Heme Detoxification Protein for New Ant - ACS Publications

Sep 26, 2017 - Translational Bioinformatics Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New. Delhi 110...
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Article Cite This: J. Med. Chem. 2017, 60, 8298-8308

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Exploring Heme and Hemoglobin Binding Regions of Plasmodium Heme Detoxification Protein for New Antimalarial Discovery Priya Gupta,† Sonali Mehrotra,† Anil Sharma,† Monika Chugh,† Rajan Pandey,‡ Abhinav Kaushik,‡ Sachin Khurana,† Neha Srivastava,† Tarushikha Srivastava,† Arunaditya Deshmukh,†,⊥ Ashutosh Panda,# Priyanka Aggarwal,∇ Neel Sarovar Bhavesh,∇ Raj K. Bhatnagar,∥ Asif Mohmmed,§ Dinesh Gupta,‡ and Pawan Malhotra*,† †

Malaria Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India ‡ Translational Bioinformatics Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India § Parasite Cell Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India ∥ Insect Resistance Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India ⊥ Centre for Biotechnology, Maharishi Dayanand University Rohtak, Haryana 123401, India # Department of Microbiology, All India Institute of Medical Sciences, New Delhi 110029, India ∇ Transcriptional Regulation Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India S Supporting Information *

ABSTRACT: Hemoglobin degradation/hemozoin formation, essential steps in the Plasmodium life cycle, are targets of existing antimalarials. The pathway still offers vast possibilities to be explored for new antimalarial discoveries. Here, we characterize heme detoxification protein, Pf HDP, a major protein involved in hemozoin formation, as a novel drug target. Using in silico and biochemical approaches, we identified two heme binding sites and a hemoglobin binding site in Pf HDP. Treatment of Plasmodium falciparum 3D7 parasites with peptide corresponding to the hemoglobin binding domain in Pf HDP resulted in food vacuole abnormalities similar to that seen with a cysteine protease inhibitor, E-64 (I-1). Screening of compounds that bound the modeled Pf HDP structure in the heme/hemoglobin-binding pockets from Maybridge Screening Collection identified a compound, ML-2, that inhibited parasite growth in a dose-dependent manner, thus paving the way for testing its potential as a new drug candidate. These results provide functional insights into the role of Pf HDP in Hz formation and further suggest that Pf HDP could be an important drug target to combat malaria.

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INTRODUCTION Malaria still remains a major parasitic disease with an estimate of 212 million cases of the disease reported worldwide in the year 2015.1 Plasmodium, an apicomplexan protozoan parasite, is the causative agent of the malarial infection, and the disease is transmitted through the bite of a female Anopheles mosquito. The main challenge to control the disease is to prevent the spread of resistance in parasites against the existing antimalarial drugs. Although artemisinin-based combination therapies are quite effective and are being widely used, early resistance against these therapies was seen first in western Cambodia, later spreading across an expanding area of the Greater Mekong subregion.2 Hence there is an urgent need for newer antimalarial drugs and an effective vaccine to prevent the © 2017 American Chemical Society

spread of disease. Malaria parasite resides in the host red blood cells during the asexual stage of its life cycle. Inside the host erythrocyte, the parasite digests hemoglobin in a specialized organelle, the food vacuole.3 The amino acids formed upon degradation are used for the parasite’s biosynthetic requirements.4 The free toxic heme generated in this process is converted into an inert insoluble polymer, hemozoin.5 The formation of hemozoin is a critical step for the survival of parasite as the free heme, which is generated during hemoglobin degradation is toxic to the parasite and can lead to the formation of reactive oxygen species, thus inducing an Received: January 19, 2017 Published: September 26, 2017 8298

DOI: 10.1021/acs.jmedchem.7b00089 J. Med. Chem. 2017, 60, 8298−8308

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Figure 1. (A) Schematics of Pf HDP protein depicting two putative heme binding domains (i) HeD1. aa191−200, (ii) HeD2. aa171−181) and a putative hemoglobin binding domain (HbD. aa154−172). (B) (i) In silico identification of heme binding loops in the structure of Pf HDP, HeD1 domain is shown in purple, and HeD2 domain is represented in blue. (ii) In silico docking of the heme molecule to the heme detoxification protein. Blue color denotes the heme molecule. Adjacent residues from Pf HDP interacting with heme moiety are highlighted in purple. (C) In silico identification of Hemoglobin binding loop in the structure of Pf HDP by aligning the hemoglobin binding loop of cysteine protease; falcipain-2. Red color marks the putative hemoglobin binding domain of Pf HDP.

showed two heme binding domains and a hemoglobin binding domain in Pf HDP. A peptide corresponding to the Hb binding domain produced food vacuole abnormalities similar to the one observed with E-64 (trans-epoxysuccinyl-L-leucylamido-(4guanidino)butane) 117 (I-1), a cysteine protease inhibitor thus advocating Pf HDP as an important target for new antimalarial discovery. We further screened a library of chemical compounds, Maybridge Hitfinder for binding to Pf HDP, and identified a compound which inhibited parasite growth in a concentration dependent manner, thus suggesting Pf HDP as an important target for new antimalarial discovery.

oxidative stress inside the parasite. Disruption of hemozoin formation appears to be an attractive target in killing the parasite as the process is indispensable for the survival of parasite. Importantly, several antimalarial drugs such as chloroquine (CQ) and artemisinin (ART) have been suggested to block the Hz formation.6 However, emerging resistance against many of these drugs has been the main reason to identify new drug targets and develop new antimalarials. Hz crystal consists of Fe(III)PPIX, and its structure is identical to β-hematin in which ferric iron of one heme is coordinated to the propionate carboxylate group of an adjacent heme.7 Lipids as well as proteins have been suggested to catalyze Hz formation inside the food vacuole.8 Pf HRPII, histidine-rich protein II, was the first protein that was shown to be involved in Hz formation.9 Interestingly, Pf HRPII knockouts still retain the ability to survive and grow normally, suggesting the role of certain other factors in the formation of hemozoin.10 It has been shown in Plasmodium and other hematophagous organisms like Schistosoma mansoni that the hydrophobic interior of the lipid bodies provides a milieu where the heme molecules can associate via the hydrogen bonds to form the hemozoin crystal.11,12 Pf HDP is one of the most potent hemozoin producing enzymes, it binds heme with a high affinity, and the histidine residues His122, His172, His175, and His197 of Pf HDP have been shown to be critical for heme binding.13,14 Unlike the parasite, the heme degradation inside the human host occurs as a series of autocatalytic oxidative reactions with heme bound to the enzyme heme oxygenase, yielding biliverdin, carbon monoxide, and iron as the final products.15 Pf HDP has been shown to exist in a ∼200 kDa complex with other proteins including falcipain2/2′, plasmepsin II, plasmepsin IV, and histo aspartic protease inside the food vacuole, and this complex known as degradosequestrome is involved in hemoglobin degradation and hemozoin formation.16 Here, we generated a number of deletion mutants of Pf HDP and characterized them for heme/Hb binding. The results

