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Characterization of lipid binding properties of Plasmodium falciparum acyl-CoA binding proteins and their competitive inhibition by mefloquine Abhishek Kumar, Debasish Kumar Ghosh, Jamshaid Ali, and Akash Ranjan ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.9b00003 • Publication Date (Web): 15 Apr 2019 Downloaded from http://pubs.acs.org on April 16, 2019

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Characterization of lipid binding properties of Plasmodium falciparum acyl-

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CoA binding proteins and their competitive inhibition by mefloquine

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Running title: Lipid binding function of PfACBPs and their inhibition by mefloquine

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Abhishek Kumar1,2, Debasish Kumar Ghosh1,2, Jamshaid Ali1,2,3, and Akash Ranjan1,*

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1. Computational and Functional Genomics Group

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Centre for DNA Fingerprinting and Diagnostics

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Uppal, Hyderabad 500039, Telangana, INDIA

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2. Graduate studies

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Manipal Academy of Higher Education

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Manipal, Karnataka 576104, INDIA

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3. Rajiv Gandhi Centre for Biotechnology Thiruvnanthapuram, Kerala 695014, INDIA

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* Corresponding author

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Email: [email protected]

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Telephone: +91-40-27216159

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Fax: +91-40-27216006

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ORCID of Dr. Akash Ranjan: 0000-0002-4582-1553

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ABSTRACT

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Malaria remains a worldwide concern in terms of morbidity and mortality. Limited

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understanding of Plasmodium proteome makes it challenging to control malaria. Understanding of

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the expression and functions of different Plasmodium proteins will help in knowing this organism’s

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virulence properties, other than facilitating the drug development process. In this study, we

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characterize the lipid binding and biophysical properties of the putative Plasmodium falciparum acyl-

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CoA binding proteins (PfACBPs), which may have intriguing functions in different stages of

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Plasmodium falciparum life-cycle. While the PfACBPs can bind to long-chain fatty acyl-CoAs with high

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affinity, their affinity for short-chain fatty acyl-CoAs is weak. Base-stacking, electrostatic and

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hydrophobic interactions between the aromatic rings, charged groups/residues and hydrophobic

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chain/residues are responsible for acyl-CoA binding to PfACBPs. PfACBPs can also bind to

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phospholipids. PfACBPs cannot bind to the fatty acids and unphosphorylated fatty acid esters.

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PfACBPs are globular/helical proteins that contain a conserved acyl-CoA binding region. They exist in

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folded or unfolded conformations without attaining any intermediate state. In a systematic high-

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throughput in silico screening, mefloquine is identified as a potential ligand of PfACBPs. Binding

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affinities of mefloquine are much higher than that of fatty acyl-CoAs for all PfACBPs. Mefloquine

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binds to the acyl-CoA binding pocket of PfACBPs, thereby engaging many of the critical residues.

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Thus, mefloquine acts as a competitive inhibitor against fatty acyl-CoA binding to PfACBPs, leading to

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prevention of Plasmodium falciparum growth and proliferation. Taken together, our study

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characterizes the functions of annotated PfACBPs and highlights the mechanistic details of their

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inactivation by mefloquine.

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KEY WORDS

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Plasmodium falciparum; Acyl-CoA Binding Proteins; Long-chain fatty acyl-CoA; Mefloquine;

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Competitive inhibition

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INTRODUCTION

83 The parasite-borne malaria disease causes significantly high fatality in developing and

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underdeveloped countries (1). Different kinds of malaria are caused by different species of

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Plasmodium, resulting in a yearly death of millions of people across the globe (2). The complexity of

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Plasmodium life-cycle creates a situation that allows this organism to tolerate many stressful

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conditions and drugs. This makes the malaria biology a challenging field of study. The adenine-

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thymine rich Plasmodium genome encodes many different proteins that help this organism to

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survive in the physiological and altered conditions (3). For example, not only Plasmodium has to

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survive in multiple hosts but it also has to overcome different stresses inside the host organisms.

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Moreover, Plasmodium evades the host surveillance mechanisms by remaining completely latent

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and undetectable inside the host cells (4). While mosquito’s midgut is the site of Plasmodium

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fertilization, the human hepatocytes and erythrocytes are the primary cells for Plasmodium survival

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(5). The ultrastructure of Plasmodium reveals that it has many different organelles, like apicoplast,

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parasitophorous vacuoles etc (6). Due to the presence of these membranous structures and lipid

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storage bodies, Plasmodium requires an abundant supply of different forms of lipids. These lipids are

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utilized in metabolic processes to produce energy, other than being used in the membrane

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formation (7). Synthesis and/or acquirement of the lipids require the functional activity of several

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enzymes, storage proteins and transporters (8). Acyl-CoA binding proteins (PfACBPs) are known to

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be present in different Plasmodium species. The ACBPs are a class of small homologous proteins

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which have evolved as conserved proteins in different eukaryotic organisms. ACBP is known to be

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essential in the membrane biogenesis and lipid storage/transport processes (8). Such functions are

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finally transduced to regulate the parasite’s lipid homeostasis (9). The maintenance of lipid

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metabolism and storage helps the parasites to remain competent in the host system. For example,

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the protozoan parasite Trypanosoma brucei exploits its ACBP to remain viable is host’s bloodstream

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(10).

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Plasmodium falciparum genome annotation reveals the presence of many paralogous genes

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which are recognized as putative acyl-CoA binding proteins (PfACBPs). The microarray data of

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Plasmodium falciparum transcriptome suggests that the three annotated genes, PF3D7_1001200

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(PfACBP16), PF3D7_0810000 (PfACBP99) and PF3D7_1477800 (PfACBP749) have the highest

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expression in the trophozoite stage of Plasmodium falciparum. Since the trophozoite rapidly invades

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to erythrocytes, it needs an affluent availability of lipids to sustain the high proliferation rate (11).

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This can be a reason for higher expression of different PfACBPs in the trophozoite stage. However,

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detailed information of the roles of PfACBPs in cell physiology is still unknown. We find it interesting

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to explore the structural and functional properties of the putative PfACBPs. Studies on the PfACBPs

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will not only help us in understanding of the complex lipid-flux regulation in the parasite but these

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will also allow us to consider PfACBPs as a potential druggable target. The rationale of this study is to

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systematically analyze the functionality of PfACBPs, followed by identification of the small molecule

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modulators(s) of PfACBPs. Although drugs are available to curb malaria (12), there is an increasing

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demand of new pharmacological molecules to mitigate the challenges of Plasmodium infection, 3 ACS Paragon Plus Environment

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resistance and virulence. The increasing information of sequence data from Plasmodium falciparum

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and human genome projects, in combination with the structural data, is facilitating the identification

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and design of novel drugs.

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In this study, we have characterized the acyl-CoA binding and biophysical properties of

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PfACBPs. We have also identified mefloquine as a potential small molecule modulator that can

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competitively inhibit the fatty acyl-CoAs binding to PfACBPs. We observe that PfACBPs have higher

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binding affinity for the long-chain fatty acyl-CoAs than the short-chain acyl-CoAs. PfACBPs can also

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bind to the phosphorylated lipids, but not to the unphosphorylated lipid esters. PfACBPs are alpha

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helical globular proteins. While PfACBPs can exist in the folded and unfolded conformations, we do

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not find them to adopt any intermediate state. This indicates that acyl-CoAs bind to PfACBPs in the

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protein’s native state without inducing any allosteric conformational changes. An in silico high-

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throughput virtual screening and in vitro binding assays have presented mefloquine as a strong

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ligand of PfACBPs. Mefloquine binding to PfACBPs prevents the association of the acyl-CoAs to the

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PfACBPs, resulting in a competitive inhibition of fatty acyl-CoA binding to the proteins. We have

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confirmed that mefloquine is lethal for Plasmodium falciparum growth and proliferation. Our studies

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indicate that one of the mechanisms in which mefloquine exerts its toxicity to Plasmodium

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falciparum is by inactivation and degradation of the mefloquine bound PfACBPs.

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RESULTS

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Plasmodium falciparum acyl-CoA binding proteins bind to a variety of lipids

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Plasmodium requires an abundant supply of lipids to survive inside the human erythrocytes

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(13). It is reported in previous studies that lipids are heavily accumulated inside the Plasmodium cells

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(14), including the apicoplast of Plasmodium (15). In this context, the lipid binding proteins,

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especially the acyl-CoA binding proteins (PfACBPs), are meant to play significant functions in storage

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and transport of lipids in the complex cellular environments (apicoplasts, lipid storage granules etc.).

