Electronic Research Journal of Engineering, Computer and Applied Sciences ISSN: 2709-3700 www.erjsciences.info Volume 3 (2021)

In silico Antisickling evaluation of 3, 4-dihydroxybenzeoic acid isolated from . thonningii leaf

Ijoma Kingsley Ikechukwu1 Department of Pure and Industrial Chemistry, Nnamdi Azikiwe University, Awka, Anambra State, Nigeria Email: [email protected]

Ajiwe Vincent Department of Pure and Industrial Chemistry, Nnamdi Azikiwe University, Awka, Anambra State, Nigeria

Abstract: Molecular docking and molecular dynamic simulation studies were performed on 3, 4- dihydroxybenzeoic acid isolated from Ficus thonningii leaf a known antisickling used in Eastern Nigeria in the management of Sickle Cell Disease patients. The Harbone method was used for extraction, whereas a combination of column chromatography and flash chromatography was used for the isolation and purification of the active principal of the leaf extract, a combination of H-NMR, C-13 NMR, COSY, TOCSY, HSQC and HMBC was used to elucidate the structure of the pure isolate. Binding affinity of -5.8kcal/mol from the molecular docking assay indicates that 3, 4-dihydroxybenzeoic acid binds to sickle Deoxyhemoglobin and was sufficient enough to interfere with the processes that trigger sickle hemoglobin polymerization in vitro. The molecular dynamic simulation analysis of the binding site amino acid residue was performed at 500 ps and it further confirmed the possible Hemoglobin allosteric effect of 3, 4-dihydroxybenzeoic acid as an effector ligand because of the observed perturbation and variations in the Holo and Apo simulations studies of Root Mean Square Deviation (RMSD), Radius of Gyration (Rg) and Solvent Accessible Surface Area (SASA), Electrostatic internal energy and van der Waal (VDW) interactions.

Keywords: Sickle Hemoglobin, Molecular docking, Molecular dynamics, Antisickling

Introduction:

Sickle cell hemoglobin (HbS) results from a point mutation in which a neutral hydrophobic Valine residue is substituted for a negatively charged Glutathione at the β6 position of normal adult hemoglobin (HbA). As a consequence, deoxygenated molecules aggregate into fibers, which form elongated bundles. These result in alteration of hemoglobin function, and reduce the flexibility of the erythrocytes (Eaton, and Hofrichter, 1987). The crystal structure of deoxy HbS was solved at 3.0 Å resolutions by Love and co- workers in a unit cell arrangement that is believed to correspond closely to the arrangement within the fiber, (Padlan and Love, 1985; Padlan and Love, 1985) using crystals grown in low ionic strength solutions containing polyethylene glycol. The low ionic strength HbS structure shows small but significant differences from that of HbA, particularly in a hinge like displacement of the β chain α helices, which are involved in intermolecular contact (Padlan and

