Glideosomal GAP50 binders that inhibit invasion and restrict malaria parasite in vitro and in vivo

Malaria continues to be a killer disease even in the modern world. Vaccines and drugs have a lot to learn from the malaria parasite before they can be successful. Here, using a lter for glideosomal anchor protein PfGAP50, we have explored a plethora of small molecules to shortlist eight GAP50 binders with promising antiplasmodial activity (IC 50 < 3 µM) that are also highly selective. Of these, Hayatinin, Bedaquiline, MMV688271, Curine, and Brilacidin with PfINDO IC 50 ≤ 1 µM were found to stall merozoites invasion by inhibiting IMC formation besides increasing ROS levels in trophozoites. Bedaquiline loaded healthy RBCs showed prophylactic ability to prevent intraerythrocytic development of malaria parasite. Synergistic activities with ΣFIC values as low as 0.22 (Curine and Artemisinin) or 0.37 (Bedaquiline and Artemisinin) augur well for the development of drug combinations to combat malaria effectively. Interestingly, orally delivered Bedaquiline (50 mg/Kg b. wt.) showed substantial suppression of parasitemia in the mouse model of malaria.


Introduction
Notwithstanding intense efforts to discover drugs and vaccines and attempts to combat it through vector control, malaria continues to be a deadly curse in Africa, Asia, and South America. With the emergence of artemisinin-resistant parasite strains and the inability of Artemisinin Combination Therapies (ACT) to clear them, there is a need to identify and aim antimalarials at novel targets with new mechanisms of action. In humans, the mosquito vector-borne malaria parasite traverses through different developmental stages, rst in the liver and then in the blood. Most importantly, exponential parasite proliferation accompanied by the malaria pathology is initiated following the invasion of RBCs by merozoites. Hence malaria can be stymied if invasion by blood-stage malaria parasite is blocked.
The invasive molecular machinery of the malaria parasite is ne-tuned for the initial attachment of merozoites, followed by the formation of a tight junction between the invading merozoite and the red blood cell membrane. However, the force required to penetrate the host RBCs is generated by the parasite's multiprotein glideosomal machinery consisting of GAP50, GAP45, MTIP, and MyoA. Of these, GAP50 plays a pivotal role by acting rst as a scaffolding protein and next by anchoring the glideosomal complex to the inner membrane leading to the positioning of the complex in the space between the plasma membrane and the inner membrane 1,2 . Among the proteins of this complex, actin-myosin and myosin A/MTIP complex have been explored as drug targets, and a few natural products such as Latrunculins, Cytochalasins and synthetic pyrazole-based urea peptides such as C416 have been shown to prevent RBC invasion by blocking actin polymerization and MTIP/MyoA association 3,4 .
In this study, we have chosen to explore small molecules of diverse chemical classes to target the hitherto neglected, highly conserved PfGAP50 anchor protein of the glideosomal machinery. Further, PfGAP50 has been found also to be a key member of PfPhil1, a newly identi ed multiprotein complex located close to glideosomal complex within the inner membrane complex and to be crucially involved in invasion 5 . We surmised that small molecules targeting PfGAP50 might disrupt the delicate organization of the glideosomal complex and the Phil1 complex and prevent invasion, thereby limiting the exponential rise in %Parasitemia. To achieve our goal, we rst screened about a thousand different compounds coming from azepino quinolines, MMV pathogen, and pandemic boxes and medicinal plant-derived natural products against PfGAP50 using Schrödinger drug discovery suite. Further, we used recombinantly expressed, and a nity-puri ed PfGAP50 to study the binding of this protein to our library of small molecules using surface plasmon resonance (SPR). Our screening has led to the identi cation of ve potent antiplasmodial lead molecules endowed with high selectivity and low resistance indices that are gifted with the ability to prevent the nefarious act of invasion by the malaria parasite. In the present work, these ve anti-invasion molecules with a potential to develop into new antimalarials have been studied for the rst time against malaria.

