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Table of Contents
RESEARCH ARTICLE
Year : 2019  |  Volume : 56  |  Issue : 3  |  Page : 189-199

A novel quinoline-appended chalcone derivative as potential Plasmodium falciparum gametocytocide


1 Department of Chemistry, Visvesvaraya National Institute of Technology, Nagpur, Maharashtra; Department of Biochemistry & Molecular Biology, Dalhousie University, Halifax, Canada
2 ICMR–National Institute of Malaria Research, New Delhi, India
3 Department of Pharmaceutical Chemistry, College of Pharmacy, Madras Medical College, Chennai, India
4 Department of Chemistry, Visvesvaraya National Institute of Technology, Nagpur, Maharashtra, India

Date of Submission03-Apr-2018
Date of Acceptance31-May-2018
Date of Web Publication09-Jul-2020

Correspondence Address:
Dr Sujit Kumar Ghosh
Department of Chemistry, Visvesvaraya National Institute of Technology, Nagpur–440 010, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0972-9062.289398

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  Abstract 

Background & objectives: Malaria has remained a global health problem despite the effective control and treatment measures. In the backdrop of drug resistance, developing novel hybrid molecules targeting the sexual stages (gametocytes) of the human malaria parasite Plasmodium falciparum is of great significance. Recently, chalcone- based polyphenols have generated a great interest in the malaria research community worldwide due to their ease of synthesis and significant biological activity. The primary objective of this study was to investigate the interaction of a newly synthesized quinoline-appended chalcone derivative (ADMQ) with gametocyte specific proteins, Pfg 27 and Pfs 25 and explore its in vitro gametocytocidal potential.
Methods: The characterization of ligand-protein interactions at the atomistic level was done by a simulation strategy that combines molecular docking and molecular dynamics (MD) simulation in a coherent workflow. The X-ray crystal structure of Pfg 27 was retrieved from protein data bank and Pfs 25 was built using the Iterative Threading ASSembly Refinement (I-TASSER) server. The detailed interaction of both ADMQ and a known gametocytocidal agent, methylene blue (MB) (used as a positive control) with gametocyte proteins Pfg 27 and Pfs 25 was studied with a 50 ns explicit MD simulation. The ligand binding pose in terms of glide score, molecular mechanics-generalized born surface area (MM-GBSA) binding energies, protein-ligand root-mean-square-deviation (RMSD) and secondary structure elements (SSE) changes were analyzed accordingly. The direct effect of ADMQ on structural integrity of P. falciparum gametocytes was also examined using in vitro microscopy.
Results: The analogous Glide score and MM-GBSA free energy of binding indicated stable interactions for both ADMQ and MB harboured in the active site of targeted gametocyte proteins, Pfg 27 and Pfs 25, separately. Explicit MD simulation by Desmond software package indicated similar distinguishable conformational changes in the active site of target polypeptide chain due to the specific accommodation of ADMQ molecule. The simulation also manifested comparable mechanistic profile in terms of protein-ligand RMSD and changes in secondary structure elements (SSE). Further, ADMQ treatment was found to adversely affect the structural integrity of gametocytes, which resulted in appearance of vesicles protruding from the gametocytes.
Interpretation & conclusion: The consolidated in silico molecular modeling and in vitro study described herein may give an insight into the interaction patterns of quinoline-chalcone hybrids with critical gametocyte proteins in the mosquito. This study will possibly pave the way for further exploration of similar heterocyclic quinoline-chalcone hybrids to open up new avenues in drug candidate development against P. falciparum gametocytes.

Keywords: Chalcone; gametocytes; malaria; molecular docking; molecular dynamics; Plasmodium falciparum


How to cite this article:
Kumar H, Wadi I, Devaraji V, Pillai C R, Ghosh SK. A novel quinoline-appended chalcone derivative as potential Plasmodium falciparum gametocytocide. J Vector Borne Dis 2019;56:189-99

How to cite this URL:
Kumar H, Wadi I, Devaraji V, Pillai C R, Ghosh SK. A novel quinoline-appended chalcone derivative as potential Plasmodium falciparum gametocytocide. J Vector Borne Dis [serial online] 2019 [cited 2020 Sep 23];56:189-99. Available from: http://www.jvbd.org/text.asp?2019/56/3/189/289398

Himank Kumar, Ishan Wadi. Authors contributed equally





  Introduction Top


Malaria is one of the oldest tropical diseases on this planet and continues to affect millions of people worldwide[1]. In 2018, approximately 228 million malaria cases were reported out of which 405,000 people succumbed to this deadly disease[2]. Apart from the problem of severity (especially in Plasmodium falciparum cases) and the complex nature of the disease, the rapidly evolving resistance against standard antimalarial drugs and insecticides have restricted our options to fight malaria[3],[4],[5],[6]. Research efforts are therefore, constantly being directed towards development of novel antimalarial drugs. Moreover, blocking transmission is one of the ways to halt the spread of drug resistant parasites and hence, focus is also on developing strategies that can help in reduction of malaria transmission[7].

