dosing based on PfSCID mouse exposure.Fig. S5. Observed (symbols) and predicted (lines) parasite load in mouse based on the direct effect model and PfSCID mouse efficacy data. Table S6. Estimated EC50, EC90, and MIC based on modelling of highest net kill rate.Table S8. Estimated PKPD parameters from PfSCID mouse parasitemias and exposure study.Table S9. Estimated PKPD parameters from PfSCID mouse parasitemias and exposures indicating highest net kill rate (red).

Section 5: In vitro and in vivo potential to reduce transmissionMaterials and Methods

P. falciparum gametocyte viability assaysGametocyte ex vivo African clinical isolatesStandard membrane-feeding assayIn vivo  transmission blockade assessed by a mouse-to-mouse population model

Table S10. Late stage gametocyte viability measured by pLDH activity in support of Figure 2B.Table S11. Early and late stage gametocyte viability measured by luminescence in support of Figure 2C.Table S12. Early and late stage gametocyte clearance rates in support of Figure 2D.Table S13. Gametocytocidal activity of MMV390048, DHA and Methylene blue against ex vivo clinical isolates of P. falciparum parasites at a concentration equivalent to their respective IC50.

Table S14. Inhibition of exflagellation in support of Figure 2E.Table S15. Standard Membrane Feeding Assay indirect mode results in support of Figure 2F.Table S16. Standard Membrane-Feeding Assay direct mode results in support of Figure 2F insert.

Section 6: In vitro and in vivo liver stage activityMaterials and Methods

In vitro P. cynomolgi liver stage assayIn vivo prophylactic and radical cure efficacy studies in the P. cynomolgi infected monkey model

Fig. S6. Prophylactic efficacy of MMV390048 in the P. cynomolgi liver stage in vitro assay.Table S17. Parasitemias during the prophylactic P. cynomolgi infected monkey study in support of Figure 3A.Table S18. Parasitemias during the radical cure P. cynomolgi infected monkey study in support of Figure 3B.Table S19. Plasma concentrations of MMV390048 during the prophylactic P. cynomolgi infected monkey study in support of Figure 3C.Table S20. Plasma concentrations of MMV390048 during the radical cure P. cynomolgi infected monkey study in support of Figure 3D.

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Table S21. MMV390048 radical cure (≥24 h addition) vs. prophylactic (3 h addition) effects in the P. cynomolgi in vitro liver stage assay.

Section 7: In vitro P. falciparum resistance generation and genome sequencingMaterials and Methods

Selecting for resistance in P. falciparum in vitroIC50 determination for MMV390048-selected and transgenic PI4K mutant P. falciparumBioinformatic analysis of Illumnia MiSeq samples

Fig. S7. MMV390048-resistant clones show a 4-5 fold shift in IC50 relative to the parental Dd2 strain. Mean (±SD) IC50 and IC90 values for clones A2 and E6 (from flask 2), and clones B9 and C7 (from flask 3).Fig. S8. Copy-number variation analysis using the program BIC-Seq (23).Table S22. Number of triplicates at different inocula that became positive after drug pressure with MMV390048 at approximately 3 x IC50, and their respective days of recrudescence.Table S23. Whole-genome sequence analysis of three cloned Dd2 parasite lines selected for resistance to compound MMV390048.

Section 8: Chemoproteomics identifies PfPI4K as the target Materials and Methods

Synthesis of MMV032185P. falciparum growth conditions and protein extraction for chemoproteomics Chemoproteomics

Fig. S9. Phylogenetic tree of the human (blue) and Plasmodium (red) lipid kinase family.Table 24. Correlation between the degree of binding to PfPI4K, activity against PcPI4K, and antiparasitic activity of MMV390048 analogues.

Section 9: Functional evaluation against recombinant P. vivax PI4KMaterials and MethodFig. S10. Correlation between whole-cell NF54 activity and inhibition of PvPI4K across a selection of MMV390048 analogues.

Section 10: In vivo pharmacokinetics Materails and MethodFig. S11. Pharmacokinetic profiles following intravenous and oral administration of MMV390048 to male Swiss outbred mice, male Sprague Dawley rats, female cynomolgus monkeys and female beagle dogs.Fig. S12. Allometric scaling of clearance from different species as a function of body weight.Table S25. Plasma PK parameters after a single intravenous dose of MMV390048 in different species.Table S26. Plasma PK parameters after a single oral dose of MMV390048 in different species.

Section 11: G6PD deficiency riskFig. S13. Assessment of haemolytic toxicity.

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Acknowledgments: S.G.D., M.J.L.M, M.B., and G.D. would like to thank K. Kammerer and T. Rudi for sample preparation, M. Boesche for operating LC-MS instruments, and T. Mathieson for the generation of the Plasmodium search database. S.W. and C.S. thank Petros Papastogiannidis and Jolanda Kamber for assistance in performing the Pb in vivo assays. We thank L.D. Shultz and The Jackson Laboratory for providing access to nonobese diabetic scid IL2Rgc null mice (NSG mice) through their collaboration with GSK Tres Cantos Medicines Development Campus.