Results
A total of 951 different compounds from diverse chemical libraries [pathogen box (400), pandemic box (400), Azepino quinolines (140), and specialized secondary metabolites puri ed from C. pareira (11)] were screened for their in silico binding to PfGAP50 by via (a) Schrödinger drug discovery suite, (b) differential scanning uorimetry (DSF) and (c) SPR. As shown (Fig-1a), employing different lters at successive stages, we shortlisted 345 (after in silico Extra Precision (XP) mode docking and DSF), 39 (after in vitro antiplasmodial assay), 8 (after consideration of both anti Pf IC 50 and selectivity indices) and 3 (after checking the disruption of inner membrane complex and invasion). Upon analysis of results, it was interesting to know that of the pathogen and pandemic box molecules shortlisted as PfGAP50 binders, 20 were those that had been pre-approved by the FDA for the treatment of fungal, bacterial, and viral infections.
For molecules selected from MMV, the PfGAP50 KD values (in parentheses, µM) observed were Me oquine (40) Pentamidine were identi ed as binders to PfGAP50, their discontinued use against malaria 6-8 led us not to subject them for detailed in vitro stage-speci c and IMC inhibition assays. Among 11 molecules from C. pareira (SI-1_Fig 2), Hayatinine, Curine, and Magnocurarine were identi ed as PfGAP50 binders with KD below 100 µM (SI-1_Table-2).
In vitro antiplasmodial potency and mammalian cell cytotoxicity Our next step for shortlisting molecules was screening for potencies of molecules for their antiplasmodial IC 50 s against chloroquine-sensitive Pf3D7, chloroquine-resistant PfINDO, and artemisinin-resistant PfCam 3.1 R539T strains. We shortlisted the best top eight molecules on the basis of IC 50s and selectivity indices ( Fig-1b Inhibition of asexual stages of P. falciparum In its replicative phase, the malaria parasite traverses through diverse cell cycle stages like the ring (R), trophozoite (T), and the Schizont (S). These three stages have morphologically identi able sub-stages like early (E), intermediate and late (L). During these transitions, there are distinct changes in chromatin remodelling, transcriptome, and proteome of the malaria parasite, making some targets fade away and some new targets emerge 9 . Hence it is not surprising to expect that IC 50 determined against different stages of the malaria parasite can be quite different. In a bid to explore the differential vulnerability of cell cycle stages of the malaria parasite to our lead antiplasmodial molecules, we subjected different stages of the parasite to each lead molecule at different concentrations. As shown (Table-1), there were marked differences in the observed IC 50s against different stages. Taking ER to LR, ET to LT, ES to LS, and LS to ER stages into consideration, the ratio of the highest to the lowest IC 50s obtained ranged from 54.3 µM (USINB4-124-8) to 0.58 µM (MMV688271). As a speci c example, USINB4-124-8 showed a much higher IC 50 (µM) (38.12) against trophozoites and schizonts (54.32) than against rings (7.2). In contrast, MMV1782353 and MMV642550 showed good activity (IC 50 < 5 µM) against early and late schizonts while showing less potency (IC 50 > 8 µM) against rings (Table-2). It is noteworthy that despite great structural resemblance (Fig-1b), Hayatinine (608.7 Da) showed a marked preference to prevent LS to ER transition whereas the preference for Curine (594 Da) was ES to LS along with ET to LT transitions.

Kill kinetics of test molecules
To understand the time kinetics of action by these test molecules on development, egression, and invasion events of P. falciparum, we treated PfINDO rings and schizonts for variable times. Brie y, rings (6 -12 h p.i), and schizonts (32 -36 h p.i), were treated with test molecules (at IC 50 and 2x IC 50 ) for variable times (D + ) as indicated (Fig-2a) Monitoring growth of malaria parasite in healthy RBCs "preloaded" with test molecules Test molecules retained inside RBCs can prevent the growth and maturation of the parasite. However, entrapment of test molecules by healthy RBCs is hitherto not a very well explored prophylactic strategy. In this experiment, healthy RBCs preincubated with different test molecules for 72 h and thereafter given centrifugal washes to remove test molecules from the medium, were exposed to mature "about to egress" schizonts. Parasitemia was monitored after 96 h. Control, Curine, Hayatinine, MMV642550, and USINB4-124-8 treated RBCs showed 100 % growth (Fig-2b). On the other hand, MMV642550, MMV1634402, and MMV688271 treated RBCs showed only 50 % growth of control indicating a decrease in growth and maturation rate of the parasite. Interestingly Bedaquiline loaded RBCs, showed necrotic trophozoites with % growth < 5 %. It appears that Curine, Hayatinine, MMV642550, and USINB4-124-8 are either not taken up or easily washed off and are not retained by healthy RBCs. On the other hand, MMV642550, MMV1634402, and MMV688271 appear to be retained partially, which causes partial retardation in parasite growth. In a prophylactic role, Bedaquiline appeared to be the best in being retained at concentrations that kill the parasite (Fig-2b).