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RESULTS In Silico Analysis of Heme and Hemoglobin Binding Domains of Pf HDP. Although Pf HDP is known to convert heme to hemozoin crystals, little is known about the region(s) or residues involved in heme binding. In the absence of a crystal structure for Pf HDP protein, here we applied in silico approaches to model the structure of Pf HDP. Because Pf HDP does not have significant sequence homology with any of the PDB structure deposited in the Protein Data Bank, we used I-TASSER, an ab initio based three-dimensional structure prediction web server, to solve 3D structure for Pf HDP. The best I-TASSER Pf HDP model has a C-score value −0.55 and Tm value of 0.64, indicating good structure prediction for a Pf HDP model (Supporting Information, Figure S1A). We next looked for putative heme binding sequences in Pf HDP based on two abundant repeat sequences: HHAHHAADA and HHAAD identified in histidine rich protein II and known to bind the heme molecules via the bishistidyl ligation.18 On the basis of the sequence alignment between Pf HRPII and Pf HDP, we could identify two putative heme binding sequences in Pf HDP (HeD2 and HeD1) spanning aa 171− 181 and aa 191−200, respectively (Figure 1A). These putative heme binding sequences possess the three His residues H172, H175, and H197 that have been earlier shown to be critical in 8299

DOI: 10.1021/acs.jmedchem.7b00089 J. Med. Chem. 2017, 60, 8298−8308

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Figure 2. (A) Schematic representation of the wild-type PfwHDP and mutant Pf HDP proteins: Pf HDPHeD1, HeD1 (aa191−205) motif deleted; Pf HDPHeD2, HeD2(aa171−205) deleted; Pf HDPHbD, HbD(aa154−170) deleted; Pf HDP-N, 1−119aa; Pf HDP-C, 88−205aa. (B) Coomassie stained SDS-PAGE and Western blot analysis of recombinant wild-type and mutant Pf HDP proteins. Western blot was probed using an anti-His antibody. (C) Upper panel: In vitro heme to hemozoin conversion activity of recombinant wild-type and mutant proteins. Lower panel: In vitro hemoglobin to hemozoin conversion activity assay of the wild-type and mutant proteins in the presence of recombinant falcipain-2. The error bars represent the mean ± SD of the triplicate experimental values.

heme binding.14 We further used F-pocket to predict the ligand binding pockets using simulated Pf HDP 3D structure and CDD to predict active residues. F-pocket predicted the presence of 11 feasible binding pockets with pocket score in the range of 6.0517−32.4021 and drug score in between 0.0083−0.3477. The predicted pocket with best score has R4, R186, F5, Y6, Y7, Y130, Y178, N8, N174, L9, L133, H172, H175, H197, C173, S176, I177, I184, I185, P187, and ASP200 residues (Supporting Information, Figure S1B,S1D). Further, we used PatchDock Web server for Pf HDP-HEME docking to retrieve information regarding binding residues involved in Pf HDP-HEME interactions. FireDock was used on best hits retrieved from PatchDock for energy refinement and free global binding energy calculation. Docking analysis showed similar residues of Pf HDP involved in the heme binding as predicted by Fpocket. This binding pocket includes three histidine residues (172, 175, and 197), which may be important for Pf HDP activity as reported in previous studies.14 On the basis of both above prediction results, we identified two heme binding domains (Figure 1B (i)) in Pf HDP, KHCNHSIYLNG and CHNGVVHIVD, containing predicted HIS residues which may be essential for Pf HDP heme to hemozoin conversion activity.

A previous report based on immunoelectron microscopy of Plasmodium infected erythrocytes has shown the presence of Pf HDP in the same transport vesicles that deliver hemoglobin to the food vacuole, thereby suggesting a possible interaction between Pf HDP and hemoglobin.13 Hence, we looked for Hb binding domain if any were present in the Pf HDP sequence. To identify putative Hb binding sequence(s) in Pf HDP, we aligned the 14aa long hemoglobin binding motif of falcipain-2 with the Pf HDP sequence and looked for the homologous sequence in Pf HDP. As shown in Supporting Information, Figure S6B, a sequence corresponding to amino acid 154−172 in Pf HDP showed partial homology with the Hb binding loop sequence of falcipain-2, thereby indicating it as a putative Hb binding domain of Pf HDP (Figure 1C). Deletion of Heme Binding Domains Impairs the Hemozoin Formation Activity of the Pf HDP Protein. To functionally characterize and validate the putative heme binding as well as Hb binding sequences identified by in silico analysis, we generated a number of Pf HDP deletion mutants: Pf HDPHeD1 encoding amino acids 1−190 (lacking the heme binding domain HeD1), Pf HDPHeD2 encoding amino acids 1− 170 (lacking the second heme binding domain HeD2), Pf HDPHbD lacking the Hb binding domain (amino acids 153−171), Pf HDP-N encoding amino acids 1−119, and 8300

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Figure 3. (A) Schematic showing the location and sequence of two heme binding peptides: peptide 2 (MKCHNGVVHIVD) and peptide 3 (GEFKHCNHSIYLNG) corresponding to the HeD1 and HeD2 motifs that were synthesized and assessed for their heme binding properties. (B) SORET assay to assess the binding of heme to synthetic peptides: 2 and 3. A peptide where the histidines in peptide 2 were replaced with alanines was used as a control peptide 4. (C) A Cartesian scatter graph showing the dose-dependent inhibition of Pf HDP heme to hemozoin formation activity by peptides 2 and 3. The error bars represent the mean ± SD of the triplicate experimental values.