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Funding: We acknowledge the Medicines for Malaria Venture (MMV) for financial support of this research (project MMV09/0002). K.C. acknowledges support from the University of Cape Town, South African Medical Research Council, and South African Research Chairs Initiative of the Department of Science and Technology. D.A.F. gratefully acknowledges support from the National Institutes of Health (R01 AI085584 and R01 AI103058). A.M.Z. and C.H.M.K. acknowledges support from MMV and the Wellcome Trust (translational research grant WT078285).

Author contributions: K.C., L.J.S., D.W., J.B., and M.J.W. supervised the work respectively as project director, leader, coordinators, and consultant providing intellectual input and direction. D.G.C., C.L.M., and T.P. were responsible for data compilation and analysis, and manuscript writing and coordination. P.P.H., T.S.A., M.C.S.L., R.B., and D.A.F. were responsible for resistant mutant generation and sequencing. S.G.D., M.J.L.M., M.B., and G.D. did all chemoproteomic studies. D.L. was responsible for transmission blocking and PfSCID mouse efficacy and related PK studies. M.J.D. and A.R. performed gamete formation assays. L.M.B., J.R., M.B. and K.J.D. performed gametocyte assays. K.J.D. and R.W.S. established the SMFA platform at TropIQ. A.M.B. and S.E.Z. established and performed mouse-to-mouse transmission studies. B.C., C.D., A.M.Z., C.H.M.K., A.T., and P.V. were responsible for in vitro and in vivo Pc studies. S.A.C., K.L.W., D.M.S., J.Ma., J.Mo., and K.C.M. performed and interpreted PK studies across all species. S.W. and C.S. performed in vitro Pf and in vivo Pb efficacy studies. L.S. and J.G. were responsible for in vitro PRR analysis. S.M.S. modelled and estimated inhibitory concentrations. B.J.D., I.A.B., M.S.M., and S.F. did all Pf SCID mouse efficacy and related PK studies, respectively. C.B. and C.S.L. conducted PvPI4K enzyme assays. K.K.H. was responsible for liver stage P. berghei phenotyping. R.R. was responsible for G6PD experiments. D.A.F., M.C.S.L., G.D., B.C., D.L., S.A.C., and S.W. also contributed to manuscript writing.

Competing interests: M.J.W. and the Medicines for Malaria Venture holds a patent on MMV390048.

[Figures and tables captions:]Fig. 1. In vitro speed of action of MMV390048. (A) 2-aminopyridine MMV390048. (B) In vitro parasite reduction ratio depicting the number of parasites (3D7) over time after treatment with 10x IC50 of MMV390048 compared to other antimalarials. (C) IC50 speed assay (NF54 unsynchronized culture; mean ± SD of n ≥ 3 independent assays) indicating activity at different incubation times. (D) Parasitemia as a function of time following once daily dosing for 4 days in the P. falciparum SCID mouse model (n = 1 per dose level). Dosing was started on day 3 post-infection. All data points are included in Supplementary Tables S2, S3, and S4.

Fig. 2. In vitro and in vivo transmission blocking potential of MMV390048. (A) P. falciparum sexual stages and the various readouts used in drug screening assays. (B) Gametocyte viability assay performed by measuring pLDH activity of non-recombinant stage IV-V parasites. MMV390048 in black and dihydroartemisinin in blue (n = 2). (C) Gametocyte viability assay performed measuring luminescence of recombinant parasites expressing luciferase. Stage I-III gametocytes in black, and stage IV-V gametocytes in red (n = 3). (D) Stage I-III (black) and stage IV-V (red) gametocyte clearance rates at IC50 concentration over a three-day period (n = 3). (E) Male Gamete Formation Assay (reference DDD107498 IC50 = 1.8 nM (7), n = 4). (F) Dose response of MMV390048 in the Standard Membrane-Feeding Assay in indirect mode, i.e. with

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24 h of exposure of P. falciparum stage V gametocytes to MMV390048. Insert displaying direct mode, i.e. MMV390048 incubated with P. falciparum stage V gametocytes with concomitant blood feeding of mosquitoes showing <50% inhibition at 1 µM (n = 2). (G) Mouse to mouse model of transmission. All data points are included in Supplementary Tables S10, S11, S12, S15, and S16.