Erythrocytic stages of the parasite are responsible for majority of the clinical symptoms of malaria but only non- replicating sexual forms of the parasite (gametocytes) are capable of transmitting the disease. Plasmodium falci- parum gametocytes mature through five morphologically distinct stages inside a human host, taking 10–12 days to fully develop[8],[9]. Stage I to stage IV gametocytes are sequestered inside host tissues, specifically the bone marrow and the spleen[10],[11] and only the stage V gametocytes are released into peripheral circulation. The stage V gametocytes are then picked up by the mosquito in their subsequent blood meal to perpetuate the life-cycle forward. Various metabolic, biochemical and genetic changes occur during the gametocyte development and are known to have a profound impact on how the antimalarial drugs act upon the gametocytes.

Most common antimalarial drugs including chloro- quine that targets parasite’s asexual erythrocytic stages have moderate activity against the highly metabolizing stage I–III gametocytes but are completely inactive against stage IV and V gametocytes. At present, primaquine is the only licensed antimalarial drug having in vivo gametocytocidal activity[12]. However, due to associated hemolytic toxicity concerns especially in G6PD deficient individuals, its use as a transmission blocking drug is limited[13]. A thiazine dye, methylene blue (MB) has shown significant in vitro gametocytocidal potential against both early and late stage gametocytes[12],[14]. Use of MB as a gametocytocidal agent is still controversial since many studies reported haemolytic toxicity of MB amongst G6PD deficient population[15],[16],[17],[18],[19]. Therefore, development of new gametocytocidal agents in order to block the transmission of P falciparum is of utmost importance.

In the context of gametocytes being the target for antimalarial drugs to limit transmission, chalcone derivatives have emerged as recent subject of great interest for their ease of synthesis and potential pharmacological ac- tivities[20],[21],[22],[23],[24],[25],[26]. In the present study, an effort has been made towards studying the in silico interactions of a newly synthesized quinoline appended chalcone derivative (ADMQ) with two sexual stage-specific proteins, Pfg 27 (RNA binding phosphoprotein that contributes to cell integrity and expressed in stage I and stage II gametocytes)[27] and Pfs 25 (late stage gametocyte protein expressed in stage V gametocytes and female gametes)[28]. The extra precision (XP) mode in molecular docking along with explicit molecular dynamics (MD) simulation is employed to ascertain the percentage changes in secondary structure elements (SSE); the stability of ADMQ in proteinaceous environment is depicted through protein-ligand root- mean-square deviation (RMSD).

Further, ADMQ was also tested in vitro against the gametocytes of P. falciparum. This blend of in silico molecular modeling and in vitro exercise is expected to open up new avenues for prospective use of chalcone-based organic entities to target P. falciparum sexual stages.

It is pertinent to mention here that ADMQ undergoes a structural change [Figure 1] from its native form (Form I) to β-hydroxy keto form (Form III) in physiological conditions[24],[25],[26].
Figure 1: Structural change of ADMQ in different environments[24],[25],[26].

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  Material & Methods Top


In silico studies

The computational molecular modeling studies were carried on 64 bit Linux Centos 6 operating system, and Windows 7, Intel core i5-2500 CPU @ 3.3 GHz system coupled with 4 GB high speed RAM. The simulation and modeling were carried out using Maestro interface of Schrödinger suite (Schrödinger 2014: Maestro, ver. 9.9, Schrödinger, LLC, NY, 2014) for both ADMQ and MB, a known gametocytocidal agent.

Ligand and protein preparation

The newly synthesized ligand ADMQ was outlined and sketched in Maestro[29],[30] 9.9. The LigPrep module (Schrödinger 2014: LigPrep, ver. 3.1, Schrödinger, LLC, NY, 2014) was utilized for ligand preparation for molecular geometry rectification of ligand and its subsequent ionization at a physiological pH 7.4. Ligprep enabled chirality retention and generated an energy minimized 3D conformation of ligand, a pre-requisite for docking. The targeted gametocyte proteins, Pfg 27 and Pfs 25 were docked with ADMQ[31]. The X-ray crystal structure of Pfg 27 was retrieved from protein data bank (PDB ID: 1N81)[32],[33] and Pfs 25 was built using Iterative Threading ASSembly Refinement (I-TASSER), a hierarchical method of predicting protein’s structure and function. Both, Pfg 27 and Pfs 25 used in the study were first prepared using protein preparation wizard (Schrödinger Suite 2014 Protein Preparation Wizard[34]; Epik[35] ver. 2.9; Schrödinger, LLC, NY, 2014). This was achieved by preprocessing the protein by adding missing hydrogen atoms, correcting transposed heavy atoms in arginine, glutamine along with histidine side chains and optimizing the protein’s hydrogen bond network by performing a restrained minimization (RMSD of 0.3Å) with optimized potentials for liquid simulations (OPLS) 2005 force-field[36],[37].