Pf HDP-C encoding amino acids 88−205 of the full length protein (Figure 2A). Wild-type and mutant Pf HDP proteins were expressed and purified to near homogeneity by a protocol described by Jani et al.13 (Figure 2B). These recombinant proteins were assessed for Hz formation activity in an in vitro assay. In an in vitro Hz formation assay where 0.5 μM wild-type Pf HDP protein and 300 μM heme were used, 50% of the heme was converted to Hz over 4h at pH 5.2 (Figure 2C, upper panel). This activity was specific to Pf HDP, as no activity was detected in the absence of Pf HDP protein. In contrast to wildtype Pf HDP protein, the efficiency of Hz production reduced by 4%, 30%, 60%, 75%, and 71% for Pf HDPHbD, Pf HDPHeD1, Pf HDPHeD2, Pf HDP-N, and Pf HDP-C proteins, respectively (Figure 2C, upper panel), suggesting a critical role of the deleted heme binding domains identified in the present study in Hz formation. Pf HDPHeD1 and Pf HDPHeD2 proteins also showed reduced heme binding in Soret assay as compared to the wild-type protein (Supporting Information, Figure S7). Interestingly, a complete inhibition of heme conversion to Hz could not be achieved with Pf HDPHeD2 protein. The striking observation though was that the Pf HDP-C protein, which contained the heme binding domains, also showed a drop in activity as compared to the wild-type protein. This could be ascribed to the fact that heme requires a hydrophobic core for binding to the protein and the Pf HDP-C protein might not be able to provide such matrix alone, suggesting a proper folding requirement for the protein activity.

These recombinant Pf HDP proteins were further assessed for Hb to Hz conversion activity in the presence of recombinant falcipain-2 protein using an assay described by Chug et al.16 In comparison to the wild-type Pf HDP protein, three mutant proteins, Pf HDP HeD1 , Pf HDP HeD2 , and Pf HDPHbD, showed reduced Hb to Hz formation activity, and among these mutant proteins, Pf HDPHbD showed the greatest reduction in comparison to the other two mutants (Figure 2C, lower panel). We further explored the heme binding properties of the two putative Pf HDP heme-binding domains identified by in silico analysis. Two peptides, a 12 mer peptide corresponding to HeD1 region 2 (heme PD1, MKCHNGVVHIVD) and a 14 mer peptide corresponding to HeD2 region 3 (heme PD2, GEFKHCNHSIYLNG), were synthesized (Figure 3A) and tested for their ability to bind to heme in vitro.19 Both the peptides were ≥95% pure (determined by HPLC), and the HPLC report has been provided in the Supporting Information. Heme binding curve was constructed by plotting the change in absorbance at the Soret peak (400 nm) versus heme concentration (Figure 3B). Both peptides at 20 μM concentration in the presence of heme produced a Soret peak at 400 nm, whose intensity increased with an increase in concentration of heme in the reaction. Both of these peptides showed considerable heme binding in comparison to a control peptide 4, GEFKAAAASIYLNG (Figure 3B). To further confirm that HeD1 and HeD2 are truly the Pf HDP heme binding domains, we tested the ability of these peptides to 8301

DOI: 10.1021/acs.jmedchem.7b00089 J. Med. Chem. 2017, 60, 8298−8308

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Figure 4. (A) Schematic representation of the wild-type (w) and hemoglobin binding domain deleted mutant (HbD) Pf HDP proteins. (B) ELISA based binding assay to show and compare the binding of PfwHDP or Pf HDPHbD mutant proteins with hemoglobin was assessed by an in vitro ELISA interaction analysis. Pf varc was used as a negative control. The error bars represent the mean ± SEM of the replicate values. (C) Peptide 5, corresponding to the Pf HDP Hb binding site, competes with Pf wHDP for hemoglobin binding in an in vitro ELISA binding studies. Peptide 6 was used as a control. (D) Effect of peptide 5 on in vitro P. falciparum culture. Treating the 3D7 P. falciparum parasite culture with peptide 5 at the late ring stage leads to food vacuole abnormalities and parasite stress.

compete with wild-type Pf HDP protein in an in vitro heme to Hz conversion assay. Each of the two peptides inhibited He to Hz conversion considerably in a concentration-dependent manner (Figure 3C). At 100 μM of peptide concentration, we observed >75% inhibition in He to Hz conversion with both the peptides. The IC50 calculation of the inhibition has been reported in Supporting Information, Figure S8. These results suggested that HeD1 and HeD2 are truly the heme binding regions of Pf HDP. Identification of a Hemoglobin Binding Domain in Pf HDP. Since we identified a sequence partially homologous to falcipain-2 hemoglobin binding loop in Pf HDP, we next studied an interaction of recombinant wild-type Pf HDP and Pf HDPHbD (Figure 4A), a mutant protein with hemoglobin in an in vitro ELISA binding assay. Recombinant Pf varC protein was used as a negative control. As shown in Figure 4B, both the Pf HDP proteins bound hemoglobin in a concentrationdependent manner, while Pf varC did not show any binding to Hb. Importantly, we observed a drop in binding affinity to hemoglobin for Pf HDPHbD protein in comparison to wild-type Pf HDP protein, thereby suggesting that the deletion of residues corresponding to aa 153−171 of Pf HDP results in substantial impairment in hemoglobin binding to Pf HDP. To further confirm the Hb binding characteristics of the identified Pf HDP Hb binding domain, we synthesized a 19-mer peptide, LRNLLNNDLIVKIEGEFKH, referred as 5 (HbP1), and performed an in vitro ELISA based Hb binding assay in the presence of the peptide. Synthetic 19-mer peptide 5 corresponding to the hemoglobin binding domain competed with wild-type Pf HDP for binding to hemoglobin. As shown in

Figure 4C, peptide 5 displaced the binding of Hb to wild-type Pf HDP in a concentration-dependent manner. 6 (HbC1), a scrambled peptide, LHRKNFLELGNENIDKL, synthesized by shuffling the 19-mer peptide sequence, did not displace the binding of Hb to wild-type Pf HDP. Together, these results showed that the motif extending from aa 153−171 of Pf HDP represents an Hb binding domain. Effect of Hb Binding Peptide on P. falciparum Development. As Pf HDP has been shown to a part of degradosequestrasome complex that participates in Hb degradation and Hz formation, the two essential steps in the Plasmodium life cycle, we next investigated the effect of peptide 5 on parasite growth and development. Briefly, highly synchronized ring stage parasites (3D7 strain) at 2% hematocrit and 1% parasitemia were treated with the peptide 5 at different concentrations (50−100 μM), respectively, in a 96-well cell culture plate. After 12 h of incubation, treated parasites were examined by microscopic examination of the Giemsa stained smears. Treatment of parasites with peptide 5 resulted in gross deformities in the food vacuole, a phenotype similar to that seen in cysteine protease inhibitor, 1, treated parasites (Figure 4D). The food vacuole of treated parasites was swollen and darkly stained. The abnormal food vacuole also showed tight clumps of malaria pigment that appeared to be different from normal untreated parasites. Together, these results advocate a role of Pf HDP in hemoglobin degradation/hemozoin formation. Identification of Small Molecules That Bind Pf HDP in Silico and Inhibit the Plasmodium Parasite Growth. Given that Pf HDP plays an important role in hemozoin 8302

DOI: 10.1021/acs.jmedchem.7b00089 J. Med. Chem. 2017, 60, 8298−8308

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Figure 5. (A) In silico docking of the compound 8 in the HeD1 motif of Pf HDP. (B) SPR interaction analysis to show that Pf HDP binds to the compound 8. The dissociation constant as found out by kinetic analysis was 2.9 μM. (C) Compound 7 interferes with P. falciparum growth. Addition of compound 8 in an in vitro P. falciparum culture inhibited parasite growth in dose-dependent manner. The error bars represent the mean ± SD of the triplicate experimental values. (D) Inhibition of Pf HDP activity by the compound 8 identified by the in silico docking of the drug in the HeD1 motif. The error bars represent the mean ± SD of the triplicate experimental values.