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Plasmodium falciparum proteins, PfACBP16 and PfACBP99, were previously annotated as the

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putative acyl-CoA binding proteins. In the first part of this study, we characterized the functional

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properties of these acyl-CoA binding proteins. All of the three PfACBPs were capable of binding to

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the fatty acyl-CoAs. We performed surface plasmon resonance (SPR) based experiments to find the

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binding pattern and affinities of the PfACBPs for different chain-length fatty acyl-CoAs. While all of

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the PfACBPs could bind to the long-chain fatty acyl-CoAs with high affinity, they showed very weak

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binding potential for the short-chain fatty acyl-CoAs (Figure 1A, 1B, 1C). High binding capacity of

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PfACBPs for different long-chain fatty acyl-CoAs like, palmitoyl-CoA, stearoyl-CoA etc., were

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observed (Table 1). It was noted that the binding affinities of PfACBPs were increasingly higher for

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the very long-chain fatty acyl-CoAs, like arachidonoyl-CoA, oleoyl-CoA etc (Figure 1D; Table 2). The

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binding affinities of different long-chain fatty acyl-CoAs did not differ considerably for the PfACBPs

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(Table 1). The Plasmodium acyl-CoA binding proteins did not bind to the short-chain fatty acyl-CoAs 4 ACS Paragon Plus Environment


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with high affinity (Table 1). The affinities decreased gradually towards the chain length of the short-

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chain fatty acyl-CoAs. Although PfACBP16 and PfACBP99 could weakly bind to the short-chain fatty

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acyl-CoAs, it was noted that the PfACBP749 did not bind to any of the short-chain fatty acyl-CoAs

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that had lower than 10-carbon chain length. A comparative analysis of the binding affinities of

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PfACBP16, PfACBP99 and PfACBP749 for myristoyl-CoA showed that PfACBP16 had the highest

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affinity for myristoyl-CoA (Table 3). PfACBP99 had the medium range binding affinity, whereas

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PfACBP749 displayed the weakest binding affinity for myristoyl-CoA (Table 3). It was interesting to

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find that all of the three PfACBPs function redundantly to bind the long-chain fatty acyl-CoAs, and

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not to the short-chain fatty acyl-CoAs. We believed that different acyl-CoA binding proteins function

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in different stages of the Plasmodium life cycle. It was also possible that different PfACBPs

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functioned in different pathways of lipid metabolism. PfACBP16 and PfACBP99 could be involved in

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processes (like lipid transport) that required temporally longer association of acyl-CoAs with the

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PfACBPs. On the other hand, PfACBP749 might be involved in processes (like lipid storage) that could

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show leniency in terms of binding affinity.

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We tested if PfACBPs could bind exclusively to the fatty acyl-CoAs, or they could also bind to

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the unmodified fatty acids. None of the acyl-CoA binding proteins showed any interaction with the

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long- or short-chain fatty acids (Supporting figure 1A, 1B). PfACBP16, PfACBP99 and PfACBP749

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failed to bind myristic acid or hexanoic acid. These findings suggested that the CoA moiety was

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indispensable for fatty acyl-CoAs binding to PfACBPs.

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To get a better understanding of the regions of PfACBPs that were responsible for binding to

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the fatty acyl-CoAs, we performed unrestrained docking studies of different fatty acyl-CoAs upon the

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acyl-CoA binding proteins. PfACBP16, PfACBP99 and PfACBP749 are paralogous proteins, and they

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share high sequence similarity with each other (Figure 2A). The structure of PfACBP749 was

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previously solved (16). It showed an abundance of alpha helical regions in its structure. Among the

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four helices, the second helix was involved in binding to the fatty acyl-CoAs. Two tyrosine residues at

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the 30th and 33rd positions (Y30 and Y33) were found to be important for binding to the CoA of the

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fatty acyl-CoAs. The interaction involved the π-stacking in between the aromatic rings of tyrosine

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and CoA. Sequence alignment of PfACBP16 and PfACBP99 with PfACBP749 showed that the residues

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of second helix were completely conserved in all three proteins (Figure 2A). Moreover, the tyr-30

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(Y30) and tyr-33 (Y33) were position-wise conserved in all of the acyl-CoA binding proteins (Figure

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2A). It indicated that individual PfACBP could exploit the same residues and mechanism to bind to

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the fatty acyl-CoAs. To investigate if all three PfACBPs accommodated the fatty acyl-CoAs at the

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binding cleft near the second helix, we generated the structures of PfACBP16 and PfACBP99 by

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advanced homology modelling [using PfACBP749 structure (PDB: 1HBK) as the template]. Like

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PfACBP749, PfACBP16 and PfACBP99 showed the abundance of helical regions in their structures.

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PfACBP16 and PfACBP99 also possessed the same binding cavity near the second helix (Figure 2B).

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Unrestrained molecular docking of myristoyl-CoA upon the modelled structures of PfACBP16 and

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PfACBP99 showed that the most favourable and stable binding of myristoyl-CoA occurred by its

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binding to the surface cleft of the second helix (Figure 2B). The tyr-30 and tyr-33 were observed to 5 ACS Paragon Plus Environment

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play important roles in stabilizing the CoA by aromatic ring stacking interactions (Figure 2B).

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Additionally, the lys-34 (K34) was observed to stabilize the negatively charged phosphate group of

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CoA through ionic interaction. We understood that the CoA mediated the primary interactions with

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the Y30 and Y33 of PfACBPs. Ionic interactions between the phosphate group of fatty acyl-CoA and

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positively charged amino acids of PfACBPs as well as hydrophobic interactions between the long acyl

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chains of fatty acyl-CoAs and hydrophobic residues of PfACBPs stabilized the overall binding of fatty

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acyl-CoAs to the acyl-CoA binding proteins. The acyl chain of the myristoyl-CoA extended towards

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the third helix of the PfACBPs (Figure 2B). The sequence and structure of the third helix is also

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conserved in all the acyl-CoA binding proteins. We speculated that the longer fatty acyl chains

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extended sufficiently to interact with the residues of the third helix, thereby stabilizing the complex.

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The shorter fatty acyl chains could not reach to the third helix. Hence, their binding affinities for

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PfACBPs were lower and they could not bind to PfACBPs for a temporally longer period of time.

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Several key hydrophobic residues, like ile-52 (I52), ala-59 (A59), and val-63 (V63), in the third helix

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had played decisive roles in mediating the hydrophobic interactions with the nonpolar acyl chains of

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the fatty acyl-CoAs.

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The importance of the Y30 and Y33 of PfACBPs in binding to the acyl-CoAs was

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experimentally proven. To demonstrate the effectiveness of these two tyrosine residues, we

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introduced two tyrosine-to-alanine mutations in the 30th and 33rd positions of PfACBPs (Figure 2C).

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These mutations had generated the PfACBP16-Y30A, Y33A, PfACBP99-Y30A, Y33A and PfACBP749-

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Y30A, Y33A constructs. All of these mutants of PfACBPs failed to show any interaction with the

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myristoyl-CoA (Figure 2D). Similarly, none of the PfACBP mutants could bind to any of the long or

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short chain fatty acyl-CoAs (data not shown). Since the indispensable aromatic rings were destroyed

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in the Y30A, Y33A mutants of PfACBPs, the acyl-CoAs could not bind to the proteins.

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Other than the acyl-CoAs, PfACBPs showed binding to the phospholipids. Using the

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conventional protein-lipid overlay (PLO) assays, we determined that binding of PfACBPs were only

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limited to the phospholipids, but not to the unphosphorylated fatty acyl-esters. While all of the

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PfACBPs could bind to varying concentrations (2-50nM) of phosphatidylcholine (PC) and

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phosphatidic acid (PA) (Figure 3A) they did not show any binding to diacylglycerol (DAG) (Figure 3A).

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Given the fact that phospholipids were the primary constituents of the membranes, we studied the

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binding abilities of PfACBPs to the plasma membranes. We pursued the fluorescence associated cell

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sorting (FACS)-coupled red blood cell (R.B.C) ghost binding assays to monitor the PfACBP binding to

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human erythrocyte plasma membrane. This technique was based on measuring the fluorescence

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intensities of the secondary antibody-attached fluorescein isothiocyanate (FITC) fluorophore. A

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positive binding was indicated by higher fluorescence intensity of FITC. While the blank-control and

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mock-control samples did not bind to the erythrocyte membrane, PfACBPs showed prominent

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binding to the plasma membrane of erythrocytes (Figure 3B). These results led us to confirm that

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phospholipids were the true binding partners of PfACBPs. Though the phospholipids lacked the CoA

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moiety, they contained the phosphate group and acyl chains which mediated the interaction with

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PfACBPs. Some conserved basic amino acid(s) (like lys-34) in PfACBPs were likely to make 6 ACS Paragon Plus Environment


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electrostatic contacts with the phosphate group of the conjugated lipids. However, the mechanism

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of phospholipid interaction with PfACBPs remained to be investigated.