Effect of lead molecules on the rupture of schizonts and invasion by merozoites
Molecules with IC 50 < 10 µM that were preventing egress or invasion during LS à ER transition were studied to examine inhibitions of egress from schizonts and invasion by merozoites into RBCs. In the presence of MMV1782353/ MMV642550, schizont maturation was found to be stalled but once these molecules were removed schizonts were able to rupture and release merozoites. However, the released merozoites were unable to invade, leading to a sharp decrease in %P with respect to -ve control (Fig-2c). In the case of Hayatinine, and MMV1634402), 8 h treatment led to arrest at 46 to 48 h p.i schizonts with few merozoites stuck to the RBC membrane. Curine allowed schizont egress, but the level of invasion was signi cantly reduced. Both Bedaquiline and MMV688271 showed schizontocidal activity on mature about to rupture schizonts (Fig-2c).

Effect of test molecules on ROS levels
Molecules showing IC 50 (LRàT) < 10 µM, were taken at 2 x IC 50 to assess their effects on ROS levels.
Bedaquiline and MMV688271 were found to elevate ROS similar to H 2 O 2 (100 µM) while MMV642550/MMV1634402 showed ROS levels similar to ART (700 nM). Further, in all samples, ROS levels were found to be quite similar at both 8 h and 12 h, suggesting 8 h treatment is su cient to have maximum ROS levels. Although the parasite is adept in regulating ROS via the formation of hemozoin, Glutathione, and Catalase, the large excess production stimulated by the small molecules could overwhelm the parasite's ROS homeostasis, leading to its death. Among the seven molecules selected, only two molecules viz. Hayatinine and Curine showed only moderate elevation in ROS (Fig-2d). This may be due to the combined effects of ROS stimulating and ROS scavenging 10 activities of these two molecules.
Putative antiplasmodial targets of Bedaquiline, Hayatinine, MMV1634402, MMV688271, and Curine To nd if there may be alternate targets other than GAP50 for the promising molecules studied by us, we performed cross-docking against 68 different malaria proteins, involved in different metabolic pathways of Plasmodium. Each lead molecule was docked into the active sites of selected proteins, and the scores for docking and dG binding were computed. Out of the 68 proteins studied (SI- Table 2), the number of the protein-ligand complexes with negative values for dG binding score were 63 (MMV688271), 47 (Bedaquiline), 18 (MMV1634402), 31 (Hayatinine), and 42 (Curine) (SI-1_Table-9). Thirteen different proteins showed binding to all the ve lead molecules (Fig-2e).

IMC formation and invasion inhibition using Phil1 marker
Since PfGAP50 is involved in the formation of IMC during schizogony 11 and anchors key proteins involved in invasion 12 , we checked whether our lead GAP50 binder molecules could perturb IMC formation and invasion by merozoites. Indeed, two of the major inner membrane multiprotein complexes viz. Glideosomal complex 13 and Phil1 complex 14 play a key role in invasion. Interestingly pull-down experiments have demonstrated the presence of GAP50 in both these complexes 2,5,11 . We hypothesized that if IMC formation is disrupted, then the regular arrangement of Phil1 seen in control cells will not be seen in parasites with disrupted protein complexes. Our results showed that untreated control parasites showed distinct punctate green uorescence marks on the periphery of schizont, indicating proper organization of Phil1 with GAP50 in IMC, and such parasites were highly invasive (Fig-3, left panel, row A). However,in Bedaquiline, Hayatinine, and MMV1634402 treatment groups, an irregular distorted, depleted pattern of Phil1 uorescence around developing merozoites was seen, suggesting a defect in IMC formation. In all instances where IMC formation was found to be distorted (Fig 3, left panel, rows C, D, and F), we noticed a blockade of invasion as well (Fig 3, right panel, rows C, D, and F). However, MMV688271 and Curine, which did not show any effect on IMC formation, nevertheless caused inhibition of invasion (Fig-3).