11 2 1 (ML-5), and HTS12239 (1,5-diphenyl-3-(2thienylcarbonyl)[1,2,4]triazolo[4,3-a]pyrimidin-7(1H)-one) 1221 (ML-6), showed significant inhibition in parasite growth at 50 and 100 μM concentrations (Supporting Information, Table S1). These compounds passed through a pan assay interference compounds (PAINS) filter.23 Among these compounds, 7, 8, and 9 bind the HeD1 domain of Pf HDP identified in the present study, 10 and 11 bind the HeD2 domain, while 12 binds the Hb binding domain of Pf HDP (Supporting Information, Figure S10). The purity of two of the compounds 7 and 9 as determined by qNMR was >94% and >95%, respectively. The compound 8 was estimated to be 90.72% pure. Two of the compounds 10 and 12 showed >86% and >72% purity. However, the purity percentage of compound 11 could not be determined. We next tested the effect of these compounds at several different concentrations on parasite growth. Five of the six selected compounds showed concentration dependent inhibition of parasite growth in an in vitro growth inhibition assay (Supporting Information, Figure S11). Incidentally, five of the identified compounds precipitated after repeated freeze− thawing, while one of these compounds, 8, C21H21F3N2, 1(3,4-dihydronaphthalen-2-yl)-4-[3-(trifluoromethyl) phenyl] piperazine, remained stable and inhibited parasite growth in dose-dependent manner at submicromolar range (Figure 5C) . These results were reproducible over three independent experiments, each carried out in triplicate. The IC50 value of the inhibition as calculated by GraFit7 software was 59.6 ± 1.8

formation, an essential step in Plasmodium blood stage cycle, we performed virtual screening studies of the Maybridge library of diverse drug-like 14400 compounds targeting Pf HDP modeled structure. The Hitfinder database includes 14400 premier compounds representing the drug-like diversity of the Maybridge Screening Collection, prescreened for Lipinski rules,20 and is commercially available for purity greater than 90%.21 The purity was further estimated by qNMR according to a protocol described in ACS guidelines,22 and the results have been provided as separate Supporting Information. On the basis of binding affinities for the heme/hemoglobin binding sites as well as drug likeliness scores, we selected 13 compounds and tested their ability to inhibit the parasite growth in an in vitro assay (Supporting Information, Table S1). Briefly, the highly synchronized ring stage parasites (3D7 strain) at 2% hematocrit and 1% parasitemia were treated with two different concentrations of compounds (50 and 100 μM), respectively, in a 96-well cell culture plate. The parasitemia was estimated after an incubation of 60 h both in control and treated samples using flow cytometry. Six of these compounds, RH00035 (N′9[3-(trifluoromethyl)benzoyl]-9H-xanthene-9-carbohydrazide) 721 (ML-1), HTS01276 (1-(3,4-dihydronaphthalen-2-yl)-4-[3(trifluoromethyl)phenyl]piperazine) 821 (ML-2), HTS09502 (N-{2-[(7-methyl-2,3-dihydro-1H-inden-4-yl)oxy]-3-pyridinyl}-3,4-dihydro-2H-1,5-benzodioxepine-7-carboxamide) 921 (ML-3), BTB02953 (5-({[1-(2,4-dimethylphenyl)-1H-1,2,3,4tetraazol-5-yl]thio}methyl)-3-(3-nitrophenyl)-1,2,4-oxadiazole) 1021 (ML-4), DSHS00186 ((2,4-dinitrophenyl)hydrazone) 8303

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μM (Supporting Information, Figure S12). The binding of the compound 8 to Pf HDP was confirmed by utilizing the surface plasmon resonance based approach. The sensogram showed a proportionate increase in binding of the drug with time to the immobilized HDP, resulting in the equilibrium association constant KA 3.37 × 105 M−1 with a corresponding equilibrium dissociation constant of KD 2.96 × 10−6 M (Figure 5B). To mimic the elevated response due to DMSO, solvent correction dilutions were passed over the surface of the chip and were found to be in the acceptable range (as per manufacturer’s instructions). The kinetic analysis and binding experiments thus confirmed the binding of the drug to Pf HDP. We next tested the ability of compound 8 to inhibit heme to hemozoin conversion mediated by Pf HDP. As shown in Figure 5D, compound 8 considerably reduced heme to hemozoin conversion activity of Pf HDP. Together; our results identified a number of compounds targeting Pf HDP, which form basis for the development of new antimalarial.