Molecular docking and free energy of binding studies

ADMQ was docked at the selected active sites of target proteins using Glide’s XP module (Glide, ver. 6.3, Schrödinger, LLC, NY, 2014]. These sites were identified by SiteMap program that located the binding sites and also predicted the druggability of those sites. The scoring function, SiteScore was used to assess a site’s propensity for ligand binding. Sites bearing the highest SiteScore were selected as potential active sites for subsequent grid generation and docking experiments. Receptor grids were generated using receptor grid generation tool and a receptor grid scaling encompassing van der Waals radii of 0.8 and a partial charge cut-off 0.15 was employed. The pose viewer file containing the best pose of ligand in the particular site was generated and used for calculating the free energy of binding using Prime[38] molecular mechanics-generalized born surface area (MM-GBSA) module (Prime, ver. 3.6, Schrödinger, LLC, NY, 2014] by employing variable-dielectric generalized born (VSGB) solvation model with force field 0PLS-2005.

Molecular dynamics simulation

The MD simulation was performed using Desmond (Desmond Molecular Dynamics System, Ver. 2.2, D.E. Shaw Research, NY, 2009), an explicit-solvent molecular dynamics program[39]. A predefined solvent model TIP3P was used with orthorhombic shape boundary box. Na+ salt was added to neutralize the system using force field OPLS–2005. The model system was minimized using a hybrid method of steepest-descent and the limited-memory Broyden-Fletcher Goldfarb-Shanno (LBFGS) algorithms. A total of 2000 iterations were carried out with convergence threshold of 1kcal/mol/D to minimize the solvated ligand-receptor complex. The MD simulation was carried out with a simulation time of 50 ns and a recording interval of 1.2 ps for the energy and 4.2 ps for the trajectory, respectively. A detailed separate analysis for the secondary structural changes on Pfg 27 and Pfs 25 was carried out to assert the possibility of ADMQ as potential antiplasmodial agent.

In vitro cultivation of P. falciparum asexual stages

Briefly, asexual erythrocytic stages of P falciparum field isolate (RKL-9, collected from Rourkela, Odisha) were cultivated in vitro according to the protocols of Trager and Jensen[40] with minor modifications[41],[42],[43]. Cryo- preserved parasites were revived using standard protocols and were maintained in freshly collected A+ human erythrocytes suspended in RPMI 1640 media (GIBCO Technologies, USA) containing 25 mM of HEPES supplemented with 10% human AB+ serum, 2 g/l dextrose, 2 g/l sodium bicarbonate and 40 μg/ml gentamycin sul-fate (Ranbaxy Laboratories, India). Parasite cultures were maintained in an incubator at 37 °C continuously flushed with 5% CO2. Synchronic cultures were obtained by treatment with 5% sorbitol to get uniform population of ring stage parasites. Media was aspirated out daily and replaced with pre-warmed fresh media. Parasitaemia was routinely monitored by examination of Giemsa-stained smears.

In vitro cultivation of P. falciparum gametocytes and biological assays

Gametocyte production was initiated using our own protocol as described elsewhere[14]. Gametocyte culture was set up in 10% haematocrit by sorbitol-synchronizing the culture and reducing the asexual erythrocytic stage parasitaemia to 0.5% (by addition of fresh erythrocytes) (Day 0). For the course of next two weeks, RPMI 1640 media supplemented with 50 mg/l hypoxanthine (Sigma Aldrich, USA) was used during daily media changes. Haematocrit was halved on Day 8 and no fresh erythrocytes were added during the entire course of culture. N-Acetyl- Glucosamine was added from Days 9–12 to enrich the culture for gametocytes by eliminating the asexual erythrocytic stages. Gametocytaemia was monitored on every alternate day by examination of Giemsa-stained thin blood smears. ADMQ was synthesized as described elsewhere[24]. Drug assays were carried out on Days 13–14, when majority of gametocytes were late stages (Stage IV–V) (overall gametocytaemia 2–3%). Gametocytes were examined thoroughly the day before the experiment and on the experimental day to rule out any morphological de- formability. ADMQ and MB were dissolved in DMSO and double distilled water, respectively and dilutions were made afresh with incomplete RPMI 1640 media to achieve a working dilution of 1 mM (final DMSO concentration <0.5%). Gametocytes were treated with 1 mM of ADMQ and MB at a final volume of 100 μl in 96 well plates and kept inside an incubator at 5% CO2 for 48 h. Separate wells were maintained containing gametocytes with RPMI 1640 without addition of any drug. These treatments was carried out in duplicates and repeated thrice in similar conditions. Methylene blue acted as a positive control in our experiments because of its reported antimalarial activity against all stages of P falciparum gametocytes[14]. Untreated gametocytes in standard culture media, ADMQ treated uninfected erythrocytes in standard culture media and uninfected erythrocytes with standard culture media acted as viability and background monitor. Giemsa- stained smears of ADMQ and MB treated gametocytes were examined under a microscope.

Ethical statement

The study was approved by the Institutional Ethics Committee (IEC) of ICMR–National Institute of Malaria Research, New Delhi and bears Ethical Committee Reference No. – ECR/NIMR/EC/2016/276.