Fig. 3. Efficacy of MMV390048 in the P. cynomolgi infected Rhesus model. Measured daily parasitemia for individual monkeys used during the prophylactic (A) or radical cure (B) experiments. Black arrow indicates the day at which CQ treatment (10 mg base/kg p.o) was started to clear the developing parasitemia due to primary infection. Treatment in panel (A) was started at Day -1 (not shown in this figure), and dark green arrow indicates when treatment with MMV390048 (20 mg/kg once daily for 5 days) was started in panel (B). Red and blue arrows in panel (A) represent the days at which control group monkeys 1 and 2 respectively were taken out of the study due to their radical cure rescue following administration of a treatment regimen consisting of CQ and primaquine (10 and 1.78 mg base/kg p.o) in order to treat parasitemia due to relapse. (C) Mean MMV390048 plasma concentration time curves (20 mg/kg, p.o, single dose) for the 3 monkeys tested in the prophylactic group experiment performed in the Pc infected Rhesus model. The red arrow represents the time above the EC99 ± SEM (353 ± 32 nM or 138 ± 13 ng/mL) measured in vitro in the Pc liver stage assay in a prophylactic mode and corrected for both Albumax (54.3%) and Protein binding (86.1%). (D) Mean MMV390048 plasma concentration time curves (20 mg/kg, p.o, 5 doses regimen as represented by dark green arrows) for the 3 monkeys tested in the radical cure group experiment performed in the Pc infected Rhesus model. The black line represents the blood stage minimum inhibitory concentration (11.5 ng/mL free concentration) calculated from the in vivo SCID mouse efficacy model, and the red arrow represents the time above the EC99 ± SEM (353 ± 32 nM or 138 ± 13 ng/mL) measured in vitro in the Pc liver stage assay in a prophylactic mode. Both the MIC and the liver stage IC99 were corrected for protein binding (86.1%), blood to plasma partitioning (0.86), and Albumax (54.3%) respectively. Finally, the blue arrow represents the time at which monkeys 3, 4 and 5 relapsed. LLOQ define the lower limit of quantification obtained. All data points are included in Supplementary Tables S17, S18, S19, and S20.

Fig. 4. Phenotype of MMV390048 treated P. berghei liver stages. (A) P. berghei early liver stages treated with 460 nM MMV390048 from 2-8 hpi, displaying mislocalization of the parasitophorous vacuole membrane (PVM) protein UIS4 to the parasite interior (red, goat polyclonal antibody) compared to treatment with an equivalent amount of DMSO (1:1000) as control (B). Cytoplasmic P. berghei heat shock protein 70 (PbHSP70) is shown in green (2E6 monoclonal antibody). Images are single confocal sections.

Fig. 5. Identification of Plasmodium PI4 Kinase as target of MMV390048 by chemoproteomics. (A) MMV390048 binds to P. falciparum PI4 kinase in parasite extracts. An analogue of MMV390048, MMV666845, was covalently immobilized on Sepharose beads and used for affinity capturing of potential target proteins from a P. falciparum blood-stage extract. The addition of excess MMV390048 (10 µM) to the P. falciparum extract competitively inhibited the binding of P. falciparum PI4 kinase to the beads. No other protein was reproducibly inhibited. The addition of MMV034137, a closely related structural analogue devoid of anti-

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malarial activity, had no effect on the capturing of PfPI4K. (B) MMV390048 binds to the ATP-binding site of Plasmodium PI4K but not human PI4Kα or PI4Kβ. A set of 7 promiscuous ATP-competitive kinase inhibitors was covalently immobilized to beads (“Kinobeads”) and used for affinity capturing of potential kinase targets from a P. falciparum blood-stage extract, and from human erythroleukemia (K562) cells. The plot illustrates pIC50 values obtained in two independent replicates of the experiment. The addition of MMV390048 to the extract competitively inhibited the binding of PfPI4K to the beads. PIP4K2C was the only human target protein affected with an IC50 value in the same range as PfPI4K. (C) The addition of MMV390048 to the extract over a range of concentrations yielded an apparent dissociation constant (Kd

app) of 0.3 µM using MMV666845 for affinity capturing and a calculated Kdapp of 0.1

µM using Kinobeads for affinity capturing. (D) Structure-activity relationship (SAR) of the MMV390048 lead series revealed excellent correlation of anti-malarial activity and PfPI4K binding in parasite extracts. 14 compounds from the aminopyridine (MMV390048) series with increasing activity against P. falciparum were subjected to profiling on immobilized analogue-beads (see also A). With increased potency against P. falciparum, the compounds showed increased competition and thus reduce the PfPI4K binding to the bead matrix. r: Pearson correlation coefficient; p: p-value (calculated probability). All data points are included in Supplementary proteomics spreadsheet.

Table 1. Transmission blocking effects of MMV390048 (2 mg/kg) measured in the mouse-to-mouse transmission model.

Effect on transmission to the

mosquito host

Tr Bl effect – oocyst intensity (%)(95% Cls)

69.3%(65.7-73.1)

Tr Bl effect – oocyst prevalence (%)(95% Cls)

30.3%(28-32.6)

Tr Bl effect – sporozoite intensity (%)(95% Cls)

37.2%(23.6-48.4)

Tr Bl effect – sporozoite prevalence (%)(95% Cls)

46.5%(19.9-65.6)

Effect on subsequent

transmission to the vertebrate host

Inhibition of infection of naive mice (%)(P-value – Fishers’s exact)

10.1%(0.075)

Pre-patency (± SEM)compared to 6.1 days (± 0.28) in control

mice

6.8 days(± 0.28)

Effect size(95% Cls)

28.5%(22.8-33.7)

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