In silico stabilities of complexes of PfGAP50 with Bedaquiline, Hayatinine, MMV688271, Curine, and MMV1634402 Studying the ner nuances of the mode of binding of small molecule ligands to the target proteins of interest can facilitate the development of structure-based novel therapeutics. Towards this goal, Bedaquiline and MMV688271 (pathogen box), Hayatinine and Curine (Cissampelos pareira), and MMV1634402 (pandemic box) were shortlisted based on the in silico screening followed by MD simulation to assess the dynamic stability of their intermolecular interactions with amino acid residues constituting the respective binding pockets in PfGAP50 (Fig-4).
Bedaquiline interaction was found to be primarily through hydrophobic interactions (W35, I69, H256, M278), hydrogen bonding (G65), and water bridge (N221), as seen through MD simulation (Fig-4a). These interactions were found to be maintained over 30 % of 100 ns simulation time and appeared to help in stabilizing the binding of Bedaquiline with GAP50, resulting in -36.9 kcal/mol binding energy. The sensorgram pro le obtained using one-on-one interaction with GAP50 suggested incomplete dissociation with a binding a nity (KD) 9.27 µM. Curine, that shares structural similarity with Hayatinine, was also found to bind to GAP50, but at a completely different site from that of Hayatinine (SI-1_Fig-11). Residues interacting with Curine via hydrogen bonds, hydrophobic interactions, and water bridges were with Y96, L134, D135, D137, A138, V350, and E351 (Fig-4b). On the other hand, Hayatinine was found to interact with G119, Q120, E133, M146, P147 and H152. The Hydrogen bonds, hydrophobic bonds, and water bridges with backbone and side chains of these amino acid residues persisting for more than 30 % of simulation time seem to prevent surface binder Hayatinine from moving out of its binding site. Interactions of MMV688271 (Fig-4d) with GAP50 were found to be stabilized through Hydrogen bonds (E123, H152, F344, L347, P348), water bridges (E123, H152, F344, L347), and hydrophobic interactions (H152, F344, L347, V350). MMV1634402 (Brilacidin) binding with GAP50 was found to be majorly driven by water bridges mediated via side chains and the backbone of N127, E129, N148, D201, I322, N349, E351, and L352 between ligand and protein which were further strengthened by Hydrogen bond interactions involving E129, E351, and L352. Amongst these interactions, the ones with E129 and E351 were maintained for > 50 % of 100 ns simulation time (Fig-4e). A large number (sixteen) of water bridges between Brilacidin and GAP50, may have contributed to the lowest -dG of binding -64.82 kcal/mol, with a KD 2.81 µM.
In vitro effects of the combination of lead molecules with antimalarial drugs on ART resistant P. falciparum Drug combinations help in the assessment of synergistic, additive, or antagonistic activities. This, in turn, helps in the selection of combinations that retard the development of resistance, reduce toxicity, and increase the e cacy of the drugs used. Herein, we tested our lead molecules (Hayatinine, Bedaquiline, MMV688271, Curine, and MMV1634402) in combination with standard antimalarial drugs (Me oquine, Pyrimethamine, and ART) for their activity against intraerythrocytic development of ART resistant P. falciparum. In different test molecule and antimalarial drug combinations, strong synergy (∑FIC < 1) was observed at a 1:4 molar ratio (Table-2). These results show the importance of our lead molecules in providing synergistic combinations for effective antimalarial therapy with a reduced probability of drug resistance. In vivo toxicity and antimalarial study of Bedaquiline, and USINB4-124-8 USINB4-124-8 at 100 mg/kg b.wt did not show any signs of acute toxicity (SI-1_Table-10) and till day 16 mice were healthy with respect to control mice. Bedaquiline acute toxicity was not carried out as this molecule is already FDA approved for tuberculosis treatment. Treatment of P. berghei ANKA infected mice with vehicle solution (-ve control) led to the progressive increase in %Parasitemia with a median survival of 19 days and death of all 7 mice by day 22. These mice exhibited increased uctuations in body temperature and a decrease in mean body weight with the progression of the disease. Interestingly, Bedaquiline (50 mg/kg b. wt.) resulted in the complete suppression of %Parasitemia by day 19, reduced uctuations in temperature and body weight as compared to -ve control, and survival of three out of seven mice till 34 days (Fig-5). On the other hand, USINB4-124-8 (100 mg/kg b. wt.) showed no difference from -ve control in terms of survival, % rise in parasitemia, uctuation in body weight, and temperature (Fig-5).