deletion of these two domains did not result in complete loss of heme to hemozoin formation activity of Pf HDP, therefore indicating that there are probably other heme binding regions in Pf HDP besides HeD1 and HeD2. These results are in line with a previous report, which showed that deletion of all the histidine residues of Pf HDP still retained its 49% activity as compared to the wild-type protein.14 Similar observation has been reported in Rhodnius prolixus, where the modification of histidine residues in α-glucosidase did not completely abolish its activity.25 Interestingly, complete loss of heme to hemozoin conversion activity was seen in two Pf HDP mutants, Pf HDP-N or Pf HDP-C, where C-terminal and N-terminal regions are deleted, respectively, thereby indicating that full Pf HDP is required for maximum heme to hemozoin conversion activity. Surprisingly, peptide(s) corresponding to either of the two heme binding regions inhibited >75% heme to hemozoin conversion activity of wild-type Pf HDP. It is possible that either of the two peptides competed for all the heme with Pf HDP. Besides existing in the food vacuole, Pf HDP has been shown to exist in transport vesicles along with hemoglobin in Plasmodium infected erythrocytes.13 To know whether Pf HDP also binds hemoglobin and has a role in trafficking of hemoglobin to food vacuole, we looked for the hemoglobin binding region within a Pf HDP sequence by aligning the hemoglobin binding sequence identified in falcipain-226 with the Pf HDP sequence. The analysis identified a hemoglobin binding sequence (HbD) partially homologous to a unique 14aa sequence of falcipain-2 within Pf HDP sequence. A Pf HDP mutant protein (Pf HDPHbD) that lacks this putative HbD sequence was generated, and its binding to hemoglobin was compared with the wild-type Pf HDP. Pf HDPHbD protein showed a significant reduced binding to hemoglobin in comparison to wild-type Pf HDP, thereby confirming the identity of HbD in Pf HDP. A synthetic peptide, 5, corresponding to the Pf HDP HbD domain, effectively competed with Pf HDP for hemoglobin binding in an in vitro ELISA based binding study. To know the functional relevance of Pf HDP HbD domain in hemoglobin transport or degradation, Plasmodium 3D7 culture at ring stage was treated with peptide 5, and surprisingly these treated parasites showed food vacuole abnormalities similar to the abnormalities observed with hemoglobinase(s) inhibitors.27,28 Together, these results demonstrated that Pf HDP is a multifunctional protein having a role in heme to hemozoin formation as well as an unknown role mediated by its interaction with hemoglobin. Processes of hemoglobin degradation/hemozoin formation and proteins associated with these processes are targets of existing antimalarials and are also targets for new antimalarial discovery.29,30 Given the fact that Pf HDP has multiple roles and is essential for malaria parasite survival,13 we further exploited this protein for identifying new compounds against malaria. For the same, we carried out screening of a library of chemical compounds in Maybridge library to identify the compounds that bound at high affinity at the heme/ hemoglobin binding sites of Pf HDP. This library has been successfully used to identify compounds for a number of diseases and pathogens.31 The compounds having the highest activation energy for Pf HDP heme/hemoglobin sites were selected for experimental validation, inhibition of parasite growth. The compound 8 inhibited the Pf HDP heme to hemozoin conversion activity and inhibited the growth of the parasite at submicromolar concentrations. The compound also

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DISCUSSION Hemoglobin degradation is an essential step during the asexual stages of Plasmodium life cycle, and the heme generated during the process is highly toxic to the parasite. The detoxification of heme is indispensable to the survival of the parasite as inhibitor(s)/drugs that inhibit heme to hemozoin conversion kill the parasite.2,6 A number of Plasmodium proteases and a heme detoxification protein (Pf HDP) have been shown to be involved in hemoglobin degradation as well as in hemozoin formation.8,13 It has been recently shown that Pf HDP is colocalized in transport vesicles with hemoglobin and is highly efficient in the conversion of heme to hemozoin.13 In the present study, we characterized Pf HDP for heme/Hb binding sites as well as an essential protein for antimalarial drug discovery. Given that Pf HDP does not show homology with any known protein and its crystal structure has not been solved, we applied in silico approaches to model Pf HDP structure in order to identify heme/Hb binding residues/regions. Because the Cterminal region of Pf HDP is homologous to fasciclin-1, whose structure is known, we built a 3D structure of Pf HDP using ITASSER, an ab initio based three-dimensional structure prediction web server. I-TASSER has been used successfully to build many such models of proteins, which have not been crystallized so far.24 Having modeled the structure of Pf HDP, we next looked for heme binding sites in Pf HDP structure based on the two characterized histidine rich heme binding sequences: HHAHHAADA and HHAAD identified in Pf HRPII, a well-known Plasmodium heme binding protein.18 Two putative heme-binding sequences referred to as HeD1 and HeD2 in Pf HDP, which possess four histidine residues, H172, H175, H192, and H197, were identified in Pf HDP. A recent study has shown the involvement of three of these four histidine residues of Pf HDP in heme binding,14 therefore confirming the identity of two heme binding regions identified in present study. To know whether HeD1 and HeD2 regions of Pf HDP are actually involved in heme binding and subsequently are involved in heme to hemozoin conversion mediated by Pf HDP, we generated two Pf HDP deletion mutants: Pf HDPHeD1 and Pf HDPHeD2. Comparison of heme to hemozoin conversion activity among Pf HDPHeD1, Pf HDPHeD2 and wild-type Pf HDP proteins showed that these mutant proteins have significantly reduced hemozoin formation activity in comparison to the wild-type Pf HDP protein. However, the 8304

DOI: 10.1021/acs.jmedchem.7b00089 J. Med. Chem. 2017, 60, 8298−8308

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100 using a sonicator, pelleted again, and then washed four times in wash buffer without Triton X-100. The inclusion bodies were solubilized for 30 min in 50 mM CAPS buffer at pH 11.0 containing 1.5% N-laurylsarkosine and 0.3 M NaCl and centrifuged at 10000g for 30 min. The protein was purified using Ni-NTA packing where the protein solution was allowed to bind Ni-NTA for 3−4 h and then washed in 5 mM imidazole in 50 mM CAPS and 0.3% Nlaurylsarkosine. The protein was eluted in varying concentrations of imidazole (75, 100, and 150 mM) in 50 mM CAPS and 0.3% Nlaurylsarkosine. The protein fractions were dialyzed against 50 mM CAPS buffer containing 135 mM NaCl and 20% glycerol. Hemozoin Formation Assay. Heme to hemozoin formation assay was performed as described previously.9 Briefly, a stock solution of 10 mM heme was prepared in DMSO. Heme was used at a concentration of 300 μM in 500 mM sodium acetate buffer pH 5.2. Then 0.5 μM of Pf HDP was added to each reaction. The reaction was allowed to proceed for 4 h at 37 °C. After the completion of the reaction, 10 μL of 10% SDS was added to stop the reaction and the mixture was vortexed and centrifuged at 13000 rpm for 30 min. The pellet was resuspended in 2.5% SDS and incubated at 37 °C for 1 h. The mixture was then centrifuged at 13000 rpm for 30 min. The pellet was washed in 50 mM sodium bicarbonate at pH 9.1 to remove the heme. The pellet was finally washed in distilled water, and the hemozoin formed was resuspended in 0.1N NaOH containing 2.5% SDS. The absorbance was recorded at 405 nm. In Vitro Hz Formation Assay from Hb. Hemoglobin to hemozoin formation assay was used as described earlier.16 Briefly, hemoglobin (800 μg) along with falcipain 2 (0.5 μM) was used as a substrate in 500 mM sodium acetate buffer pH 5.2. The reaction was allowed to take place for 2 h. Then 0.5 μM of Pf HDP was added to each reaction. The reaction was allowed to proceed for 4 h at 37 °C. After the completion of reaction, 10 μL of 10% SDS was added to stop the reaction, and the mixture was vortexed and centrifuged at 13000 rpm for 30 min. The pellet was resuspended in 2.5% SDS and incubated at 37 °C for 1 h. The mixture was then centrifuged at 13000 rpm for 30 min. The pellet was washed in 50 mM sodium bicarbonate at pH 9.1 to remove the heme. The pellet was finally washed in distilled water, and the hemozoin formed was resuspended in 0.1N NaOH containing 2.5% SDS. The absorbance was recorded at 405 nm. Heme Binding Assay (Soret Assay). Soret assay has been used routinely to study the interaction of heme with heme binding protein.19 For the Soret assay, 20 μM of each peptide 2 and 3 was used for analysis. Peptide 4 was used as a control. Heme was added in increments of 3 μM to the peptide solution in 100 mM sodium acetate buffer pH 5.2. After each addition of heme, the absorption spectra were recorded and change in absorbance at 400 nm was plotted against the concentration of heme. Spectra were recorded on a Hitachi 557 double beam double wavelength spectrophotometer. The curve depicts the nonlinear fit of the data. The peptides used in this study were synthesized from GL Biochem (Shanghai) Ltd. The peptides possess purity of more than 95% (determined by HPLC), and the details of the purity of these peptides as provided by the supplier is available in the Supporting Information (Figures S13, S14, S15). The HPLC report of each peptide is provided in Supporting Information, purity profile characteristics of all the peptides used in the study. Analysis of Protein Interaction by ELISA. ELISA based protein−protein interaction analysis was performed as described previously.16 Briefly, a 96-well microtiter plate was coated overnight at 4 °C with 200 ng of recombinant Pf wHDP or Pf HDP mutant protein. After blocking the wells with 3% BSA in PBS for 2 h, hemoglobin was added in different amounts ranging from 0 to 2400 ng, and the plate incubated for 3 h at 37 °C. Interaction was detected using antibodies against Hb (1:500). Incubation with HRP conjugated antirabbit antibodies (1:5000) was done for 1 h and quantified after adding the substrate OPD by measuring the resulting absorbance at 490 nm. For competition experiments, 400 ng of Hb was preincubated with varying concentrations of the synthetic peptide 5 corresponding to the predicted Hb binding motif for 30 min at 37 °C and then added to Pf HDP coated ELISA plate. Peptide 6 was used as the negative control. Interaction was detected using antibodies against Hb (1:500).