  Results Top


Antiplasmodialpotential of ADMQ by in silico approach The use of MD simulation generated 10 different conformers based on SiteScore, size, druggable score (D- score) and volume. Out of the 10 conformers, site 1 of conformer 6 was observed as the best suited active site [Table 1].
Table 1: Ten different conformers generated from 50 ns MD simulation of target protein Pfs 25

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Molecular docking study results

Molecular docking exercise (XP mode) was carried out to assess the selected binding site of ADMQ (Form III) and MB, it’s preferred orientation and detailed mechanism of interaction with the target proteins (Pfg 27 and Pfs 25) individually using Glide program. The minimum energy conformation of MB and ADMQ (Form III) inside the scaffold of Pfg 27 was obtained and depicted in [Figure 2]a and [Figure 2]b, respectively. The Glide protocol yielded the Glide score of –3.3 kcal/mol with Form III of ADMQ as compared to –3.7 kcal/mol with MB. This indicates a reasonably good interaction and stability for both the compounds in the active site of Pfg 27. Moreover, the MM-GBSA free energy of binding of –56.6 kcal/mol for ADMQ and –47.3 kcal/mol for MB demonstrates that both of these ligands are stable while sitting in the active site of Pfg 27.
Figure 2: Minimum energy conformation of: (a) Methylene blue (MB); and (b) β-hydroxy keto form of ADMQ inside the scaffold of Pfg 27 (a sexual stage-specific phosphoprotein PDB ID: 1N81).

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Similarly, inside the scaffold of Pfs 25, a Glide score of –4.1 kcal/mol was observed for Form III of ADMQ as compared to –3.0 kcal/mol for MB [Figure 3]a and [Figure 3]b. This indicated a reasonably good interaction for both the ligands in the active site of Pfs 25. Further, the corresponding prime MM-GBSA free energy of binding was found to be –61.8 kcal/mol for ADMQ and–46.2 kcal/mol for MB. This indicated that the stability of both ADMQ and MB are comparable and both exhibited almost similar binding energies while sitting in the active site of Pfs 25.
Figure 3: Minimum energy conformation of: (a) Methylene blue (MB); and (b) β-hydroxy keto form of ADMQ (Form III) inside the scaffold of Pfs 25 (built from I–TASSER), an early expressed sexual stage protein.

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Molecular dynamics simulation

In addition to XP docking, MD simulation was also carried out to ascertain the interaction pattern of ADMQ with targeted proteins. The best pose out of four complexes (ADMQ-Pfg 27, MB-Pfg 27, ADMQ-Pfs 25 and MB- Pfs 25) generated during regular XP docking was further considered for real-time MD simulation by Desmond, where system builder used explicit aqueous medium followed by complex minimization to bring down protein- ligand complex to the least energy level. All the four real time simulations of 50 ns each expounded the stability and interaction pattern of the respective probe molecules in terms of the protein-ligand RMSD and protein SSE. Moreover, RMSD was measured for both MB and ADMQ on each Pfg 27 and Pfs 25, during the entire 50 ns run. The RMSD evaluation as a function of time for Pfg 27 and Pfs 25, with MB are depicted in [Figure 4]a and [Figure 4]b, respectively and; for ADMQ are shown in [Figure 5]a and [Figure 5]b, respectively.
Figure 4: RMSD plot of protein and ligand (MB) during 50 ns simulation: (a) Protein Pfg 27; and (b) Protein Pfs 25. Lig Fit Prot shows the RMSD of a ligand when the protein-ligand complex is first aligned on the protein backbone of the reference followed by measuring the RMSD of the ligand heavy atoms. C-alphas (Cá) refers to the alpha carbon atom of protein.

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Figure 5: RMSD plot of protein and ligand (ADMQ) during 50 ns simulation: (a) Protein Pfg 27; and (b) Protein Pfs 25. Lig Fit Prot shows the RMSD of a ligand when the protein-ligand complex is first aligned on the protein backbone of the reference followed by measuring the RMSD of the ligand heavy atoms. C-alpha (Cα) refers to the alpha carbon atoms of protein.

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The RMSD analysis indicated that the simulation was equilibrised and changes ranged in between 1 and 3 A, acceptable for small globular proteins. This signifies that the proteins Pfg 27 and Pfs 25 had not undergone large conformational change during the simulation. The ligand RMSD plot (right Y-axis) indicates that the ligands MB and ADMQ were stable with respect to proteins and in their binding pockets.

In order to investigate the conformational changes in terms of percentage helix, percentage strand and percentage total SSE elements, the protein SSE like α-helices and β-strands were also monitored throughout the simulation to get a clear picture. The plot for MB [Figure 6]a and ADMQ [Figure 6]b for Pfg 27 depicted the SSE distribution by residue index throughout the protein structure. The results so obtained can be attributed to the similar change in percentage of a-helices (59.40% in MB and 59.48% in ADMQ) throughout the complete residue index. Correspondingly, for Pfs 25, the percentage change in a-helix was found to be almost similar, i.e. 1.42% for MB [Figure 7]a and 1.62% for ADMQ [Figure 7]b which depicted similar change in a-helix throughout the length of meticulous 50 ns simulation. The β-strands in Pfs 25 also shared the same juncture with almost similar change in the percentage strand, i.e. 25.93% for MB and 25.56% for ADMQ.
Figure 6: Secondary structure elements (SSE) distribution by residue index of: (a) MB; and (b) ADMQ throughout the protein structure, Pfg 27 during the course of 50 ns simulation.