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We tested the expression levels of PfACBPs in different life-cycle stages of Plasmodium

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falciparum. According to the available microarray data (PlasmoDB), expression of PfACBPs was found

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to be high in the trophozoite stage. We confirmed this phenomenon by showing the abundant

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presence of PfACBPs in the trophozoite stage of Plasmodium falciparum (Figure 3C). We found that

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the level of PfACBP proteins was also high in the schizont stage (Figure 3C). However, we did not see

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any PfACBP expression in the ring stage of Plasmodium (Figure 3C). Thus, PfACBPs were expressed

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more in the later stages of Plasmodium life-cycle that were associated with the proliferation and

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invasion properties of Plasmodium. In the proliferation and infection phases, Plasmodium required

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higher content of lipids for the membrane biogenesis and metabolism. Hence, the upregulated

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PfACBP expression was correlated to the higher lipid usage by schizont and trophozoite stage

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parasites.

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PfACBPs are globular proteins

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Before we studied further to find the small molecule modulators of PfACBPs, it was

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necessary to know the structural and biophysical properties of different PfACBPs. The actual or

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modelled structures of PfACBPs were required to rationally conduct the ligand binding studies. As

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we mentioned in the previous section, we had generated the molecular models of PfACBP16 and

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PfACBP99 based on the template structure of apo-PfACBP749 protein (Figure 2B). We

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experimentally validated that all PfACBPs were alpha helical globular proteins. The high content of

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alpha helical regions in PfACBPs was observed in the circular dichroism (CD) spectroscopic studies

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(Figure 4A, 4B). Far-UV wavelength scanning of the PfACBPs in the CD spectroscopy showed two

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wavelength dips at 222nm and 208nm. Since the maximum negative ellipticity at 208nm and 222nm

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in CD spectrum represented the helical nature of proteins, we understood that PfACBPs were helical

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proteins (Table 4). This corroborated with the molecular model structures that showed the presence

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of helical regions (and not any beta sheets) in the PfACBPs. The globular nature of PfACBPs was also

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prominent in the model structures. PfACBPs contained some short patches of hydrophobic regions

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(Figure 4C). These regions were buried in the core of the PfACBPs (Figure 4C). PfACBPs had high

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solubility in aqueous (buffer) medium (reaching the concentration as high as 6mg/ml without

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forming aggregates). PfACBPs were heat-labile. The CD spectroscopy based thermal unfolding

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experiments showed that the melting temperatures (Tm) of PfACBPs were ranged in between 47oC to

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59oC (Supporting figure 2A, 2B, 2C). The completely denatured PfACBPs were unable to acquire the

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fully-folded (native) state in the renaturation conditions (Supporting figure 2A, 2B, 2C). However,

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the PfACBPs were insensitive to the pH variation of environment. Robustness of PfACBP structures

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remained intact during the gradual decrease of the pH from 8.5 to 5.5 (Supporting figure 3A).

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PfACBPs could undergo a two-state folding-unfolding process. The gradual thermal denaturation associated CD spectroscopic studies and convex constraint algorithm (CCA) 7 ACS Paragon Plus Environment

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deconvolution of the CD spectra showed the presence of only folded and unfolded conformations of

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the PfACBPs (Figure 5A, 5B, 5C). No transition-state intermediate conformation was observed in any

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of the PfACBPs. Three-component CCA deconvolution of the spectra set showed the presence of

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three curves, among which two were almost superimposed (Figure 5A, 5B, 5C). This implied the

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presence of only folded and unfolded structures (and not any intermediate structure) of PfACBPs.

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We had previously used this method to understand the intermediate-state conformations of human

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aggregation-prone protein(s) (17). The absence of conformational intermediate(s) of PfACBPs was

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putatively linked to non-allosteric binding to acyl-CoAs to PfACBPs. Acyl-CoAs bound to the PfACBPs

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in the protein’s native folded conformation. No allosteric changes were associated with the proteins

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during acyl-CoA binding to PfACBPs. However, it remained to be a subject of experimental validation.

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Mefloquine is a competitive inhibitor of acyl-CoA binding to PfACBPs

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In the next part of our study, we screened the small molecule ligand(s) of PfACBP749 by

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using a high-throughput computational docking approach. The aim of this study was to identify

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potential regulator(s) and its/their mechanism(s) of action against different PfACBPs. We took the

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apo-PfACBP749 as the substrate protein for docking of PubChem and GSK-TresCantos anti-malarial

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set (TCAMS) of compounds. A total of 4,58,897 PubChem compounds and 26,784 TCAMS

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compounds were tested for their abilities to bind to PfACBP749 (Figure 6A, 6B). The acyl-CoA

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binding cavity was decided as the position for molecular docking of the ligands. The binding

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potentials of ligands were measured by the docking scores that ranged in between -8 to -16.6 for the

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PubChem compounds and -1.4 to -20.6 for the TCAMS compounds (Figure 6A, 6B). More negative

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docking scores indicated the stronger bindings. We curated the ten best docked molecules from the

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PubChem compounds (Figure 6C). While these compounds varied in their chemical structures and

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compositions, we found that mefloquine was a potential binding partner of PfACBP749. Since

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mefloquine was a food and drug administration (FDA) approved drug, we were particularly

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interested to see if mefloquine had inhibitory activity against the PfACBPs. Mefloquine binding with

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the PfACBPs were measured by isothermal titration calorimetry. It was observed that mefloquine

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could strongly interact with the PfACBPs (Figure 7A) (Table 5). The binding affinities of mefloquine

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for PfACBPs were much stronger than the affinities of myristoyl-CoA bindings to PfACBPs. We

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investigated the specificity of mefloquine binding to PfACBPs by comparing with the binding of

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chloroquine (another quinoline-based drug) to PfACBPs. Since chloroquine did not bind to any of the

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PfACBPs (Figure 7B), we were quite certain about the specific high affinity binding of mefloquine

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with Plasmodium acyl-CoA binding proteins. Though mefloquine and chloroquine were quinoline-

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based drugs, they had significant differences in their composition. Other than the quinoline ring,

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mefloquine contained electronegative fluorine atoms. On the contrary, chloroquine lacked

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electronegative atoms. Instead, chloroquine contained secondary amine and alkyl chain. We

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believed that electronegative atoms/groups were required for the quinoline drugs to bind to the

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PfACBPs.

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Mefloquine binding to PfACBPs had occurred in the same region where acyl-CoAs bound

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PfACBPs (Figure 8A, 8B, 8C). The aromatic quinoline ring of mefloquine was involved in the primary

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base-stacking interactions with the cavity-residing tyr-30 (Y30) and tyr-33 (Y33) amino acids. There

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were additional stabilizing interactions which occurred between the fluorides of mefloquine and the

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lysine residue of PfACBPs. Though mefloquine was volumetrically less than acyl-CoAs and it also

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lacked the long hydrophobic chain, we speculated that mefloquine mediated the hydrophobic

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interactions through its piperidine ring. Since mefloquine engaged the important Y30 and Y33

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residues of PfACBPs (Figure 9A, 9B, 9C), we were enthusiastic to see if mefloquine could partially or

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completely reduce the acyl-CoA binding to PfACBPs. Indeed, we found that mefloquine binding to

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PfACBPs completely abolished the myristoyl-CoA binding to PfACBPs (Figure 10A, 10B, 10C). This

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phenomenon was also true for other acyl-CoAs binding to PfACBPs. In the SPR-based interaction

338

studies, none of the acyl-CoAs showed any binding to the preformed mefloquine-PfACBP complexes.

339

Hence, it was certain that mefloquine acted as a competitive inhibitor against the acyl-CoA binding

340

to PfACBPs.

341 It would be relevant here to mention that another compound, CID 3106719, showed high

342 343

binding ability to PfACBP749. In the computational studies, the docking score of this compound was

344

even higher than mefloquine. The presence of benzo[de]isoquinolin ring, quinazoline ring, and oxo

345

groups in CID 3106719 could lead to its high-affinity binding to PfACBP749. This compound could be

346

tested as an interesting target against PfACBPs and other Plasmodium proteins/machineries.