showed a dose-dependent interaction with Pf HDP as determined by SPR interaction analysis. The dissociation constant for the binding was 2.9 μM. The study paves the way for new molecules targeting Pf HDP, an essential protein required for Plasmodium development and for new antimalarial development.

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CONCLUSION In conclusion, here we characterize Pf HDP, an essential protein in Plasmodium life cycle for heme/hemozoin binding sites and show that a peptide corresponding to hemoglobin binding domain of Pf HDP produced food vacuole abnormalities in P. falciparum, similar to one seen with falcipain-2/plasmepsin inhibitors. We further identify unique compounds from a Maybridge Hitfinder library of drug-like compounds targeting Pf HDP. The study thus reveals new unique scaffolds by targeting Pf HDP using computational, structural, and experimental approaches for rational drug design to combat drug resistant malaria.

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EXPERIMENTAL SECTION

In Silico Modeling of Pf HDP Structure. We retrieved Pf HDP sequence from PlasmoDB. SWISSMODEL32 and I-TASSER24 was used to generate three-dimensional structure for Pf HDP. Gromacs version 4.6.333 was used to perform molecular dynamic (MD) analysis to check the stability of modeled structure. CHARMM27 force field34 was used for energy minimization and MD production run. CDD35 was used to predict active site residues. Fpocket36 was used to predict active pocket present in Pf HDP. Crystal structure of human hemoglobin (Hb) (1BUW) and heme structure was retrieved from the Protein Data Bank (PDB, http://www.rcsb.org/pdb/home/home. do). PatchDock37 was used for Pf HDP-heme docking and FireDock38 energy refinement on docked complexes. All generated and molecular dynamic simulated models were subjected to Structural Analysis and Verification Server (SAVES).39−43 Pymol was used to visualize structures. Cloning, Recombinant Expression, and Purification of Mutant Pf HDP Proteins. The forward and reverse primers for the cloning of different mutant proteins are: Pf HDPHeD1, 5′-CCCATGGGATCCATGAAAAATAGATTTTAT-3′ and 5′-CCGGTACCCTTCATATTTGGTCTTAT-3′, respectively; Pf HDPHeD2, 5′CCCATGGGATCCATGAAAAATAGATTTTAT-3′ and 5′CCGGTACCAAATTCCCCCTCAATTTTTACT-3′, respectively; Pf HDP-N, 5′-CGGGATCCTTAAATCACGTTACGAAAGA-3′ and 5′-CGGTCGACTAAAACAAATTCTGATAATTT-3′, respectively; Pf HDP-C, 5′-GCCCATGGCATTTTTTCCCTCCAACGAAGCC-3′ and 5′-GCCTCGAGAAGCTTTCAAAAAATGGATGGGCTTATC3′, respectively. The PCR product of Pf HDPHeD1 and Pf HDPHeD2 were cloned into a pQE30 vector. The gene for Pf HDP-C was amplified and cloned in a pTEM-30 vector. The gene for Pf HDP-N was amplified and cloned in a pET-28a vector. The Pf HDPHbD gene was synthesized from genscript in a pUC vector and further subcloned in a pQE30 vector. The genes cloned in pQE30 and pTEM-30 vectors were expressed in M15 Escherichia coli cells. The gene cloned in a pET-28a vector was expressed in BL21 Escherichia coli cells and protein localized to inclusion bodies, which were isolated as described.13 Briefly, cultures of bacteria-containing plasmid pHDP were grown to mid log phase, induced with isopropyl-1-thio-β-Dgalactopyranoside (IPTG, 1 mM) for 4 h at 37 °C, and harvested by centrifugation at 4000g for 20 min. The total cell pellet was resuspended in wash buffer (50 mM Tris·HCl at pH 7.5, 20 mM EDTA) containing 0.5 mg/mL lysozyme and incubated for 1 h at room temperature with intermittent shaking and then with vigorous shaking for an additional 30 min. The washed cell pellet was lysed after adding wash buffer containing 0.5 M NaCl and 2.5% Triton X-100. The inclusion bodies were pelleted by centrifugation at 13000 rpm for 50 min at 4 °C, resuspended in wash buffer containing 1% Triton X8305

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injection of 10 mM glycine pH2.5 (GE Healthcare) was used to regenerate the surface after each cycle. A total of eight dilutions of DMSO solvent correction solutions were prepared fresh and were run along the experimental cycles. The experiment was carried out at 25 °C, and the kinetic analysis was performed on the Biacore T200 Kinetics Evaluation Software (GE Heathcare).