Discussion
Control measures against the vector Anopheles mosquito and extensive use of artemisinin-based combination therapy (ACT) against the parasite have resulted in a marked decrease in malaria cases in the last decade 15 . However, the surge in malaria cases in developing Asian countries which have reported resistance to CQ, sulfadoxine-pyrimethamine, ACT, and other antimalarial combinations, demands new antimalarials against new targets to combat the threat of drug-resistant strains. An emerging strategy to kill Plasmodium is to target its Achilles' heel, viz invasion by sporozoites into hepatocytes, merozoites into erythrocytes, and ookinetes into mosquito gut wall. As many proteins of the merozoites bear similarity with proteins found in sporozoites and gametocytes 12,16 , a small molecule preventing invasion by binding to a common target could block entry of malaria parasite into the safe milieu of different cells in man and mosquito. This could enable the immune system to kill the "out of cell" malaria parasite and not allow it to create havoc by the exponential rise in its numbers upon successful invasion into a cell. Once dead, resistance is not possible either. Thus, invasion into the host cell is the malaria parasite's greatest strategy to escape from the host immune responses because the immune system fails to be provoked into action by an intracellular parasite. We aimed to block invasion so as not to give the parasite a chance for the exponential rise in %Parasitemia that malaria parasite ensures from within the safe con nes of the host cell. Despite its vital importance to the parasite, only the major surface proteins of merozoite have been targeted for both vaccine and drug development while the highly conserved glideosomal proteins have remained largely neglected 17 . These motor proteins are involved in the meticulously organized development of the inner membrane complex that includes the IMC resident multiprotein complexes that are known to facilitate the highly e cient and extremely fast process of invasion by merozoites, sporozoites, and ookinetes. Among these proteins, glideosome associated protein 50 aka GAP50 is highly conserved in apicomplexan parasites and acts as an anchor for other motor proteins. Further GAP50 bears low homology to human protein homologs 18 which makes it a suitable target for drugs. The presence of conserved regions on the surface of GAP50 19 , zygote motility inhibition by anti-GAP50 antibodies 20 , and the ability of GAP50 to bind human complement factor H 21 suggest that this protein is of vital importance to the parasite. Hence GAP50 is well suited for designing the next generation of novel drugs against malaria.
In the absence of known inhibitors against this protein, our study has focussed on compounds belonging to diverse chemical classes for screening using in silico molecular docking and DSF with PfGAP50 as the target. These two tools helped us in hit enrichment in a high throughput manner and narrowed down compounds for in vitro SPR and in vitro antiplasmodial activity. Even as the structural homology between GAP50 and hPAP (human purple acid phosphatases) is only 30% 19 , we selected only those compounds which showed Selectivity Indices > 100 against HUH 7 and HEK293 human cell lines. This criterion helped to eliminate all those compounds that might be toxic by binding with hPAP or any other protein targets in the host. Since GAP50 is expressed in the late trophozoite and schizont stages (30 -35 h p.i) of Plasmodium and is involved in IMC formation 11 , we checked whether the inhibitors identi ed by us were able to prevent successful schizogony and egress of merozoites. In this attempt, we found some GAP50 binders to be schizontocidal. This inhibitory activity was further veri ed by setting up a 57 h stagespeci c assay. This assay revealed that Hayatinine, Curine, USINB4-124-8, MMV688271, MMV1634402, MMV642550, and MMV1782353 were most active against trophozoites and schizonts which represent the mature replicative stages of Plasmodium. Bedaquiline, on the other hand, spared these mature stages and showed the highest activity against the step of invasion by merozoites. Moreover, most of these GAP50 binder molecules caused elevation of ROS levels that were at par with the levels elicited by the endoperoxide harboring Artemisinin's. High ROS levels induced by these molecules can overwhelm the ROS handling mechanisms of parasites leading to their death by oxidative damage. Our results suggest a possibility for protein targets other than GAP50 in the malaria proteome, which can, upon binding to the compounds studied by us, cause the death of the mature stages.