1 Corresponding author

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Love, 1985; Padlan and Love, 1985). The HbS crystal structure is usually obtained under ionic strength conditions that approximate physiological conditions (Perutz, 1968 and Prabhakaran and Johnson, 1993), thus, it is important to evaluate the stability of regions that participate as contact sites and energy of association between HbS molecules and HbS molecules-ligands interactions. Molecular dynamics has been highly successful in simulating the various motions within protein structures (McCammon and Harvey, 1987). Understanding the magnitudes and time scales of atomic fluctuations in proteins is essential for characterizing the internal motions that play important roles in their biological activity. The inter subunit contacts of the four Hb subunits within the Hb tetramer are stabilized by many weak nonbonded interactions. Thus, we have compared molecular dynamics simulations of DeOxyHbS (Holo) and DeOxyHbs-FTH2 (Apo), and have studied the fluctuations in Root mean square deviation, radius of gyration, solvent accessible surface area as well as the potential energy and van der waal interactions between Apo and Holo simulations, because several researches have established that the capability of biomolecules to prevent in vitro polymerization depends on tendency and efficiency to bind to the complimentary contact region/site of deoxyHbS monomers (Bianchi 2007, Abdulmalik et al.,2005); modification of amino acid residues that contribute to the three dimensional structures of HbS contact region and other critical sites (Oyewole, 2008); and Stabilization of the R (relaxed) state of HbS molecule (Ibraheem 2010; Chikezie, 2011; Oyewole, 2008). Similarly, it has been shown that any innocuous agent that can be bound to the region involved in sickling should alter the binding site significantly enough to prevent the formation of rods, i.e., prevent sickling (Schoenborn, 1965). Finch et al.,1973; Josephs et al.,1976; Fermi et al.,1984 and Magdoff-Fairchild et al.,1972 showed that in vitro; the sickling fibers are rods composed of four, six or eight monofilaments which are helically wound around each other, each monofilament being a string of stacked hemoglobin molecules. This suggests that sickling is not simply caused by a single complementary site, but that other molecular binding sites play a significant role. Interference with any of these contact points might, therefore, prevent sickling. Certain anesthetics and some other relatively chemically inert gases bind to myoglobin and hemoglobin (Schoenborn, 1976). The use of traditional medicine can’t fade out in the treatment and management of an array of diseases in the African continent. Several studies demonstrated the anti-sickling property of various extracts (Pauline, 2013) and the bioactivity of plant extracts is attributed to the presence of phytochemicals (Ijoma and Ajiwe, 2017; Ijoma et al.,2017; Ijoma et al.,2016). The antisickling characterization of Ficus species have been reported (Mpiana et al.,2008) Protein aggregation is a complex biological process associated with various diseases, including neurodegenerative diseases such as Alzheimer and Parkinson, as well as sickle cell disease (SCD), a red blood cell disorder (Horwish, 2002; Sami et al.,2017; Ross et al.,2004). Understanding the thermodynamics and the molecular mechanisms of protein aggregation is, therefore, critical for the development of therapeutic strategies and the design of protein aggregation inhibitors. From a molecular perspective, protein aggregation depends on a complex balance of electrostatic and hydrophobic interactions mediated by water and osmolytes (Bellissent-Funel et al.,2016; Ball, 2017), which in turn, influence protein function. A remarkable example concerns the human hemoglobin mutant, HbS, or sickle cell Hb, responsible for SCD (Pauling et al.,1949; Ingram, 1956; Murayama, 1966; Padlan and Love, 1985; Padlan and Love, 1985; Harrington et al.,1997; Eaton and Hofrichter., 1987; Eaton and Hofrichter, 1990; Noguchi and Schechter, 1985). The molecule of Hb is formed by 4 polypeptide chains,

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specifically, 2α chains and 2β chains (Petruz, 1970). HbS differs from normal adult HbA by a single amino acid, namely, the substitution of Glu (charged) in the 6th position of the β polypeptide chains, by Val (hydrophobic) (Ingram, 1956). This substitution, associated with the mutation of a single nucleotide in the gene for the β-chain6, has great biological implications, being responsible for the polymerization of Deoxy- HbS into 14-stranded helical fibers, which distort the shape of erythrocytes, causing several problems associated with vaso-occlusion (Eaton and Hofrichter, 1987; Eaton and Hofrichter, 1990; Rees et al.,2010; Ware et al.,2017). This mutated Val-β6 has its hydrophobic side chain lodged in a pocket of a neighbor HbS tetramer, formed by several hydrophobic residues, noteworthy, but not exclusive, Ala-β70, Phe-β85, and Leu-β88, as well as Heme (Padlan and Love, 1985; Padlan and Love, 1985; Harrington et al.,1997). Furthermore, since the replaced Glu-β6 → Val-β6 are at the Hb surface, the structure of Deoxy-HbS is not significantly perturbed, relative to that of Deoxy-HbA (Padlan and Love, 1985; Padlan and Love, 1985; Harrington et al.,1997; Perutz et al.,1986). HbS polymerization is characterized by a delay time (Adachi and Asakura, 1978; Adachi and Asakura, 1979; Adachi and Asakura, 1982) and is believed to occur via a double nucleation mechanism, initiated by the formation of HbS fibers (homogeneous nucleation), followed by fiber growth, though nucleation of additional polymers on the surface of existing ones (heterogeneous nucleation) (Ferrone et al.,1985 and Ferrone et al.,1985). Furthermore, it was proposed that homogenous nucleation proceeds through a two-step mechanism where metastable dense clusters play the role of nucleation precursors (Galkin et al.,2007). Thus, delaying or preventing the formation of such precursors could represent a possible solution to hemoglobin gelation.