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Figure 7: Distribution of SSE by residue index of: (a) MB; and (b) ADMQ throughout the protein structure, Pfs 25 during the course of 50 ns simulation.

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Further, while exploring the conformational changes in the active site of Pfg 27 and Pfs 25 by MB and ADMQ separately, SSE elements like a-helices, β-strands, turns and loops were monitored throughout the simulation trajectory. The top plots in [Figure 8], [Figure 9] summarized the percent SSE composition for each trajectory frame over the course of the simulation and the bottom plots monitored each residue and its SSE assignment over time. Remarkably, the changes in α-helix and β-strands in SSE elements in both the target proteins were almost similar indicating that the active site of both the proteins underwent almost similar conformational changes, when the ligands (both MB and ADMQ) were incorporated in the same active site.
Figure 8: Composition of SSE for each trajectory frame over the course of simulation with: (a) MB; and (b) ADMQ on Pfg 27. The top panel shows the percent SSE composition whereas the bottom panel monitors each residue and its SSE assignment over time.

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Figure 9: Composition of SSE for each trajectory frame over the course of simulation with: (a) MB; and (b) ADMQ on Pfs 25. The top panel shows the percent SSE composition whereas the bottom panel monitors each residue and its SSE assignment over time.

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In vitro gametocytocidal potential of ADMQ

Gametocytes are those stages of malaria parasites that are directly responsible for malaria transmission. In the present study, gametocytocidal potential of a newly synthesized quinoline appended chalcone derivative (ADMQ) is demonstrated by first producing a heterogeneous population of gametocytes and then assessing the response of these gametocytes towards the test compound ADMQ.

Production of gametocyte population

Gametocyte production was carried out in a field isolate of P. falciparum, collected from one of the most malaria endemic zones of India, Rourkela (Odisha). This isolate was successfully revived in vitro from the cryo- preserved stabilate using the standard protocols and was adapted to lab conditions. Further, the asexual stages were cultivated using a procedure previously described. Gametocyte production was carried out and a heterogeneous population of gametocytes was obtained with majority (>70%) of late stage gametocytes and was used for experimentation. Gametocytes were found to be perfectly healthy based on the developmental pattern and the staining characteristics, upon microscopic assessment every alternate day until the day of the experiment. Gametocytes were found to mature through all the five stages and were found to be morphologically uncompromised with intact chromatin (stained pink) and cytoplasm (stained blue) with absolutely no morphological aberrations. Moreover, male gametocytes were found to exflagellate which confirmed functional viability of gametocytes. Therefore, it was inferred that the cultivated gametocytes were morphologically healthy and functionally viable at the time of experimentation.

In vitro gametocytocidal activity of ADMQ

After successful mass production, the gametocytes were incubated with 1 mM of the test compound ADMQ and the same concentration of MB (positive control) in 96 well microtiter plates for 48 h. At 1 mM, >99% of gametocytes were either found to be knocked out or developed morphological aberrations in ADMQ group as depicted in the right column of [Figure 10]a, [Figure 10]b, [Figure 10]c, [Figure 10]d, [Figure 10]e. These morphological aberrations were found to occur in almost all the gametocytes, regardless of stage (I to V) and sex (male or female). These aberrations appeared in the form of protruding vesicles in the erythrocyte’s cytoplasm. However, it is difficult to ascertain which stage of gametocytes or which sex was more vulnerable to ADMQ treatment. Interestingly, these morphological abnormalities in the form of protruding vesicles or bulges were only observed in ADMQ-treated gametocytes (not in untreated gametocytes and in ADMQ-treated uninfected erythrocytes). This finding serves as a cornerstone for our further studies to ascertain the origin of these vesicles. Further transmission blocking studies are underway to see whether vacuolated stage V gametocytes are infective when fed to Anopheles stephensi mosquitoes.
Figure 10: Microscopic images (a–e) of untreated vs. ADMQ-treated gametocytes. Vesicles staining pink can be seen originating from gametocyte residing inside the erythrocyte's cytoplasm in ADMQ-treated group (not observed in the untreated control). Gametocytes are marked by arrows.

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Methylene blue also proved to be effective against P. falciparum gametocytes. This known gametocytocidal agent was also found to either completely knock out the gametocytes or render them morphologically abnormal. Rather than causing vacuole formation as seen with ADMQ, methylene blue either caused unusual shrinkage in exposed gametocytes or caused loss of actual shape. Many gametocytes were found to develop membrane deformations as evident from the comparative microscopic analysis shown in [Figure 11]. Detailed morphological deformations induced by MB are described in our previous study[14].
Figure 11: Comparative microscopic analysis of morphological abnormalities in gametocytes (a) perfectly healthy and morphologically normal gametocytes (untreated); (b) vacuolar formation in ADMQ-treated gametocytes; and (c) morphological deformations in MB-treated gametocytes. Gametocytes are marked by arrows.