Our results on invasion inhibition showed Bedaquiline, Hayatinine, Curine, MMV688271, and MMV1634402 as potent inhibitors with % inhibition around 90%. Further, to nd if the invasion was inhibited due to disruption of IMC and the GAP50 containing complexes therein, we did immuno uorescence-based confocal microscopy to study IMC formation using Phil1(a member of the Phil1 complex present in IMC) as an IMC marker. In independent experiments, the same compounds were also tested for their ability to inhibit invasion. Our results indicated that Bedaquiline, MMV1634402, and Hayatinine, which prevented IMC formation, also caused invasion inhibition. On the other hand, the schizontocidal compounds Curine and MMV688271, which did not perturb IMC formation, did not inhibit invasion and may have preferential binding with proteins other than GAP50 involved in schizogony.
Data on the in silico binding of our lead molecules, i.e. MMV688271, Hayatinine, Curine, Bedaquiline, and MMV1634402 with GAP50 has shed light on the binding site residues, which can help in the optimization of these inhibitors to increase their potency and selectivity. Interestingly while Hayatinine and MMV688271 binding sites were found to consist predominantly of non-polar residues, the sites that recognized Bedaquiline and MMV1634402 were rich in polar residues. In contrast, the MMV688271 binding site contained an equal proportion of polar and non-polar residues. The involvement of water molecules in stabilizing Hayatinine, Curine, MMV688271, and MMV1634402 binding in their respective grooves shows the importance of these water bridges present in these regions. It is tempting to hypothesize that binding by the above-mentioned lead compounds may perturb the protein-protein interactions that are the hallmarks of the multiprotein complexes found in the IMC.
In a bid to explore what proteins other than GAP50 may bind to these lead compounds, we have found that these molecules may have thirteen different alternate protein targets besides GAP50. The binding of our lead compounds to one or more of these targets may well be the explanation for their multi-stage activity. Coupled with over hundred-fold selectivity, the presence of multi targets in malaria proteome could be a plus point for these compounds since multi-targeting drugs could have low chances of losing e cacy due to the development of resistance 22 .
The longer half-life of small molecules in vivo is usually associated with their association with serum proteins or active uptake in cells from where they are released in small amounts over a period of time 23 . Since malaria parasites actively target RBCs, we explored if healthy RBCs could be preloaded with some of our lead compounds such that these "loaded" RBCs could gain competence to restrict the growth of malaria parasites. Such retention of compounds in RBCs offers a prophylactic strategy that can be useful for passengers prone to traveler's malaria. Our data showed Bedaquiline as the most promising molecule that is retained by RBCs in a concentration su cient at preventing the growth of the parasite. Similar but slightly less potent effects were also observed in RBCs preloaded with MMV642550, MMV1634402, and MMV688271.
The nding of potent antiplasmodial activity in Bedaquiline is highly signi cant since it is a drug already in use against MDR TB 24 . A similar antiplasmodial activity was found in Brilacidin (MMV1634402), a peptidomimetic mimicking the human defensin peptides. Incidentally, Brilacidin has recently been shown to possess antiviral activity against SARS-CoV-2 25 and is currently under human clinical trials for COVID-19 treatment 26 . Our nding of Bedaquiline's ability to prevent IMC formation and subsequent invasion of merozoites became more interesting when we found that Bedaquiline could cure the mice of malaria infection with mitigation of associated symptoms like pyrexia and weight loss.
As a bonus, we found that combinations of MMV688271, MMV1634402, Hayatinine, Bedaquiline, and Curine with standard antimalarial drugs in speci c ratios showed synergistic activity. This seems to open new prospects for use of novel synergistic combinations for the formulation of new ACTs. In conclusion, we have identi ed GAP50 binder molecules with high selectivity and multistage activity, which can be taken for future drug optimization and development as novel drugs against malaria.