Taxonomy:

FICUS THONNINGII Kingdom: Plantae Phylum: Tracheophyta Class: Magnoliopsida Order: Family: Genus: Ficus L. Species: Ficus thonningii Blume (Global Biodiversity Information Facility Secretariat, 2019, Roskov et al.,2020)

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Plate1: Picture of Ficus thonningii leaf

Method for Extraction, Purification and Structural Elucidation:

10kg of the pulverized leaf of the part was weighed and soaked in a Methanol: Water mixture in a ratio of 4:1 for about 72 hours resulting in a volume of 2000ml:500ml. The mixture was filtered, and the filtrate heated in a water bath to one-tenth (1/10) of the volume at a temperature of about 40°C. The filtrate was then acidified with 2ml of 2M H2SO4 and then extracted with chloroform. The mixtures were separated using a separating funnel. The chloroform extract was heated to dryness and re-dissolved with chloroform given the chloroform extract (Harbone, 1998). This extract was thereafter subjected to column chromatography. The crude extract was separated by column chromatographic technique. The glass column (150 x 1.5cm.ID) was packed with two-third (2/3) the length with silica gel (70-230 mesh). The glass column was plugged with cotton wool at the bottom and a polytetrafluoroethylene (PTFE) stop cork, 100ml of chloroform, and methanol mixture (80:5 v/v) was poured into the column and allowed to drain to the level of the gel bed in order to condition the system. 15g of the crude extract was subjected to column chromatography and eluted with hexane-ethyl acetate (80:20, 70:30, 60:40, 50:50.), ethyl acetate (100%), and methanol (100%) gradients. Slurry of silica gel 70-230 mesh (600g) was made with the eluting solvent and packed into the glass column. The tap was opened to allow the excess solvent to run off. 15 g of the hexane leaf extract was dissolved in the eluting solvent and packed on top of the silica gel slurry with a pipette. As soon as the cake began to form on the column, glass wool fiber was placed on top of the extract and the eluting solvent was added. The collection of the eluent was done with 50 mL and 100 mL conical flasks. Further elution was done with increasing concentration gradients.

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For the methanol leaf crude extract, elution was carried out using dichloromethane-ethyl acetate (80:20, 70:30), ethyl acetate (100%), ethyl acetate-methanol (50:50), and methanol (100%) gradients. For the fractionation of chloroform leaf crude extract, elution was done with hexane- dichloromethane gradients (60:40, 50:50), ethyl acetate (100%), ethyl acetate-methanol (50:50), and finally with 100% methanol. Elution of methanol-water leaf extract was carried out with dichloromethane-ethyl acetate (80:20), ethyl acetate (100%), methanol (100%). Fractions collected were monitored with spotting on Thin Layer Chromatographic (TLC) plates and viewed under the visible U.V light (254 nm). Plates were also placed in iodine chroma-tanks to view the spots. A spray of 0.5% vanillin and 10% sulphuric acid was used on the plates, and the plates were dried in a hot air oven at 110 oC for 1 hour and color changes observed. The pure fractions were subjected to further separation and purification on silica gel column chromatography and flash chromatography. The pure isolate in chloroform leaf extract was FTH2.