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  Discussion Top


The start of gametocyte production in the host bloodstream represents a transition period that includes several morphological and biochemical changes. These changes in gametocytes may be accompanied by distinct patterns of sexual stage-specific gene expression. In this regard, the effect of a strategically designed and synthesized quinoline appended chalcone, ADMQ and a well-known gametocytocidal agent methylene blue was dealt herewith exhaustively in terms of their respective binding orientation at the active sites of targeted gametocyte proteins Pfg 27 and Pfs 25.

The underlying basis of the present study stemmed from the fact that chalcones are well-known antiplasmodial agents and plethora of potential antiplasmodial analogues can be identified by only tuning the substitution pattern on the two aromatic rings[21]. A quinoline ring is a common entity in established antimalarials and different structural arrangements in chalcone skeleton to yield hydroxylated and alkoxylated chalcones, hydroxychalcones and dihydroxychalcones, methoxychalcones have added value in guiding lead compound design[21]. Among numerous structural possibilities in the general chalcone framework, we have substituted an aryl group by heterocyclic quinoline moiety and another aryl group by bioactive anthracene moiety with intent to obtain a hybrid molecule with enhanced antiplasmodial activity.

Although quinolinyl chalcones have shown antimalarial activity[21] but quinoline appended anthracenyl chalcones are still unexplored as per the best of our knowledge. This paved way for the present study to decipher the prospective use of quinoline appended anthracenyl chalcone as a potential antimalarial candidate against gametocyte stages of P falciparum using in silico molecular docking and MD simulation technique supplemented with in vitro microscopy.

The comparable Glide score and MM-GBSA free energy of binding demonstrated good stability of both ADMQ and MB in the active site of Pfg 27 and Pfs 25, respectively. The explicit 50 ns simulation by Desmond software provided information about protein-ligand contacts and rendered similar distinguishable conformational changes in terms of protein-ligand RMSD and SSE. This molecular modeling approach demonstrated similar secondary structure changes in the homogeneity or structural integrity of active site of gametocyte proteins Pfg 27 and Pfs 25 by ADMQ and MB. Further, ADMQ treatment resulted in generation of protruding vesicles from gametocytes. Such vesicles were observed uniformly in gametocytes irrespective of the stage or sex. Presence of similar vesicles has been reported elsewhere as a characteristic of dying gametocytes[44]. This consolidated in vitro microscopic and in silico study exemplified a novel use of quinoline-appended chalcone derivative as a potential antiplasmodial drug candidate and opened new avenues for chalcone-based drug design.


  Conclusion Top


The study highlighted the interactions of the test compound ADMQ and known gametocytocidal agent methylene blue with two sexual stage (gametocyte) specific target proteins, Pfg 27 and Pfs 25. The comparable glide score, MM-GBSA free energy, low RMSD and similar changes in SSE suggested analogous activity in the binding pockets of target proteins. Further, ADMQ was found to introduce morphological aberrations in all the stages of gametocytes in vitro. The aberrations observed were in the form of small vesicles, which could be seen in the cytoplasm of the erythrocyte. From this comprehensive molecular modeling and in vitro exercise, it could be concretized that the newly synthesized quinolinyl chalcone (ADMQ) warrants further investigation in terms of its transmission-blocking ability in standard membrane feeding assays (SMFAs) to further establish its potential as a prototype of next generation of transmission-blocking antimalarials.

Conflict of interest

The authors declare no conflict of interest.


  Acknowledgements Top


SKG gratefully acknowledges the financial support from CSIR, New Delhi, Scheme No. 37 (1493)11/EMR-II. One of the authors IW acknowledges University Grants Commission, New Delhi for his Research Fellowship and the Indian Council of Medical Research, New Delhi for partial research support.



 
  References Top

1.
Breman JG, Egan A, Keusch GT. The intolerable burden of malaria: A new look at the numbers. Am J Trop Med Hyg 2001; 64(1-2 Suppl): iv–vii. doi: https://doi.org/10.4269/ajt- mh.2001.64.iv.  Back to cited text no. 1
    
2.
World malaria report 2019. Geneva: World Health Organization 2019; p. 232.  Back to cited text no. 2
    
3.
Tanner M, de Savigny D. Malaria eradication back on the table. Bull World Health Organ 2008; 86(2): 82.  Back to cited text no. 3
    
4.
Breman JG. Resistance to artemisinin-based combination therapy. Lancet Infect Dis 2012; 12: 820-2.  Back to cited text no. 4
    
5.
Cheeseman IH, Miller BA, Nair S, Nkhoma S, Tan A, Tan JC, et al. A major genome region underlying artemisinin resistance in malaria. Science 2012; 336(6077): 79-82.  Back to cited text no. 5
    
6.
Rosenthal PJ. The interplay between drug resistance and fitness in malaria parasites. Mol Microbiol 2013; 89(6): 1025-38.  Back to cited text no. 6
    
7.
Wadi I, Anvikar AR, Nath M, Sinha A, Valecha N. Critical examination of approaches exploited to assess the effectiveness of transmission-blocking drugs for malaria. Future Med Chem 2018; 10: 2619-39.  Back to cited text no. 7
    