347 348

Mefloquine binding causes degradation of PfACBPs and it restricts Plasmodium growth and

349

survival

350 351

In the final section of our study, we checked the effect of mefloquine on the spatiotemporal

352

existence of PfACBPs inside the Plasmodium cells. Mefloquine treatment to the culture medium

353

(final concentration: 10nM) caused significant reduction of PfACBPs in the Plasmodium falciparum

354

cells. We traced the confocal microscopy images and immunoblotting of Plasmodium cell lysates to

355

confirm the reduction of intracellular PfACBPs in mefloquine-treated conditions (Figure 11A, 11B,

356

11C). We believed that this reduction was due to the enhanced degradation of the mefloquine-

357

PfACBP complex in the cells. The strong binding of mefloquine with PfACBPs could have irreversibly

358

transformed the PfACBPs into non-functional forms. These non-functional PfACBPs were degraded

359

by the cellular degradation system(s). It was, indeed, observed that the degradation of the

360

mefloquine-PfACBP complex was prevented in the presence of MG132 in the medium (Figure 11B).

361

This proved the fact that the mefloquine-PfACBP complex was subjected to degradation in the

362

proteasomal pathway.

363 364

As we mentioned in a previous section, the expression of PfACBPs were high in trophozoite

365

and schizont stages, but not in the ring stage of Plasmodium falciparum. We checked if mefloquine

366

binding to PfACBPs were correlated to degradation of PfACBPs in the trophozoite and schizont

367

stages of Plasmodium falciparum. It was observed that mefloquine treatment could cause the 9 ACS Paragon Plus Environment

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368

degradation of PfACBPs in the trophozoite and schizont stages of Plasmodium falciparum (Figure

369

11D). It showed that mefloquine activity against PfACBPs and the clearance of mefloquine-PfACBP

370

complex was not stage specific, but mefloquine binding to PfACBPs could cause degradation of the

371

PfACBPs in all cellular stages of Plasmodium falciparum.

372 373

Mefloquine mediated degradation of PfACBPs had adverse effect on the physiology and

374

survival of Plasmodium. Mefloquine treatment caused significant death of the parasites (Figure 11E),

375

leading to a reduction of parasitemia (Figure 11F). This was also manifested in the time-dependent

376

manner. The temporal decline of parasite count was observed in the mefloquine-treated parasite

377

cultures. Though mefloquine was known to cause lethality to parasites in many different ways (18,

378

19), we understand that the inhibition and removal of PfACBPs from the cell system was another

379

mode of mefloquine action in terms of restricting the growth and proliferation of Plasmodium.

380 381

TABLES

382 383

Table 1: Dissociation constant (Kd) values (μM) of binding of different fatty acyl-CoAs to PfACBPs

384

(PfACBP16, PfACBP99 and PfACBP749) in surface plasmon resonance binding experiments.

385 PfACBP16

PfACBP99

PfACBP749

Hexanoyl-CoA

235.294

272.727

No binding

Octanoyl-CoA

3.5

208.823

No binding

Decanoyl-CoA

1.652

48.437

No binding

Myristoyl-CoA

0.637

1.661

3.111

Palmitoyl-CoA

0.122

7.368

5.238

Stearoyl-CoA

0.062

1.408

0.147

Arachidonoyl-CoA

0.022

0.02

0.082

Oleoyl-CoA

0.002

0.000003

0.007

386 387

Table 2: Free energy (ΔG, kcal/mol) of binding of different fatty acyl-CoAs to PfACBPs (PfACBP16,

388

PfACBP99 and PfACBP749) in molecular docking studies.

389 Arachidonyl-CoA

Myristoyl-CoA

Hexanoyl-CoA

PfACBP16

-104.39

-81.93

-67.97

PfACBP99

-99.81

-87.26

-61.41

PfACBP749

-97.45

-79.4

-19.53

390 391

Table 3: Thermodynamic parameters of myristoyl-CoA binding to different PfACBPs (PfACBP16,

392

PfACBP99 and PfACBP749) in isothermal titration calorimetry based binding studies.

393 Protein – Fatty acyl-CoA

Kd (μM)

ΔH (cal/mol)

complex

ΔS (cal/mol/deg) 10

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Page 12 of 41

PfACBP16 – Myristoyl CoA

0.68

-2.94 X 104

-69.9


1.70

-1.57 X 104

-26.4

2.97

104

-7.01


-2.61 X

394 395

Table 4: Proportion of secondary structure components in different PfACBPs.

396 PfACBP16

PfACBP99

PfACBP749

(ratio in %)

(ratio in %)

(ratio in %)

Alpha helix

61.3

56.5

48.7

Beta sheet

0.0

0.0

0.0

Turn

9.6

12.1

17.9

Disorder

29.1

31.4

33.4

Total

100

100

100

397 398

Table 5: Different thermodynamic parameters associated with mefloquine binding to PfACBPs

399

(PfACBP16, PfACBP99 and PfACBP749) in isothermal titration calorimetry based binding studies.

400 Protein – mefloquine

Kd (μM)

ΔH (cal/mol)

complex

ΔS (cal/mol/deg)

PfACBP16 – Mefloquine PfACBP99 – Mefloquine PfACBP749 – Mefloquine

0.12

-3.92 X 104

-105

0.42

-1.79 X

104

-30.9

-9.61 X

103

-61.2

1.74

401 402

DISCUSSIONS

403 404

The life-threatening malaria disease is epidemiologically widespread in geographically

405

diverse regions. Different species of Plasmodium cause malaria in human. The extent of this disease

406

can be severe in malignant and cerebral malaria. The life-cycle of Plasmodium parasite is bipartite.

407

Considering the fact that the host and vector have many differences in the physiological parameters

408

(like body temperature etc.), it can be said that the survival of Plasmodium in both human and

409

mosquito has been an evolutionary triumph of this organism. Needless to say, Plasmodium has

410

selectively maintained and flourished many of its key proteins to perform the challenging tasks.

411

Efficient exploitation of lipids in cellular structures and energy production is remarkably high in

412

Plasmodium (20). This is intrinsically correlated to the optimal expression and activity of lipid

413

binding, storage and transporter proteins. Many studies have revealed the importance of acyl-CoA-

414

synthetase, acyl-carrier proteins etc. in production and maintenance of steady-state level of cellular

415

lipids (21, 22). In this respect, testing of the functional roles of acyl-CoA binding proteins in

416

Plasmodium has remained a debacle. In this study, we have characterized the PfACBPs in terms of

417

their lipid binding and biophysical/structural properties. We have also demonstrated that

418

mefloquine acts as a competitive inhibitor of the PfACBPs that restrict the growth and proliferation

419

of Plasmodium falciparum. 11 ACS Paragon Plus Environment

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420 421

Acyl-CoA binding proteins are redundant in Plasmodium falciparum. Three PfACBPs typically

422

bind various kinds of lipid molecules. Different acyl-CoAs and phospholipids are the substrates of

423

these acyl-CoA binding proteins. Though PfACBPs can bind to a wide range of long-chain fatty acyl-

424

CoAs, they fall short to mediate high affinity binding with the short-chain acyl-CoAs. The residues of

425

the acyl-CoA binding pocket and surrounding regions determine the strength of the binding to

426

different fatty acyl-CoAs. The co-complex structures of acyl-CoA-PfACBPs (like myristoyl-CoA-

427

ACBP16/99/749) show that the CoA moiety is stacked with the critical tyrosine residue(s). The

428

hydrophobic acyl chains of acyl-CoAs extend beyond the binding cavity to make interactions with the

429

hydrophobic residues of the nearby helice(s). While the CoA and tyrosine interaction is the primary

430

mode of contact, the hydrophobic interactions stabilize the binding between acyl-CoAs and PfACBPs.

431

This explains why short-chain fatty acyl-CoAs have lower binding affinities for PfACBPs. Although the

432

CoA of short-chain fatty acyl-CoAs can bind to the tyrosine(s) in the binding pocket, the smaller

433

hydrophobic chains fail to interact with the helix associated hydrophobic amino acids. Therefore,

434

PfACBP16, PfACBP99 and PfACBP749 can be classified as the long-chain fatty acyl-CoA binding

435

protein. The short-chain acyl-CoA binding proteins in Plasmodium are yet to be identified. Since

436

Plasmodium falciparum possess some other acyl-CoA binding domain containing proteins, like

437

PfACBP14 (PF3D7_1001100.1), PfACBP15 (PF3D7_1001100.2) and PfACBP197 (PF3D7_111900), it

438

will be interesting to test if they are the short-chain acyl-CoA binding proteins.