Further purification of the pure fractions:

Fractions from column chromatography were further purified using a solvent system of petroleum ether: chloroform (4:1) as the mobile phase, this revealed one major spot with minor spots, these fractions were further purified using flash chromatography (silica gel, mesh 230-400, 30g) prior to each analysis. Elution was carried out with varying proportions of petroleum ether: chloroform. Elution with a solvent mixture of petroleum ether: chloroform (5:1) yielded a major single spot on TLC with some minor impurities at the origin. The process was repeated and similar for all fractions. Concentration, drying and washing of the fractions severally with methanol afforded FTH2.

Recrystallization of the fractions:

This fraction was dissolved in 50ml of methanol and heated slightly at a temperature of 40oC for 15 minutes in a water bath. Then the fraction was removed and allowed to cool in a refrigerator and filtered using a filter paper. The process was repeated three times and the melting point of the pure isolate was determined.

Melting point determination:

The fraction was placed in a thin-walled capillary tube and closed at one end. The capillary tube, which contained the sample, was attached to a thermometer and heated slowly. The temperature range over which the sample was observed to melt is taken as the melting point.

Nuclear magnetic resonance (h1-nmr and c13-nmr):

The H1-NMR spectral analyses were carried out using Bruker 500MHz Nuclear magnetic Resonance spectrometer to determine types of protons in the isolate using the chemical structures as well as the joules coupling constant. The C13-NMR analyses were carried out using the same Bruker 500MHz Nuclear Magnetic Resonance Spectrometer, the samples were dissolved in D2O.

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This was used to determine the types and number of carbon atoms present in the isolate. The structural elucidation was done using a combined NMR analysis of H-NMR, C-NMR, DEPT- 135, TOCSY, COSY, HSQC and HMBC. Method for Molecular Docking:

I. Retrieval Of Target Protein Structures: Protein Data Bank (Bernstein et al.,1977) was used for retrieving the structure of 2HBS (Harrington et al.,1997). The criteria for selection of the indicated structure were based on PDB advance BLAST analysis and the structure used in this study were those displaying the maximum score and query cover in BLAST. II. Ligand Preparations: The structures of the ligands were drawn, 3D optimization was performed in Chemsketch software and energy minimization was carried out using Avogadro software. The structures were prepared in Mol2 format and later converted to PDB format using Avogadro. Further shape complementarity principle was applied. III. Docking Procedures: The in silico molecular docking analysis was done by the method described by Trott and Olson, 2010. The docking analysis included retrieval of the 3D structure of target protein (2HBS) from the PDB database, then drawing, optimization, and energy minimization of the structures of the ligands using Chemsketch and Avogadro software (Hanwell et al.,2012). Molecular Docking studies were carried out using prepared hemoglobin target macromolecule and natural compound ligand by employing Autodock Vina programs. Docking was performed to obtain a population of possible conformations and orientations for the ligand at the binding site. The protein was loaded in Autodock tool software, creating a PDBQT file that contains a protein structure with hydrogens in all polar residues. All bonds of ligand were set as rotatable. All calculations for protein-fixed ligand-flexible docking were done using the Lamarckian Genetic algorithm (LGA) method. The docking site on the protein target was defined by establishing a grid box with a default grid spacing of 1.000Å (Trott and Olson, 2010). The grid center was set to 21.5323, 47.0331, and 39.9478 for x, y, and z respectively while the box was set to 101.0182x99.5713x71.0608 Å3for x, y and z respectively. The exhaustiveness was set to a default value of 8. The best conformation with the lowest binding energy was chosen, after the docking search completed (Ferreira et al.,2015; Morris et al.,2008; Godsell et al.,1996; Trott and Olson, 2010).

The interactions of the complex protein-ligand conformations, including hydrogen bonds and the bond lengths were analyzed using Discovery Studio 3.0 (Biovia, 2020) and Pymol (Delano, 2002).