8.
Baker DA. Malaria gametocytogenesis. Mol Biochem Parasitol 2010; 172(2): 57-65  Back to cited text no. 8
    
9.
Sinden RE. Gametocytogenesis of Plasmodium falciparum in vitro: An electron microscopic study. Parasitology 1982; 84(1): 1-11.  Back to cited text no. 9
    
10.
Farfour E, Charlotte F, Settegrana C, Miyara M, Buffet P. The extravascular compartment of the bone marrow: A niche for Plasmodium falciparum gametocyte maturation? Malar J 2012; 11: 285.  Back to cited text no. 10
    
11.
Smalley ME, Brown J. Plasmodium falciparum gametocyto- genesis stimulated by lymphocytes and serum from infected Gambian children. Trans R Soc Trop Med Hyg 1981; 75(2): 316-7.  Back to cited text no. 11
    
12.
Wadi I, Nath M, Anvikar AR, Singh P, Sinha A. Recent advances in transmission-blocking drugs for malaria elimination. Future Med Chem 2019; 11: 3047-89.  Back to cited text no. 12
    
13.
Baird JK, Hoffman SL. Primaquine therapy for malaria. Clin Infect Dis 2004; 39(9): 1336-45.  Back to cited text no. 13
    
14.
Wadi I, Pillai CR, Anvikar AR, Sinha A, Nath M, Valecha N. Methylene blue induced morphological deformations in Plasmodium falciparum gametocytes: Implications for transmission-blocking. Malar J 2018; 17(1): 11.  Back to cited text no. 14
    
15.
Youngster I, Arcavi L, Schechmaster R, Akayzen Y, Popliski H, Shimonov J, et al. Medications and glucose-6-phosphate dehydrogenase deficiency: An evidence-based review. Drug Saf 2010; 33(9): 713-26.  Back to cited text no. 15
    
16.
Manganelli GFico A, Martini G, Filosa S. Discussion on phar- macogenetic interaction in G6PD deficiency and methods to identify potential hemolytic drugs.Cardiovasc Hematol Disord Targets 2010; 10(2): 143-50.  Back to cited text no. 16
    
17.
Beutler E. G6PD deficiency. Blood 1994; 84(11): 3613-36.  Back to cited text no. 17
    
18.
Elyassi AR Rowshan HH. Perioperative management of the glucose-6-phosphate dehydrogenase deficient patient: A review of literature. Anesth Prog 2009; 56(3): 86-91.  Back to cited text no. 18
    
19.
Meissner PE, Mandi G, Witte S, Coulibaly B, Mansmann U, Rengelshausen J, et al. Safety of the methylene blue plus chloroquine combination in the treatment of uncomplicated falciparum malaria in young children of Burkina Faso. Malar J 2005; 4: 45.  Back to cited text no. 19
    
20.
Khatib S, Nerya O, Musa R, Shmuel M, Tamir S, Vaya J. Chalcones as potent tyrosinase inhibitors: The importance of a 2,4-substituted resorcinol moiety. Bioorg Med Chem 2005; 13(2): 433-41.  Back to cited text no. 20
    
21.
Liu M, Wilairat P, Go ML. Antimalarial alkoxylated and hydroxylated chalcones: Structure-activity relationship analysis. J Med Chem 2001; 44(25): 4443-52.  Back to cited text no. 21
    
22.
Refaat HM, Khalil OM, Kadry HH. Synthesis and anti-inflammatory activity of certain piperazinylthienylpyridazine derivatives. Arch Pharm Res 2007; 30(7): 803-11.  Back to cited text no. 22
    
23.
Sugamoto K, Kurogi C, Matsushita Y, Matsui T. Synthesis of 4-hydroxyderricin and related derivatives. Tetrahedron Lett 2008; 49(47): 6639-41.  Back to cited text no. 23
    
24.
Kumar H, Chattopadhyay A, Prasath R, Devaraji V, Joshi R, Bhavana P, et al. Design, synthesis, physicochemical studies, solvation, and DNA damage of quinoline-appended chalcone derivative: Comprehensive spectroscopic approach toward drug discovery. J Phys Chem B 2014; 118(26): 7257-66.  Back to cited text no. 24
    
25.
Kumar H, Devaraji V, Prasath R, Jadhao M, Joshi R, Bhavana P, et al. Groove binding mediated structural modulation and DNA cleavage by quinoline appended chalcone derivative. Spectrochim Acta Part A Mol Biomol Spectrosc 2015; 151: 605-15.  Back to cited text no. 25
    
26.
Kumar H, Devaraji V, Joshi R, Jadhao M, Ahirkar P, Prasath R, et al. Antihypertensive activity of a quinoline appended chalcone derivative and its site specific binding interaction with a relevant target carrier protein. RSC Adv 2015; 5(80): 65496-513.  Back to cited text no. 26
    
27.
Olivieri A, Camarda G, Bertuccini L, van de Vegte-Bolmer M, Luty AJ, Sauerwein R. The Plasmodium falciparum protein Pfg27 is dispensable for gametocyte and gamete production, but contributes to cell integrity during gametocytogenesis. Mol Microbiol 2009; 73(2): 180-93.  Back to cited text no. 27
    