439 440

PfACBPs also bind to phospholipids. Though we have not pursued extensive studies to find

441

the mechanistic details of this interaction, we assume that the phosphate group helps the

442

phospholipids to bind to the PfACBPs. This assumption is strengthened by the fact that phosphate

443

group lacking fatty acid esters do not bind to the acyl-CoA binding proteins. Two conserved basic

444

amino acids (lys-31 and lys-34) in PfACBPs can be involved in the electrostatic contacts with the

445

negative charges of the phosphate group. PfACBP’s capacity to bind long-chain fatty acyl-CoAs and

446

phospholipids is advantageous in storing various kinds of lipids in different stages of Plasmodium

447

life-cycle. Previous studies find that the rapidly growing parasites require a large quantity of

448

phosphatidylcholine for their increasing membrane structures (23). Higher expression of PfACBPs in

449

the trophozoite stage allows the storage and release of phospholipids (like phosphatidylcholine) to

450

maintain the supply of lipids.

451 452

PfACBPs are globular proteins that have a large content of alpha helical regions. Among the

453

four alpha helices, the second helix forms the acyl-CoA binding cleft. The small hydrophobic patches

454

of PfACBPs are buried in the core to avoid their exposure to aqueous environment. Some of the

455

hydrophobic residues are also located in the acyl-CoA binding pocket. All PfACBPs are heat labile

456

proteins. They are rapidly and irreversibly denatured at lower temperatures (98% pure and they were

623

concentrated by centrifugation-filtration method. The yields of recombinant proteins were in the

624

range of 6-14mgL-1.

Page 18 of 41

625 626

Circular dichroism spectroscopy

627 628

Circular dichroism (CD) spectroscopy experiments were done in JASCO-810

629

spectropolarimeter instrument. Temperature adjustments in the instrument were done by Peltier

630

temperature controller. Proteins were kept in 20mM NaH2PO4/50mM NaF buffer (pH: 7.5).

631

Secondary structures of proteins were analysed by measuring the changes in ellipticity values (Δε) in

632

the far-UV wavelength of light (190-260nm). In different experiments, protein concentrations varied

633

from 2μM to 15µM. Instrument/experiment parameters were as follows: light path length in cuvette

634

= 0.1cm, scan speed = 100nm/minute, response time = 1 second, data pitch = 1nm, band width =

635

2nm, data recording = triplicate. In the slow thermal unfolding or refolding experiments, proteins

636

were continuously heated or cooled from 20oC↔90oC (1oC/minute) and the Δε values were recorded

637

at λ = 222nm. In the pH dependent experiments, PfACBPs were kept in 20mM NaH2PO4/50mM NaF

638

buffer of variable pH (5.5 to 8.5) and the Δε of spectra of the proteins were recorded at the

639

wavelength range of 190-260nm. The secondary structure of proteins were also monitored at

640

discrete temperatures (from 20oC to 90oC, 5oC interval) by measuring the Δε values at 190-260nm.

641

From the CD spectra, the secondary structure components of different proteins were analyzed by

642

using CDSSTR algorithm. The deconvolution of spectra set was done by CCA.

643 644

Isothermal titration Calorimetry

645 646

Isothermal titration calorimetry (ITC) experiments were carried out for analyzing the binding

647

of PfACBPs with acyl-CoAs, myristic acid, mefloquine, and chloroquine. The ITC experiments were

648

conducted in MicroCal-iTC 200 instrument that was equipped with a Microcal-Thermovac

649

temperature control system. Proteins and different ligands were in kept in phosphate buffer saline

650

(PBS). The heat titrations for the binding events were done at 25o C. The cell contained 20µM of the

651

protein(s) and the syringe contained 200µM of the ligand(s). The instrument parameters were as

652

follows: reference power of instrument = 10µcal/sec, number of injections = 20 (for the first

653

injection – volume = 0.4μl, duration = 0.8 second; for the remaining nineteen injections – volume =

654

2.0μl, duration = 4.0 seconds), injection spacing time = 120 seconds, filter period = 5, rotation speed

655

of syringe = 300rpm, initial delay period = 1 minute. Microcal-LLC ITC-200 module implemented in

656

Origin graphing software was used for the data acquisition and analysis. Thermodynamic parameters

657

were generated by following 1:1 binding stochiometry of ligand to protein.

658 659

Surface plasmon resonance

660 661 662

Binding studies of the recombinant PfACBPs (PfACBP16, PfACBP99 and PfACBP749) and their mutants (PfACBP16-Y30A, Y33A, PfACBP99-Y30A, Y33A and PfACBP749-Y30A, Y33A) with the acyl17 ACS Paragon Plus Environment

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663

CoAs, fatty acids, mefloquine and chloroquine were conducted by surface plasmon resonance

664

experiments in Biacore 3000 instrument. 30 nanomole of each ligand (PfACBPs and their mutants)

665

was individually immobilized onto CM5 chips by amine coupling method [immobilization buffer:

666

acetate buffer (pH: 4.0)]. The concentrations of analytes (acyl-CoAs, fatty acids, mefloquine and

667

chloroquine) were in the range of 20nM to 10μM for the binding reactions. Acyl-CoA, fatty acid,

668

mefloquine and chloroquine were solubilized in PBS. PfACBPs were also kept in PBS. Analytes were

669

injected in the cell with a flow rate of 30µl/min at 25oC, followed by allowing the dissociation of

670

analytes from the ligands for a time period of 10 minutes. The sensograms were analyzed in

671

BIAevaluation software (version 4.1). The thermodynamic parameters (Ka, Kd etc.) of bindings were

672

analyzed by following 1:1 Langmuir binding/non-cooperative binding of ligand to analyte.

673 674

R.B.C ghost binding assay

675 676

The R.B.C ghosts were prepared from fresh human blood according to the standard

677

procedure. Blood was collected in an anti-coagulant tube, followed by centrifugation of the blood at

678

2,000rpm for 15 minutes at room temperature. The supernatant/buffy layer was discarded and the

679

erythrocyte pellet was washed with PBS. The erythrocyte pellet was poured in hypotonic buffer

680

(5mM phosphate buffer [pH 7.4]). The buffer was pre-chilled at 0°C and erythrocyte pellet was

681

dropped in the buffer from a reasonable height. This suspension was stirred for 1 hour in cold room.

682

Finally, this suspension was centrifuged at 10,000rpm for 10 minutes at 4°C. The pellet was washed

683

with an isotonic buffer (0.9% NaCl solution) until white-to-pale yellow colored R.B.C. ghosts were

684

obtained. These R.B.C ghosts were used for FACS experiments. Purified PfACBPs were incubated

685

with freshly prepared R.B.C. ghosts at 4°C for 16 hours. The R.B.C. ghosts were then washed with

686

PBS and incubated with anti-PfACBP749 antibody for 1 hour at 37°C. This was followed by washing of

687

the R.B.C. ghosts with PBS for few times and subsequent incubation with FITC conjugated anti-

688

mouse secondary antibody (anti-mouse IgG whole molecule FITC antibody, SIGMA F0257). In the

689

blank control experiment, no protein was incubated with the R.B.C ghosts. In mock control

690

experiment, an Escherichia coli protein YqeA was used to incubate with the R.B.C ghosts.

691 692

Protein lipid overlay assay

693 694

Lipids (2-50nM) were spotted on PVDF membranes. The lipid-spotted membranes were

695

incubated in the blocking buffer [50mM Tris-HCl (pH: 7.5), 150mM NaCl, 0.1% Tween-20 and 2

696

mg/ml fat-free BSA] at room temperature. The membranes were then incubated with 100nM of

697

recombinant PfACBPs at 4°C for 16 hours. This was followed by repeated washing of the membranes

698

with TBST buffer [50mM Tris-HCl (pH 7.5), 150mM NaCl and 0.1% Tween-20]. The membranes were

699

incubated with anti-PfACBP749 antibody (dilution - 1:1000; raised in mouse) for 1 hour at room

700

temperature. The membranes were subsequently washed with TBST, followed by application of anti-

701

mouse IgG (whole molecule) peroxidase secondary antibody (dilution - 1:5000, SIGMA A9044).

702

Signals were detected by conventional ECL-based chemiluminescence method.