Method for molecular dynamics:

Structure of DeOxyHbS (2HbS) reported by Harrington et al.,1997 was used in this assay. MD simulations were performed using the NAMD software (Philips et al.,2005) with CHARMM27 all-force field parameters (Best et al.,2012; Jr et al.,2004) support. For

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macromolecules generalized CHARMM27 all-force field parameters were applied (Best et al.,2012; Jr et al.,2004). The models were constructed with crystallization water molecules. After that the resulting models were solvated in a cubic periodic box with water in cubic periodic boundary conditions (Harrach and Drossel, 2014). Counter ions (Na+, Cl−) neutralized the systems. The distance between the periodic boundary conditions and the closest protein atom was set to 10.0Å all through. Prior to the MD simulation, equilibration and energy minimization was carried out on each system through the steepest descent algorithm with 100,000 steps to avoid steric clashes or improper geometries and to relieve any local stress caused by non-bonded atomic overlaps and bond-length and bond angle distortions under the NVT ensemble. After the minimization, an isothermal-isobaric (NPT) simulation was run by weak coupling to a bath of constant pressure. In our study, the constant temperature control was based on Langevin dynamics (Schlick, 2002) with a damping coefficient (gamma) of 1.0 ps. The full-system periodic electrostatics was calculated by using the particle-mesh Ewald (PME) algorithm (Darden et al.,1993, Essmann et al.,1995; Cerutti et al.,2009). The Molecular Dynamics simulation was carried out for 500 ps for both the protein and protein-ligand complex under normal temperature (310 K) and pressure (1 bar) using a temperature coupling time constant of 0.01 ps and a pressure coupling time constant of 0.02 ps. A distance dependent dielectric was used to compensate for the absence of explicit solvent. Interactions between non-bonded atom pairs were calculated with a smooth cutoff radius of 10Å. Structures were saved, and the trajectory analysis was carried out on the 500 saved structures. All parameters were estimated from the trajectory analysis. Five hundred structures at intervals of 1 ps each were chosen for further analysis using Visual molecular dynamics (VMD) (Humphrey et al.,1996) Microsoft Excel. In the present study, the Velocity-rescaling (modified Berendsen’s thermostat) (Bussi et al., 2007) was used for temperature coupling in NVT equilibration while the Parinello–Rahman barostat (Parrinello and Rahman, 1981) was used for pressure coupling during NPT equilibration. Both systems were well equilibrated. The temperature of the systems reached 310 K while the pressure was maintained at 1 bar. The equilibration was then followed with a 500 ps long MD production run under NPT ensemble for each of the systems. VDW forces and short range electrostatic interactions were treated using a cutoff of 10 Å. During the MD run, the LINCS algorithm was used to constrain the lengths of all bonds (Hess et al.,1997)

Results and discussions:

Structural elucidation:

FTH2 (3, 4-dihydroxybenzeoic acid)

o 13 Gray crystalline solid; Rf 0.72; melting point 221-223 C (Sahil and Souravh, 2014) C (500 MHZ, D2O): C-1 (125.24 ppm), C-2 (119.63 ppm), C-3 (150.04 ppm), C-4 (146.07 ppm), 1 C-5 (131.39 ppm), C-6 (118.12 ppm), C-7 (177.94 ppm). H (500 MHZ, D2O): H-2 (7.42 ppm), H-5 (6.92 ppm), C-6 (7.39 ppm). The 13C, 1H, DEPT-135, TOCSY, COSY, HSQC and HMBC spectra for FTH2 matched those of 3, 4-dihydroxybenzeoic acid (figure 1) and was thus assigned (Benahmed et al., 2011). Molecular weight of 154.12g/mol

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O 7 OH

6 1 2 5 3 4 OH

OH

Figure 1: 3, 4-dihydroxybenzeoic acid (C7H6O4)

Molecular docking results:

Figure 2: 2D view of FTH2 binding interactions with 2HbS

Figure 2 above, indicates the absence of a hydrogen bond interaction, and subsequent electrostatic interactions of attractive charge and Pi-Sigma made by the O- anion of FTH2 to βHB:77, van der Waal interactions includes βASPB:79, LEUG:48, SERG:49, GLUG:30, HISG:50, ASNB:80, GLYB:83, PROH:125, and βTHRB:84. β1Asp79 and α2His50 amino acid residues have been implicated in sickle erythrocyte polymerization (Harrington et al.,1997) and thus interactions with these amino acid residues may portend antisickling activity there are many compounds that have been used to disrupt the noncovalent quaternary and tertiary structure of proteins, and it has been suggested that such compounds might be effective in inhibiting the sickling of the erythrocyte that contains the hemoglobin (Bookchin et al., 1970) and noncovalent effectors also alter the solubility of deoxyhemoglobin S thereby inhibiting sickle polymerization (Waterman et al.,1974)