Incubation with HRP conjugated antirabbit antibodies (1:5000) was done for 1 h and quantified after adding the substrate OPD by measuring the resulting absorbance at 490 nm. The peptides used in this study were synthesized from GL Biochem (Shanghai) Ltd. The peptides possess purity of more than 95% (determined by HPLC), and the details of the purity of these peptides as provided by the supplier is provided in the Supporting Information (Figure S16, S17). The HPLC report of each peptide is provided in Supporting Information, purity profile characteristics of all the peptides used in the study. Growth Inhibition Assay. The effect of HbP1peptide and the inhibitors from Maybridge library on parasite growth was evaluated on the 3D7 strains of P. falciparum. The parasite culture was synchronized using 5% sorbitol, and the assay was set at the late rings stage with a hematocrit and parasitemia of synchronized ring stage culture adjusted to 2% and 1%, respectively. The peptide 5 (50−100 μM) and the inhibitors (5−100 μM) were added to the parasite culture in 96-well plates, respectively, and giemsa stained smears were made after an incubation of 12 h in the late trophozoite stage for microscopic analysis. The parasitemia was estimated after an incubation of 48 h in the next cycle using flow cytometry. Briefly, cells from samples were collected and washed with PBS followed by staining with ethidium bromide (10 μg/mL) for 20 min at 37 °C in the dark. The cells were subsequently washed twice with PBS and analyzed on FACScalibur (Becton Dickinson) using the Cell Quest software. Fluorescence signal (FL2) was detected with the 590 nm band-pass filter using an excitation laser of 488 nm collecting 100000 cells per sample. Uninfected RBCs stained in a similar manner were used as control. Following acquisition, data were analyzed for percentage parasitemia of each sample by determining the proportion of FL2-positive cells using Cell Quest. In Silico Docking of Maybridge Library Compounds in the Pf HDP Structure. The simulated and energy minimized threedimensional modeled structure of Pf HDP protein was subjected to protein preparation steps using prepare_receptor4.py python script with default parameters.44 Three different Pf HDP surface residues were shortlisted for molecular docking that were grouped under three binding regions, whereby site 1 and site 2 are the heme binding sites HeD1 and HeD2, respectively, whereas site 3 includes hemoglobin binding residue HbD. Thus, for molecular docking, three different Pf HDP systems with a grid around different active sites were designed. The final structural models of Pf HDP systems were then subjected to high-throughput virtual screening with Maybridge drug library (∼14000 compounds) using AutoDock Vina.45 Before docking, the library compounds were also subjected for ligand preparation steps using prepare_ligand4.py autodock scripts. After molecular docking AutoDock Vina generated at-most nine different conformations for each ligand which were sorted by binding affinity (kcal/mol). For each binding sites, the top five docked compounds were retained and common drugs binding to both sites were shortlisted for experimental evaluation. The drug-likeness of the shortlisted compounds was calculated by measuring the QED score of each drug.46 The drugs were further screened through the pan assay interference compounds (PAINS) criteria.23 SPR Interaction Analysis to Show That Pf HDP Binds to the Compound 8. The surface plasmon resonance analysis was carried out on the Biacore T200 instrument (GE Healthcare). Over 9500 response units of the Pf HDP protein was immobilized on an S-series CM5 sensor chip (GE Healthcare) using 10 mM sodium acetate pH 4.0 solution (GE Healthcare). The surface of the sensor chip was blocked with 1 M ethanolamine−HCl pH 8.5 (GE Healthcare). The kinetic analysis was then carried out using phosphate buffered saline pH 7.4 supplemented with 2% DMSO (Sigma-Aldrich) and 0.05% surfactant P20 (GE Healthcare), which was used as the running buffer. The drug was previously dissolved in 100% DMSO and diluted to a concentration of 10 mM. This was further diluted to 200 μM with 1.02× PBS pH 7.4, which resulted in a final concentration of 2% DMSO in the sample. Concentration series of the drug were made in the running buffer, which were then used for the kinetic analysis. The drug was passed over the immobilized HDP at a flow rate of 30 μL−1 for a total of 180 s and was allowed to dissociate for another 180 s. An

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00089. Three dimensional structure of I-TASSER predicted Pf HDP structure; Pf HDP residues interacting with heme in HeD2 motif and HeD1 motif; Pf HDP 3D prediction (SWISSMODEL) validation using Ramachandran plot and errat score; Pf HDP 3D prediction (ITASSER) validation using Ramachandran plot and errat score; molecular dynamic analysis of Pf HDP (SWISSMODEL); molecular dynamic analysis of Pf HDP (ITASSER); sequence alignment to show the heme binding sites and hemoglobin binding site as identified by aligning the heme binding repeat sequence of Pf HRPII with Pf HDP sequence of Plasmodium falciparum and aligning the sequence of Hb binding loop in Pf fal2 with HDP sequence of Plasmodium falciparum, respectively; Soret assay to assess the binding of heme to the recombinant wild-type and mutant Pf HDP proteins; calculation of IC50 values for the competitive inhibition studies (Figure 3C) carried out in the presence of peptide 2 and 3; table showing the binding affinity score and drug likeness score of the drugs of Maybridge library; receptor (Pf HDP)-ligand ligplot images for the drugs having the highest likeliness toward the heme binding sites in Pf HDP; dose dependent growth inhibition assay in the presence of the seven drugs identified from Maybridge library, which showed best in silico binding to Pf HDP; calculation of IC50 value by GraFit7 software of compound 8 tested in growth inhibition in an in vitro P. falciparum culture; purity profile of peptide 2; purity profile of peptide 3; purity profile of peptide 4; purity profile of peptide 5; purity profile of peptide 6; growth inhibition assay in the presence of the drugs that showed binding to Pf HDP (PDF) PDB files for the coordinates of Pf HDP, Pf HDP-HeD1, and Pf HDP-HeD2, and drugs with drug IDs 702, 4817, 9868, 2878, 13258, 12899 (ZIP) Purity profile characteristics of all the peptides used in the study (ZIP) qNMR drug purity containing the purity profile characteristics of all drugs used in the study (ZIP) Molecular formula strings containing the SMILES of the drug compounds (CSV) Chemical drawing structures of the compounds containing the chemical structures of all drugs used (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone: 91 11 26741358. Fax: 91 11 26742316. E-mail: [email protected], [email protected]. ORCID