703 18 ACS Paragon Plus Environment


704

Page 20 of 41

Plasmodium falciparum culture

705 706

Plasmodium falciparum (strain: 3D7) was gifted by Dr. Puran Singh Sijwali of Centre for

707

Cellular and Molecular Biology (Hyderabad, India). The parasite culture was in vitro maintained in

708

erythrocytes (blood group: B+) at 2-3% haematocrit. Erythrocytes were washed with washing

709

medium (RPMI 1640, 2gm/litre D-glucose, and 300mg/litre L-glutamine). The culture medium (RPMI

710

1640) was supplemented with 0.5% (w/v) albumax-II, 2gmlitre-1 of D-glucose, 300mglitre-1 of L-

711

glutamine, 2gmlitre-1 of NaHCO3, 1X hypoxanthine/thymidine mixture, gentamycine and 2.5% (v/v)

712

human serum. Culture flasks were filled with calibrated gas mixture (5% O2, 5% CO2 and 90% N2)

713

before incubation at 37oC. Growth of Plasmodium falciparum in cultures was maintained by

714

maintaining standard protocols (34). Synchronization of the parasite cultures was performed by

715

alternatively applying 5% sorbitol (incubation) to the parasites and density gradient centrifugation

716

over Percoll solution (35).

717

Mefloquine treatment was given to Plasmodum cultures (60% parasitemia) by maintaining 10nM

718

mefloquine in the culture medium. Since we could achieve to induce 60% as the highest parasetemia

719

level, we used this parasetemia level for the mefloquine treatment experiments. The counts of

720

Plasmodium cells in mefloquine treated cultures were monitored at different time-points.

721

MG132 was treated to cells by mantaining 20μM of MG132 in the culture medium.

722 723

Generation of anti-PfACBP749 antibody

724 725

100µg of purified PfACBP749 protein was mixed with Complete Freund’s adjuvant (CFA) and

726

the protein suspension was injected (primary injection) into BALB/c mice. Before primary injection,

727

pre-immune bleed was collected from the mice. First booster dose (100µg of purified PfACBP749

728

protein with Incomplete Freund’s adjuvant) was given to the mice after 21 days of primary injection.

729

Second and third booster doses were administered after 42ndand 63rddays of primary injection. Test

730

bleed (200 µl) was collected on the 56th day from primary injection. Final bleed was taken after

731

exsanguination and termination of the animals. Serum was collected from the whole-blood of the

732

mice. Immunoblotting against the recombinant PfACBPs by this serum was done to test for the

733

presence and activity of anti-PfACBP749 antibody (Supporting figure 4B). Positive results in

734

immunoblotting were obtained for this serum against all PfACBPs (PfACBP16, PfACBP99 and

735

PfACBP749). We confirmed the activity of the serum-based anti-PfACBP749 antibody by comparing it

736

with the positive control anti-polyhistidine antibody. The anti-PfACBP749 antibody of serum was

737

also tested for its ability to detect the PfACBPs in the Plasmodium falciparum cell lysate. The ant-

738

PfACBP antibody could detect the PfACBPs in the Plasmodium falciparum cell lysate (Supporting

739

figure 4B).

740 741

Immunoblotting

742 743 744

The general procedure of immunoblotting followed the same steps that were described by us in earlier studies (31). Briefly, 40-60μg of proteins from solution or cell lysate was separated in 19 ACS Paragon Plus Environment

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745

14% SDS-PAGE. This was followed by transfer of proteins to PVDF membrane and incubating the

746

membrane with primary and secondary antibodies. Primary antibodies: anti-PfACBP749 antibody

747

[raised in mouse, dilution - 1:2000], anti-6XHis antibody [raised in mouse, dilution – 1:5000, SIGMA

748

H1029], anti-GAPDH antibody [raised in mouse, dilution – 1:1000; obtained from European malaria

749

reagent laboratory] and secondary antibody: anti-mouse IgG (whole molecule) peroxidase secondary

750

antibody (dilution - 1:5000, SIGMA A9044).

751 752

Immunocytochemistry

753 754

Plasmodium falciparum cells were washed with phosphate buffer saline (PBS) for two times.

755

Cells were then spread onto a glass slide to make a uniform smear of cells. This cell-smear was air-

756

dried at room temperature for 20-30 minutes, followed by fixation with ice-cold methanol for 30

757

seconds and application of 4% (w/v) paraformaldehyde /0.01% (v/v) glutaraldehyde mixture

758

(prepared in PBS) for 30 minutes. Fixed cells were washed with PBS and they were quenched with

759

10mM glycine solution for 10 minutes. The cells were then blocked with 3% BSA (prepared in PBS)

760

for 2 hours. Cell were treated with anti-PfACBP749 antibody (dilution: 1:100, dilution was done in 3%

761

BSA) for 2 hours at room temperature. After washing the primary antibody with PBS, the FITC-

762

conjugated anti-mouse IgG secondary antibody (dilution: 1:100, dilution was done in 3% BSA; SIGMA

763

F0257) was applied to the cells for two hours at room temperature. The cells were washed with PBS

764

and air-dried. Cells were mounted on DAPI-contained mounting media. Fluorescent signals were

765

detected in the LSM-700 laser scanning confocal microscope using Plan-Apochromat 63x/1.40 oil-

766

immersion objective (with DIC). Image analysis was done in ZenLite software.

767 768

Molecular modelling, high-throughput virtual screening and molecular docking

769 770

The PfACBP16 and PfACBP99 sequences were curated from the PlasmoDB database. The

771

model structures of these proteins were generated by advanced homology modelling in the stand-

772

alone version of Modeller 9.12. PfACBP749 (PDB: 1HBK) structure was taken as the template. The

773

quality of the modelled structure was checked in Molprobity for analysis of dihedral angles

774

(Ramachandran Plot) and steric clashes. The loop-structure refinement was conducted in the protein

775

preparation wizard of Maestro (Schrodinger Inc.). This was also used to add the missing hydrogen

776

atoms, assign/correct the missing bond orders. The parameters of protein preparation were as

777

follows: forcefield = OPLS_2005, convergence heavy atom to RMSD of 0.30 Å. The structures were

778

then minimized in Molecular Modeling Tool Kit program (36) with amber parameters (37) (103

779

iterations to achieve the steepest descent/conjugate gradient minimization). The entire process

780

followed the same procedure that was described by us in our previous studies (38, 39).

781 782

We screened the Pubchem and TCAMS (Tres Cantos Antimalarial Set) compounds to find the

783

potential ligand(s) of PfACBPs by using high-throughput sequential docking in AutoDock Vina. The

784

structure of PfACBP749 (PDB: 1HBK) was used as the receptor. From the 1HBK structure, the

785

coordinates of the apo-proteins (PfACBP749) was extracted in Pymol and this apo-structure was 20 ACS Paragon Plus Environment


786

further used for the docking studies. The Protein Data Bank [PDB] format of the protein was

787

converted into Protein Data Bank, Partial Charge (Q), & Atom Type (T)) format [PDBQT] by using

788

AutoDock Tools (ADT) of MGL TOOLS package. The docking scores of the ligands of TCAMS were in

789

the range of -20.6 to -1.4. In case of the PubChem compounds, the docking scores ranged in

790

between -8 to -16.6. The chosen limits of the range for the docking scores were assigned to

791

maximize the likelihood of ligand binding to the receptor protein. This range was rationally

792

determined by comparing the docking scored of known true positive and true negative ligands of

793

PfACBP749 protein.

Page 22 of 41

794 795 796

The unrestrained rigid-body docking of the myristoyl-CoA and mefloquine upon PfACBPs were conducted in the stand-alone version of Molegro Virtual Docker.

797 798

Sequence alignment

799 800 801

Sequences of PfACBPs in FASTA format were downloaded from PlasmoDB database. These sequences were aligned using MultAlin.

802 803

Statistical tests

804 805

Comparisons of mean values of groups were done by t-test.

806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824


Page 23 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

825


ASSOCIATED CONTENT

826 827 828 829

The contents of the material supplied as supporting information is available free of charge on the ACS Publications website at http://pubs.acs.org. Supporting information contains the supporting figures.

830 831

ACKNOWLEDGEMENTS

832 833

The authors thank P. S. Sijwali (CCMB, India) for providing the Plasmodium falciparum 3D7

834

strain. The DBT-IPLS of University of Calcutta is acknowledged for providing the facilities of ITC

835

experiments. The instrumentation facilities of the research support service group of CDFD are

836

appreciated for providing different instruments. This work is supported by the core grants of CDFD

837

and an extramural grant from Department of Biotechnology (DBT, Government of India). AK and

838

DKG are recipients of graduate study fellowships from the Department of Biotechnology (DBT,

839

Government of India) and the Council for Scientific and Industrial Research (CSIR, Government of

840

India) respectively. AK and DKG are the graduate students of Manipal Academy of Higher Education.