Declarations
Pf3D7 genomic DNA was used to PCR amplify the PfGAP50 gene using PfGAP50 speci c primers (5'TTTTCATATGAAATGTCAACTACGCTTTGCG-3'-FP) and (5'-TTTGGATCCTTAATCTTTATTTCCCATGGGTCC-3'-RP). The ampli ed product was cloned in pET28a + vector (SI-1_Fig-7) which was transformed into Rosetta 2 pLyS strain with ligation of PCR product downstream of His tag sequence. Using 1 % inoculum of the primary culture, secondary culture was initiated in TB broth supplemented with CoSO 4 (2 µM), Kanamycin (50 µg/ml), and Chloramphenicol 34 µg/ml 19 . Cells at A 600 2.0 were induced with isopropyl β-D thiogalactopyranoside (0.5 mM) at 18 °C for 16 hrs. Harvested cells were lysed by sonication in buffer A (50 mM Tris HCl, pH 8.3 supplemented with 10 % glycerol and 300 mM NaCl). Cellular debris was removed by centrifugation (13000g, 45 min, 4°C) and the clear supernatant was loaded on to a preequilibrated Ni-NTA column. The column was washed using buffer A with 30 column volumes (CV) and the terminal elute was found to be protein-free as tested by Bradford assay. The bound protein was eluted using buffer B (50 mM Tris-HCl, pH 8.3, 500 mM NaCl, 10 % glycerol, 0.5 M imidazole). The eluted proteins were subjected to hydrophobic interaction chromatography by loading them onto a 1M ammonium sulphate in 50 mM Tris-HCl pH 8.3 (Buffer C) preequilibrated phenyl sepharose column followed by washing with buffer C (30 CVs) and elution using 0.4 M ammonium sulphate. The puri ed protein (SI-1_Fig-8) was then dialyzed against the imidazole free buffer B and stored at 4 °C (up to 2 months).
In silico docking, molecular dynamics simulation and -dG of binding The PDB crystal structure of PfGAP50 (ID: 3TGH) was prepared for docking using the protein preparation wizard 27 of Schrödinger software (version 21-3). Ligand structures drawn using ChemSketch (version 2021.1.2) software were imported into Schrödinger software. Ligprep 27 was used to prepare ligands for docking. Grid was generated to engulf the entire protein, and docking was carried out using the Glide module 28 . Docked poses were then taken for estimation of -dG of binding using the Prime MM-GBSA module 29 . Lead PfGAP50 binders (Bedaquiline, Hayatinine, MMV1634402, MMV688271, and Curine) that showed good promise also in P. falciparum culture-based study were taken for the molecular simulation study 30 . Brie y, the protein-ligand complex was solvated using the TIP4PEW model in an orthorhombic box. Na + and Clions were added to neutralize the system. OPLS3e force eld was used to simulate for 100 ns. In a quest to determine alternate targets of lead molecules, cross-docking was performed using in silico Schrödinger tool. Brie y, malaria parasite protein structures were taken from PDB, and a grid was generated around co-crystallized ligands after preparing the protein and ligand structures. Docking was done using glide module following which binding free energies of protein-ligand complexes were calculated using Prime MM-GBSA tool.

Compounds
Pathogen and Pandemic box compounds dissolved in DMSO at 10 mM were obtained from MMV.
Azepino quinolines 31  Binding a nity determination using surface plasmon resonance PfGAP50 (50 µg/ml) was immobilized on a CM5 chip in acetate buffer, pH 4.5 using EDC-NHS coupling. Small test molecules were diluted in 1x PBS supplemented with 5 % DMSO with 0.005 % Polysorbate 20 (running buffer) to yield solutions of varying concentrations (two-fold serial dilution from 50 µM to 0.3125 µM) that were allowed to ow over the immobilized protein at a ow rate of 30 µl/min. Contact and dissociation times of 60 and 120 sec, respectively were given for each cycle. Regeneration was carried out using 10 mM glycine pH 3.0. Data obtained was tted with a 1:1 binding model to get K a (association constant), K d (dissociation constant), and KD (equilibrium binding a nity constant).
In vitro maintenance of P. falciparum cultures and assessment of antiplasmodial activity of test molecules {100 % Parasitemia (P)} were added to the washed RBCs at nal 1 % P & 2 % H. The plate was then incubated at 37 °C for 96 h with media refreshment every 24 h. Percentage growth was evaluated by SYBR green I lysis method 35 , and the cell cycle stage progressions were monitored microscopically using Giemsa-stained smears.