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Moreover, ionic interaction above (Attractive Charge) involving histidine residues has been reported to increase the solubility of Deoxyhemoglobin (Perutz et al.,1980; Perutz, 1990; Adachi et al.,1993) and thus shift the hemoglobin transition equilibrium to the Relaxed state, reducing the time spent in the deoxy state (T-state) and ionic interactions are known to stabilize Hemoglobin tertiary and quaternary structure (Perutz et al.,1980, Perutz, 1990) Analysis of the protein-protein contacts shows that both electrostatic and van der Waal interaction plays an important role in the aggregation process, aggregation is highly favored by a few protein-protein interactions involving LSY-GLU, LYS-ASP, and Heme-LSY salt bridges (Galamba and Pipolo, 2018) therefore antisickling drugs candidates should be able to inhibit/delay these interactions. FTH2 had strong electrostatic interactions and van der Waal interaction with DeoxyHbS, which according to Galamba and Pipolo, 2018 play a vital role in the aggregation of HbS. Fth2 made van der Waal interaction with the salt bridge reported by Galamba and Pipolo, 2018, at βASPB:79 though these interactions are small the sum of all van der Waal interactions are always sufficient enough to lower the potential energy at the bound region and subsequently prevents erythrocyte polymerization (Galamba and Pipolo, 2018). The strong electrostatic interactions by FTH2 are sufficient enough to counter any form of protein-protein interaction and moreover, the structural changes associated with Histidine interactions result in possible changes in solubility of deoxyhemoglobin as reported by Perutz et al.,1990 and Adachi et al.,1993.

Figure 3: Pose view of FTH2 with 2HbS (Binding affinity = -5.9kcal/mol)

The binding affinity for FTH2 interaction with 2HBS is -5.9kcal/mol (figure 3), the binding affinity of Deoxyhemoglobin polymerization has been reported (Ross et al.,1975 and Ross et al.,1977). In this research, we postulate that the presence of strong electrostatic interaction of FTH2 with Deoxyhemoglobin exert pull on the molecules of HIS77 and LEU34 (figure 2) creating small crevice where water molecules on the surface of the Deoxyhemoglobin can possibly fit in, this effect is stabilized and maximized by the sum of all van der waal interactions exerted by the

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surrounding amino acid residue and since protein-protein interaction is the major cause of Deoxyhemoglobin S polymerization (Galamba and Pipolo, 2018), the presence of these crevice alters the structure of DeOxyHbS and according to the protein structure-function relationship (Berg et al., 2002) it is expected that overall function of DeOxyHbS at the contact region is affected, and this in turn delay/distort the process and/or the progress of gelation in vitro, moreover ligands can destabilize deoxyhemoglobin by forming bonds with it (Fermi, and Perutz, 1981). Studies do suggest that the use of compounds such as FTH2 and other chemicals that do not modify hemoglobin S covalently should be explored in efforts to prevent deoxyhemoglobin S aggregation (Waterman et al., 1974) although the chemical nature of these compounds is quite different, the effect of these compounds must be to prevent the noncovalent bond formation necessary to produce the insoluble hemoglobin precipitate, perhaps by altering the water structure around the deoxyhemoglobin S molecules (Waterman et al.,1974).