841 842

AUTHOR CONTRIBUTIONS

843 844

AR and AK made the hypothesis and rationale of the study. AK, DKG and JA performed the

845

experiments. AR was responsible for obtaining funds, resource management and supervising the

846

experiments. Data analysis and manuscript writing was done by AR and AK with the help of DKG.

847 848

CONFLICT OF INTEREST

849 850

The authors declare that they have no potential conflict of interests to disclose.



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4. Bertolino P, Bowen DG. 2015. Malaria and the liver: immunological hide-and-seek or subversion of immunity from within? Front Microbiol 6: 41. 5. Aly AS, Vaughan AM, Kappe SH. 2009. Malaria parasite development in the mosquito and infection of the mammalian host. Annu Rev Microbiol 63: 195-221. 6. Aikawa M. 1971. Parasitological review. Plasmodium: the fine structure of malarial parasites. Exp Parasitol 30: 284-320.

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8. Gulati S, Ekland EH, Ruggles KV, Chan RB, Jayabalasingham B, Zhou B, Mantel PY, Lee MC,

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Spottiswoode N, Coburn-Flynn O, Hjelmqvist D, Worgall TS, Marti M, Di Paolo G, Fidock DA.

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11. Mitamura T, Palacpac NM. 2003. Lipid metabolism in Plasmodium falciparum-infected

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erythrocytes: possible new targets for malaria chemotherapy. Microbes Infect 5: 545-552.

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Expert Rev Anti Infect Ther 7: 999-1013. 13. Tran PN, Brown SH, Rug M, Ridgway MC, Mitchell TW, Maier AG. 2016. Changes in lipid

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14. Tran PN, Brown SH, Rug M, Ridgway MC, Mitchell TW, Maier AG. 2016. Changes in lipid

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15. Sherman IW. 1985. Membrane structure and function of malaria parasites and the infected erythrocyte. Parasitology 91 ( Pt 3): 609-645.

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16. Van Aalten DM, Milne KG, Zou JY, Kleywegt GJ, Bergfors T, Ferguson MA, Knudsen J, Jones

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TA. 2001. Binding site differences revealed by crystal structures of Plasmodium falciparum

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17. Ghosh DK, Roy A, Ranjan A. 2018. Disordered Nanostructure in Huntingtin Interacting

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18. Wong W, Bai XC, Sleebs BE, Triglia T, Brown A, Thompson JK, Jackson KE, Hanssen E,

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Marapana DS, Fernandez IS, Ralph SA, Cowman AF, Scheres SHW, Baum J. 2017. Mefloquine

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targets the Plasmodium falciparum 80S ribosome to inhibit protein synthesis. Nat Microbiol

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19. Walter RD. 1986. Plasmodium falciparum: inhibition of dolichol kinase by mefloquine. Exp Parasitol 62: 356-361. 20. Mi-Ichi F, Kita K, Mitamura T. 2006. Intraerythrocytic Plasmodium falciparum utilize a broad

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range of serum-derived fatty acids with limited modification for their growth. Parasitology

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21. Matesanz F, Tellez M, Alcina A. 2003. The Plasmodium falciparum fatty acyl-CoA synthetase

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family (PfACS) and differential stage-specific expression in infected erythrocytes. Mol

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22. Sharma SK, Kapoor M, Ramya TN, Kumar S, Kumar G, Modak R, Sharma S, Surolia N, Surolia

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A. 2003. Identification, characterization, and inhibition of Plasmodium falciparum beta-

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hydroxyacyl-acyl carrier protein dehydratase (FabZ). J Biol Chem 278: 45661-45671.

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23. Itoe MA, Sampaio JL, Cabal GG, Real E, Zuzarte-Luis V, March S, Bhatia SN, Frischknecht F,

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Thiele C, Shevchenko A, Mota MM. 2014. Host cell phosphatidylcholine is a key mediator of

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malaria parasite survival during liver stage infection. Cell Host Microbe 16: 778-786.

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24. Chen VB, Arendall WB, 3rd, Headd JJ, Keedy DA, Immormino RM, Kapral GJ, Murray LW,

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Richardson JS, Richardson DC. 2010. MolProbity: all-atom structure validation for

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25. Schaer CA, Laczko E, Schoedon G, Schaer DJ, Vallelian F. 2013. Chloroquine interference with

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macrophages: the paradox of an antimalarial agent. Oxid Med Cell Longev 2013: 870472.

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26. Meshnick SR. 1998. Artemisinin antimalarials: mechanisms of action and resistance. Med

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Trop (Mars) 58: 13-17. 27. Croft A, Garner P. 1997. Mefloquine to prevent malaria: a systematic review of trials. BMJ 315: 1412-1416.

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28. Dow G, Bauman R, Caridha D, Cabezas M, Du F, Gomez-Lobo R, Park M, Smith K, Cannard K.

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2006. Mefloquine induces dose-related neurological effects in a rat model. Antimicrob

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29. Croft AM, Herxheimer A. 2002. Adverse effects of the antimalaria drug, mefloquine: due to primary liver damage with secondary thyroid involvement? BMC Public Health 2: 6. 30. Gribble FM, Davis TM, Higham CE, Clark A, Ashcroft FM. 2000. The antimalarial agent mefloquine inhibits ATP-sensitive K-channels. Br J Pharmacol 131: 756-760. 31. Ghosh DK, Roy A, Ranjan A. 2018. The ATPase VCP/p97 functions as a disaggregase against toxic Huntingtin-exon1 aggregates. FEBS Lett 592: 2680-2692.



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32. Ghosh DK, Roy A, Ranjan A. 2018. Aggregation-prone Regions in HYPK Help It to Form Sequestration Complex for Toxic Protein Aggregates. J Mol Biol 430: 963-986.

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33. Roy A, Reddi R, Sawhney B, Ghosh DK, Addlagatta A, Ranjan A. 2016. Expression, Functional Characterization and X-ray Analysis of HosA, A Member of MarR Family of Transcription Regulator from Uropathogenic Escherichia coli. Protein J 35: 269-282. 34. Schuster FL. 2002. Cultivation of Plasmodium spp. Clin Microbiol Rev 15: 355-364.

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35. Radfar A, Mendez D, Moneriz C, Linares M, Marin-Garcia P, Puyet A, Diez A, Bautista JM.

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2009. Synchronous culture of Plasmodium falciparum at high parasitemia levels. Nat Protoc

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4: 1899-1915.

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36. Hinsen, K. 2000. The Molecular Modeling Toolkit: A New Approach to Molecular Simulations. J. Comp. Chem. 21 (2): 79–85. 37. Case, D. A., Cheatham T. E., Darden, T., Gohlke, H., Luo, R., Merz, K. M. Jr., Onufriev, A.,

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Simmerling, C., Wang, B., and Woods, R. J. 2005. The Amber biomolecular simulation

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programs. J. Comput. Chem. 26 (16): 1668-1688.

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38. Ghosh DK, Kumar A, Ranjan A. 2018. Metastable states of HYPK-UBA domain's seeds drive

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the dynamics of its own aggregation. Biochim Biophys Acta Gen Subj 1862: 2846-2861.

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39. Ghosh DK, Shrikondawar AN, Ranjan A. 2019. Local structural unfolding at the edge-strands

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of beta sheets is the molecular basis for instability and aggregation of G85R and G93A

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mutants of Superoxide dismutase 1. J Biomol Struct Dyn: 1-17. DOI:

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10.1080/07391102.2019.1584125

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FIGURES

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989 990 991

Figure 1. PfACBPs bind to fatty acyl-CoAs. The binding affinities of PfACBPs are higher for long-

992

chain fatty acyl-CoAs than that of short-chain acyl-CoAs. (A, B) Surface plasmon resonance studies

993

of binding analysis of PfACBP16 and PfACBP99 with myristoyl-CoA and hexanoyl-CoA. (C) Surface

994

plasmon resonance studies of binding of PfACBP749 with myristoyl-CoA and steroyl-CoA. (D)

995

Analysis of binding affinities of PfACBPs for different chain-length fatty acyl-CoAs. These affinity

996

values are measured by the Kd values that are obtained in the SPR studies. All PfACBPs bind to long-

997

chain fatty acyl CoAs with high affinities [low Kd values]. PfACBP16 and PfACBP99 can weakly bind to



998

short-chain fatty acyl-CoAs [high Kd values]. PfACBP749 cannot bind to any of the short-chain fatty

999

acyl-CoAs.