ROS measurement using CM-H2DCFDA dye ROS measurement was carried out using the protocol provided by the manufacturer (Invitrogen). This technique employs the cell-permeant 2',7'-dichlorodihydro uorescein diacetate (H 2 DCFDA), which in its native ester form is reduced and non-uorescent. However, inside cells, it is trapped and undergoes esterase catalyzed de-esteri cation followed by oxidation to the highly uorescent 2',7'dichloro uorescein (DCF  37 . Schizont enriched parasites were diluted to 1 %P at 2 % H in cRPMI and treated with test molecules at 2x IC 50 for 8 h. This was followed by removal of test molecules by three centrifugal washes with 1x PBS and incubation of the washed cells in fresh cRPMI for 48 h. %P was calculated at the end of the 2 nd cycle using SYBR Green staining and counting 10 5 cells via FACS. Evaluation of GAP50 binder antiplasmodial molecules as disruptors of inner membrane complex formation and invasion Saini et al 14 have described in malaria parasite the presence of yet another multiprotein complex called Phil1 in close proximity of the glideosomal complex. Interestingly both these complexes present in the space between the plasma membrane and inner membrane have GAP50 as one of the constituents and both complexes appear to play a crucial role in the process of merozoite invasion. In the present work, we have used Phil1 antibodies to track the development of Schizont's inner membrane complex (IMC) in the presence vs absence of the small test molecules that bind GAP50. Early schizonts (30 to 34 h p.i) were treated with test compounds for 12 h, followed by indirect immuno uorescence assay (IFA) to assess IMC development. For this, cells were rinsed once with 1x PBS and xed using a solution containing paraformaldehyde (4 % v/v) and glutaraldehyde (0.0075 % v/v) in PBS for 30 min. The xed cells were treated with Triton X-100 (0.1 % v/v) in 1x PBS (twice, 15 min each) and the so permeabilized cells were blocked using BSA (4 % w/v in 1x PBS). Parasites were then incubated with 1°Ab (anti-Phil1, rabbit raised) at 1:50 dilution overnight at 4 °C, followed by washing and incubation with 2 °Ab (Alexa conjugated anti-rabbit antibody 488, Invitrogen) at 1:500 dilution for 3 h at RT. In the end, DAPI (10 µg/mL) was used to stain the nucleus (10 min at RT). Samples were imaged using Nikon A1-R confocal microscope. Similarly, segmented schizont cultures (42 to 46 h. p.i) were treated with test molecules for 8 h and evaluated via IFA for their ability to egress and the resulting merozoites to invade healthy red blood cells leading to the formation of rings.

In vitro potency of the combinations of lead anti-plasmodial molecules
To determine the effect of combining drugs on potency, different drug combinations were made to determine IC 50 of combinations against synchronous ring stage (6 to 12 h p.i, at 1% P and 2 % H) of P. falciparum (MRA1240) in culture. 8x IC 50 of each test molecule in 10 % DMSO / 1x PBS was taken as stock (A) and mixed with 8x IC 50 of standard antimalarials (B) in four different molar ratios (4:1, 3:2, 2:3, and 1:4). Each ratio sample was further serially two-fold diluted in 10 % DMSO/ 1x PBS. Four microliters of each dilution were mixed with 96 µl of parasite culture (1% P, 2% H). Thus, each experimental well contained a total volume of 100 µl with or without test molecules. Control (0.4 % DMSO (v/v)) that is nontoxic to the parasite was used as -ve control (100 % growth) whilst Chloroquine (4 µM) was used as + ve control (0 % growth). The plates were incubated (37°C, 72 h) followed by an estimation of parasite proliferation using the SYBR Green I lysis method 35 . Data analysis was done as described by Thapar et al 38  In vivo acute toxicity study In vivo acute toxicity was done as per OECD 423 guideline 40 . Brie y, mice fasted for 4 h before oral dosing of test molecules. After dosing, mice were kept under observation for the next 45 min for any acute toxicity symptoms followed by resumption of their feed and a daily check over 16 days for any long term toxicity symptoms.

In vivo antimalarial study
In vivo antimalarial study was carried out as per ARRIVE guidelines 2.0 with prior permission from ICGEB animal ethical committee (ICGEB/IAEC/30012021/MPB-8). Brie y, 10 5 P. berghei ANKA infected RBCs were injected into mice and grouped randomly with seven mice/group. After 24 h of infection, test molecules were orally administered in vehicle solution (2% (Hydroxypropyl) methylcellulose with 2 % Tween 80 in Normal saline) for four consecutive days followed by daily monitor of %P, body weight, and surface temperature day 5 to day 30 41 . Chloroquine (50 mg/kg b.wt) was taken as + ve control. Tables   Tables 1-2