Neel Sarovar Bhavesh: 0000-0002-7248-4978 8306

DOI: 10.1021/acs.jmedchem.7b00089 J. Med. Chem. 2017, 60, 8298−8308

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(HRPII) reveals sorting of soluble proteins in the periphery of the host erythrocyte and disrupts transport to the malarial food vacuole. J. Biol. Chem. 2002, 277 (32), 28923−28933. (11) Pisciotta, J. M.; Sullivan, D. Hemozoin: oil versus water. Parasitol. Int. 2008, 57 (2), 89−96. (12) Correa Soares, J. B.; Maya-Monteiro, C. M.; Bittencourt-Cunha, P. R.; Atella, G. C.; Lara, F. A.; d’Avila, J. C.; Menezes, D.; VannierSantos, M. A.; Oliveira, P. L.; Egan, T. J.; Oliveira, M. F. Extracellular lipid droplets promote hemozoin crystallization in the gut of the blood fluke Schistosoma mansoni. FEBS Lett. 2007, 581 (9), 1742−1750. (13) Jani, D.; Nagarkatti, R.; Beatty, W.; Angel, R.; Slebodnick, C.; Andersen, J.; Kumar, S.; Rathore, D. HDP-a novel heme detoxification protein from the malaria parasite. PLoS Pathog. 2008, 4 (4), e1000053. (14) Nakatani, K.; Ishikawa, H.; Aono, S.; Mizutani, Y. Identification of essential histidine residues involved in heme binding and Hemozoin formation in heme detoxification protein from Plasmodium falciparum. Sci. Rep. 2015, 4, 6137. (15) Kikuchi, G.; Yoshida, T.; Noguchi, M. Heme oxygenase and heme degradation. Biochem. Biophys. Res. Commun. 2005, 338 (1), 558−567. (16) Chugh, M.; Sundararaman, V.; Kumar, S.; Reddy, V. S.; Siddiqui, W. A.; Stuart, K. D.; Malhotra, P. Protein complex directs hemoglobin-to-hemozoin formation in Plasmodium falciparum. Proc. Natl. Acad. Sci. U. S. A. 2013, 110 (14), 5392−5397. (17) Rosenthal, P. J.; McKerrow, J.; Aikawa, M.; Nagasawa, H.; Leech, J. A malarial cysteine proteinase is necessary for hemoglobin degradation by Plasmodium falciparum. J. Clin. Invest. 1988, 82 (5), 1560. (18) Schneider, E. L.; Marletta, M. A. Heme binding to the histidinerich protein II from Plasmodium falciparum. Biochemistry 2005, 44 (3), 979−986. (19) Pisciotta, J. M.; Coppens, I.; Tripathi, A. K.; Scholl, P. F.; Shuman, J.; Bajad, S.; Shulaev, V.; Sullivan, D. J., Jr. The role of neutral lipid nanospheres in Plasmodium falciparum haem crystallization. Biochem. J. 2007, 402 (1), 197−204. (20) Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv. Drug Delivery Rev. 2001, 46 (1−3), 3−26. (21) Thermofisher Scientific. (22) Pauli, G. F.; Chen, S. N.; Simmler, C.; Lankin, D. C.; Godecke, T.; Jaki, B. U.; Friesen, J. B.; McAlpine, J. B.; Napolitano, J. G. Importance of purity evaluation and the potential of quantitative (1)H NMR as a purity assay. J. Med. Chem. 2014, 57 (22), 9220−9231. (23) Baell, J. B.; Holloway, G. A. New substructure filters for removal of pan assay interference compounds (PAINS) from screening libraries and for their exclusion in bioassays. J. Med. Chem. 2010, 53 (7), 2719− 2740. (24) Zhang, Y. I-TASSER server for protein 3D structure prediction. BMC Bioinf. 2008, 9, 40. (25) Mury, F. B.; da Silva, J. R.; Souza Ferreira, L.; dos Santos Ferreira, B.; de Souza-Filho, G. A.; de Souza-Neto, J. A.; Martins Ribolla, P. E.; Peres Silva, C.; Veiga do Nascimento, V.; Tavares Machado, O. L.; Berbert-Molina, M. A.; Dansa-Petretski, M. Alphaglucosidase promotes hemozoin formation in a blood-sucking bug: an evolutionary history. PLoS One 2009, 4 (9), e6966. (26) Pandey, K. C.; Wang, S. X.; Sijwali, P. S.; Lau, A. L.; McKerrow, J. H.; Rosenthal, P. J. The Plasmodium falciparum cysteine protease falcipain-2 captures its substrate, hemoglobin, via a unique motif. Proc. Natl. Acad. Sci. U. S. A. 2005, 102 (26), 9138−9143. (27) Rosenthal, P. J.; Wollish, W. S.; Palmer, J. T.; Rasnick, D. Antimalarial effects of peptide inhibitors of a Plasmodium falciparum cysteine proteinase. J. Clin. Invest. 1991, 88 (5), 1467−1472. (28) Korde, R.; Bhardwaj, A.; Singh, R.; Srivastava, A.; Chauhan, V. S.; Bhatnagar, R. K.; Malhotra, P. A prodomain peptide of Plasmodium falciparum cysteine protease (falcipain-2) inhibits malaria parasite development. J. Med. Chem. 2008, 51 (11), 3116−3123. (29) Feng, T. S.; Guantai, E. M.; Nell, M.; van Rensburg, C. E.; Ncokazi, K.; Egan, T. J.; Hoppe, H. C.; Chibale, K. Effects of highly

Pawan Malhotra: 0000-0002-7384-6280 Author Contributions

P.G. performed literature searches and research experiments. R.P. and A.K. performed bioinformatics analysis. S.K. performed the SPR interaction analysis experiment. S.M., A.S., and M.C. helped with experimental design. N.S., T.S., A.P., and A.D. helped with the experiments. P.A. and N.S.B. performed and analyzed the qNMR experiments. P.G., R.P., and P.M. performed the analysis and wrote manuscript. R.B. provided the Maybridge library. A.M., P.M., and D.G. supervised the study. All authors read and approved the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by Department of Biotechnology, Government of India (BT/01/CEIB/11/V/01 and BT/PR5267/MED/15/87/2012). We also thank the Rotary Blood Bank for providing human red blood cells for Plasmodium culture. P.G. and A.K. are recipients of Senior Research Fellowship of the CSIR, Government of India. R.P. is a recipient of a Senior Research Fellowship of the UGC, Government of India. P.A. is a recipient of Junior Research Fellowship of the DBT, Government of India. We also thank Dr. Sailesh Bajpai, Application Specialist, GE-Healthcare, for guidance with the SPR interaction experiment.

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ABBREVIATIONS USED (CQ) chloroquine; (ART) artemisinin; (Hz) hemozoin; (HDP) heme detoxification protein; (His) histidine; (Hb) hemoglobin

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REFERENCES

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