Page 28 of 41

1000 1001 1002 1003 1004 1005 1006 1007 1008 1009 1010 1011 1012 1013 1014 1015 1016 1017 1018 1019 1020 1021 1022 1023 1024 1025 1026 1027 1028 1029 1030 1031 1032 1033 1034 1035 1036 1037 1038 27 ACS Paragon Plus Environment

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1039 1040 1041

Figure 2. PfACBPs are paralogous proteins. Binding of myristoyl-CoA to PfACBPs occur in the same

1042

binding pocket through the stabilizing activity of conserved residues. (A) Sequence alignment of

1043

the three PfACBPs shows the existence conserved sequences in the proteins. (B) Myristoyl-CoA binds 28 ACS Paragon Plus Environment


Page 30 of 41

1044

to a specific cavity in all the PfACBPs. Two conserved tyrosine amino acids, tyr-30 and tyr-33, interact

1045

with the CoA by base staking interactions. (C) Mutants of PfACBP16, PfACBP99 and pfACBP749, in

1046

which the Y30 and Y33 were mutated to alanine. These mutations generate the PfACBP16-

1047

Y30A,Y33A, PfACBP99-Y30A,Y33A and PfACBP749-Y30A,Y33A constructs that are used in the study.

1048

(D) Surface Plasmon resonance based binding studies of PfACBP16-Y30A, Y33A, PfACBP99-

1049

Y30A,Y33A and PfACBP749-Y30A,Y33A with myristoyl-CoA show no binding of these proteins with

1050

myristoyl-CoA.

1051 1052 1053 1054 1055 1056 1057 1058 1059 1060 1061 1062 1063 1064 1065 1066 1067 1068 1069 1070 1071 1072 1073 1074 1075 1076 1077 1078 1079 1080 1081


Page 31 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1082 1083 1084

Figure 3. PfACBPs bind to phospholipids. Expression of PfACBPs is prominent in the trophozoite

1085

and schizont stages of Plasmodium. (A) Protein-lipid overlay assays show that PfACBPs bind to the

1086

phospholipids – phosphatidylcholine [PC] and phosphatidic acid [PA]. PfACBPs do not bind to the

1087

unphosphorylated lipid ester diacylglycerol [DAG]. (B) FACS studies show that PfACBPs can bind to

1088

the R.B.C. ghost membrane, implying that PfACBPs can bind to the membranous phospholipids. (C)

1089

Confocal microscopy studies reveal the differential expression of PfACBPs in different life-cycle

1090

stages of Plasmodium falciparum. Acyl-CoA binding proteins are abundantly expressed in

1091

trophozoite and schizont stages of Plasmodium. PfACBPs do not express in ring state of parasites.

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Page 32 of 41

1094 1095 1096

Figure 4. PfACBPs are alpha helical globular proteins. (A) Circular Dichroism spectroscopic studies

1097

demonstrate that the PfACBPs are alpha-helical proteins. The far-UV wavelength spectra of all three

1098

PfACBPs show ellipticity difference [Δε] value dip at 222nm and 208nm. This indicates the alpha-

1099

helical nature of the proteins. (B) The secondary structure components of PfACBPs show the

1100

presence of high proportion of alpha-helical structures in the PfACBPs. No beta sheet structure is

1101

present in any of the PfACBPs. (C) Hydropathy plots show the presence of small patches of


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hydrophobic regions in PfACBPs. These hydrophobic regions [yellow highlighted] are buried in the

1103

globular structure of PfACBPs.

1104 1105 1106 1107 1108 1109 1110 1111 1112 1113 1114 1115 1116 1117 1118 1119 1120 1121 1122 1123 1124 1125 1126 1127 1128 1129 1130 1131 1132 1133 1134 1135 1136 1137 1138 1139 1140 1141 1142 32 ACS Paragon Plus Environment


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1143 1144 1145

Figure 5. PfACBPs do not acquire any intermediate conformation in between their folded and

1146

unfolded conformations. (A, B, C) Far-UV circular dichroism spectra of PfACBPs at different

1147

temperatures in the range of 18oC - 90oC. [Inset] Three component CCA deconvolution of each

1148

spectra set shows presence of three principle curves, among which two curves almost merge with

1149

each other. This indicates that there is no intermediate structure present in between the folded and

1150

unfolded conformations of PfACBP structures.

1151 1152 1153 1154 1155 1156 1157 1158 1159 1160 1161


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1162 1163 1164

Figure 6. High-throughput in silico virtual screening for the identification of potential small

1165

molecule ligands of PfACBPs. (A, B) High-throughput docking of Pubchem and GSK-TCAMS

1166

compounds upon PfACBP749 present several potential small molecule ligands of PfACBP749. The

1167

docking scores vary in the range of -8 to -16.6 for Pubchem compounds and -1.4 to -20.6 for GSK-

1168

TCAMS compounds. A higher negative docking score represents stronger binding affinity of the

1169

compound to PfACBP749. (C) The first ten small molecule ligands [of Pubchem compounds]

1170

according to their docking scores on PfACBP749. The anti-malarial drug mefloquine appears as one

1171

of the potential ligands of PfACBP749 [red arrow].

1172 1173 1174 1175 1176 1177 1178 1179 1180 1181 1182 34 ACS Paragon Plus Environment


Page 36 of 41

1183 1184

Figure 7. Mefloquine binds to all three paralogs of PfACBPs. (A) Calorimetric titration isotherms

1185

show high affinity binding of PfACBPs with mefloquine. (B) Another quinoline containing compound,

1186

Chloroquine, does not bind to any of the PfACBPs.

1187 1188 1189 1190 1191 1192 1193 1194 1195 1196 1197 1198 1199 1200 1201 35 ACS Paragon Plus Environment

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1202 1203 1204

Figure 8. Mefloquine binds to the PfACBPs in the acyl-CoA binding pocket. (A, B, C) [Left panels]

1205

Structures of mefloquine docked structures upon the PfACBPs. [Right panels] Superimposed

1206

structures of mefloquine and myristoyl-CoA in the binding cavity of PfACBPs.



Page 38 of 41

1221 1222 1223

Figure 9. Mefloquine binding to PfACBPs at the acyl-CoA binding pocket uses several of the acyl-

1224

CoA binding residues. (A, B, C) Ligand interaction diagrams showing the residues that are engaged in

1225

bindings of [left panels] myristoyl-CoA to PfACBPs, and [right panels] mefloquine to PfACBPs. The

1226

tyr-30 and tyr-33 residues of PfACBPs are important for binding to myristoyl-CoA and mefloquine.

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1236 1237 1238

Figure 10. Mefloquine is a competitive inhibitor of myristoyl-CoA binding to PfACBPs. (A, B, C)

1239

Myristoyl-CoA does not bind to mefloquine-bound PfACBPs [mefloquine-PfACBP complex]. The

1240

sensograms show positive response for binding of mefloquine to PfACBPs. The myristoyl-CoA was

1241

incapable of binding to the any of the PfACBPs in their mefloquine-bound form.

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Figure 11. Mefloquine binding causes clearance of PfACBPs from Plasmodium cells. Mefloquine

1257

treatment causes death of the Plasmodium cells. (A) Confocal microscopy studies show that 39 ACS Paragon Plus Environment

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1258

mefloquine treatment causes degradtaion/clearance of PfACBPs from Plasmodium falciparum cells.

1259

(B) Immunoblot showing the reduction of PfACBPs in the cell lysate that was obtained from the

1260

mefloquine treated Plasmodium falciparum cells. The mefloquine mediated degradation of PfACBPs

1261

is prevented in the proteasome inhibited [MG132 treated] condition. (C) Quantitative estimation of

1262

PfACBP level in untreated versus mefloquine treated Plasmodium falciparum cells. [Left panel for the

1263

results of confocal images] The bar graph represents the mean PfACBP [FITC] intensities in untreated

1264

and mefloquine-treated cells [paired t-test of untreated vs. 10nM mefloquine treated cells: p>0.005,

1265

n = 30, df = 28]. [Right panel] The bar graph represents the blot intensities of PfACBPs in untreated

1266

and mefloquine treated parasites. [paired t-test of untreated vs. 10nM mefloquine treated cells:

1267

p>0.001, n = 6, df = 4]. (D) Immunoblots showing that mefloquine reduces the cellular level of

1268

PfACBPs in the trophozoite and schizont states of Plasmodium falciparum. (E) Parasitemia of

1269

Plasmodium falciparum reduces in the mefloquine treated Plasmodium culture (in vitro). Many of

1270

the Plasmodium cells die when 10nM of mefloquine is present in culture medium. (F) Temporal

1271

parasite count in normal and mefloquine-treated conditions shows inhibition of proliferation in

1272

presence of mefloquine.


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