28.
Eksi S, Morahan BJ, Haile Y, Furuya T, Jiang H, Ali O, et al. Plasmodium falciparum gametocyte development 1 (Pfgdv1) and gametocytogenesis early gene identification and commitment to sexual development. PLoS Pathog 2012; 8(10): e1002964.  Back to cited text no. 28
    
29.
Friesner RA, Banks JL, Murphy RB, Halgren TA, Klicic JJ, Mainz DT, et al. Glide: A new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy. J Med Chem 2004; 47(7): 1739-49.  Back to cited text no. 29
    
30.
Friesner RA, Murphy RB, Repasky MP, Frye LL, Greenwood JR, Halgren TA, et al. Extra precision glide: Docking and scoring incorporating a model of hydrophobic enclosure for protein- ligand complexes. J Med Chem 2006; 49(21): 6177-96.  Back to cited text no. 30
    
31.
Barr PJ, Green KM, Gibson HL, Bathurst IC, Quakyi IA, Kaslow DC. Recombinant Pfs25 protein of Plasmodium falciparum elicits malaria transmission-blocking immunity in experimental animals. J Exp Med 1991; 174(5): 1203-8.  Back to cited text no. 31
    
32.
Gardner MJ, Hall N, Fung E, White O, Berriman M, Hyman RW, et al. Genome sequence of the human malaria parasite Plasmodium falciparum. Nature 2002; 419(6906): 498-511.  Back to cited text no. 32
    
33.
Sharma A, Sharma I, Kogkasuriyachai D, Kumar N. Structure of a gametocyte protein essential for sexual development in Plasmodium falciparum. Nat Struct Biol 2003; 10(3): 197-203.  Back to cited text no. 33
    
34.
Sastry GM, Adzhigirey M, Day T, Annabhimoju R, Sherman W. Protein and ligand preparation: Parameters, protocols, and influence on virtual screening enrichments. J Comput Aided Mol Des 2013; 27(3): 221-34.  Back to cited text no. 34
    
35.
Greenwood JR, Calkins D, Sullivan AP, Shelley JC. Towards the comprehensive, rapid, and accurate prediction of the favorable tautomeric states of drug-like molecules in aqueous solution. J Comput Aided Mol Des 2010; 24(6-7): 591-604.  Back to cited text no. 35
    
36.
Shivakumar D, Williams J, Wu Y, Damm W, Shelley J, Sherman W. Prediction of absolute solvation free energies using molecular dynamics free energy perturbation and the OPLS force field. J Chem Theory Comput 2010; 6(5): 1509-19.  Back to cited text no. 36
    
37.
Jorgensen WL, Tirado-Rives J. The OPLS [Optimized Potentials for Liquid Simulations] potential functions for proteins, energy minimizations for crystals of cyclic peptides and crambin. J Am Chem Soc1988; 110(6): 1657-66.  Back to cited text no. 37
    
38.
Jacobson MP, Pincus DL, Rapp CS, Day TJ, Honig B, Shaw DE, et al. A hierarchical approach to all-atom protein loop prediction. Proteins 2004; 55(2): 351-67.  Back to cited text no. 38
    
39.
Guo Z, Mohanty U, Noehre J, Sawyer TK, Sherman W, Krilov G. Probing the alpha-helical structural stability of stapled p53 peptides: Molecular dynamics simulations and analysis. Chem Biol Drug Des 2010;75(4): 348-59.  Back to cited text no. 39
    
40.
Trager W, Jensen JB. Human malaria parasites in continuous culture. Science 1976; 193(4254): 673-5.  Back to cited text no. 40
    
41.
Batra N, Rajendran V, Agarwal D, Wadi I, Ghosh PC, Gupta RD, et al. Synthesis and antimalarial evaluation of [1,2,3]- triazole tethered sulfonamide-berberine hybrids .Chemistry Select 2018; 3(34): 9790-93.  Back to cited text no. 41
    
42.
Batra N, Rajendran V, Wadi I, Lathwal A, Dutta RK, Ghosh PC, et al. Synthesis, characterization and antiplasmodial efficacy of sulfonamide-appended [1,2,3]-triazoles. J Heterocycl Chem 2020; 57(4): 1625-36. doi: https://doi.org/10.1002/jhet.3888.  Back to cited text no. 42
    
43.
Wadi I, Prasad D, Batra N, Srivastava K, Anvikar AR, Valecha N, et al. Targeting asexual and sexual blood stages of human malaria parasite P. falciparum with 7-chloroquinoline-based [1,2,3]-triazoles. Chem Med Chem 2019; 14: 484-93.   Back to cited text no. 43
    
44.
Furuya T, Mu J, Hayton K, Liu A, Duan J, Nkrumah L, et al. Disruption of a Plasmodium falciparum gene linked to male sexual development causes early arrest in gametocytogenesis. Proc Natl Acad Sci USA 2005; 102(46): 16813-8.  Back to cited text no. 44
    


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