Figure 4: 3D Binding interaction of FTH2 with 2HbS amino acid binding site residue showing distinct bond length

The bond length between FTH2 and HIS77 is 3.83Å and 3.35Å representing Pi-Anion and attractive charge interaction while LEU34 made two interactions with the FTH2 aromatic group at 3.90Å and 3.99Å representing pi-sigma interactions (figure 4)

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Figure 5: Binding interaction of FTH2 with 2HbS in the presence surrounding amino acid residues. Bonding and van der Waal interactions is ≤ 3.99Å

Figure 5, shows the interaction of FTH2 in the presence of surrounding amino acid residue exerting maximum van der Waal interactions at ≤ 3.99Å. The van der Waal interaction though may be small but has a maximum summative impact on deoxyhemoglobin aggregation (Galamba and Pipolo, 2018).

Results of molecular dynamic simulation:

4.5 4 3.5

3 2.5 2

RMSD (Å) RMSD 1.5 1 DeOxyHBS 0.5 DeOxyHBS-FTH2 0 0 100 200 300 400 500 Time (ps)

Figure 6: Fluctuation in Root Mean Square Deviation (RMSD) as a function of time for both the Holo-DeOxyHbS (Black) and Apo-DeOxyHbs-FTH2 (Gray)

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The results of the RMSD above (figure 6) show the difference between the starting structure (Holo-DeOxyHbS) and the final structure (Apo-DeOxyHbS-FTH2). The overall RMSD of the Apo-DeOxyHbS-FTH2 model was larger than that of the Holo-DeOxyHbS simulation. This is expected to correspond to the structural changes due to FTH2 docking. The mean of the RMSD for the Holo and Apo forms were calculated as 2.54Å and 2.83Å respectively; giving a deviation of approximately 0.3Å, this indicates induced perturbation due to structural variation during the simulation run. Also, from virtual inspection it was observed that there exist variations in the structure of the Apo-DeOxyHbS-FTH2 and Holo-DeOxyHbS, this offers an insight into the possible antisickling potentials of FTH2 because ligands can destabilize deoxyhemoglobin by forming bonds with it (Fermi, and Perutz, 1981).

18 16 14 12 10

Rg (Å) Rg 8 6 4 DeOxyHBS 2 DeOxyHBS-FTH2 0 0 100 200 300 400 500 Time (ps)

Figure 7: Fluctuation in Radius of Gyration (Rg) as a function of time for both the Holo- DeOxyHbS (Black) and Apo-DeOxyHbs-FTH2 (Gray)

Figure 7 above, shows the fluctuations in the radius of gyration for the Holo-DeOxyHbS and Apo-DeOxyHbs-FTH2, the radius of gyration denotes the degree of compactness of the protein structure (Gupta et al., 2016). Radius of gyration for Apo-DeOxyHbs-FTH2 increased from the minimized starting structure showing variable amplitude oscillations throughout the simulation run. From virtual inspection the Radius of gyration for Holo-DeOxyHbS were different from those of Apo-DeOxyHbS-FTH2. The contraction of Holo-DeOxyHbS could be attributed to the absence of FTH2, because Apo-DeOxyHbS-FTH2 showed a modest expansion during the simulation with the same set of potential functions.

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350

300

250

) 2 200

150 SASA (Å SASA

100 DeOxyHBS 50 DeOxyHBS-FTH2 0 0 100 200 300 400 500 Time (ps)

Figure 8: Fluctuation in Solvent Accessible Surface Area (SASA) as a function of time for both the Holo-DeOxyHbS (Black) and Apo-DeOxyHbs-FTH2 (Gray) throughout the simulation trajectory

The time course of Holo-DeOxyHbS and Apo-DeOxyHbs-FTH2 solvent-accessible surface area exhibited dissimilar behavior, as shown in Figure 8. The exposed area was derived primarily from the surface atoms, which are generally more flexible under thermal perturbation than interior atoms (Prabhakaran and Johnson, 1993). The solvent-accessible surface area for Apo-DeOxyHbs-FTH2 exhibited relatively variable oscillations about a mean area of 184.77Å2. For Apo-DeOxyHbs-FTH2, it is interesting to note that the increase in the radius of gyration reduced the clustering of surface residues which consequently led to an increase in surface area due to the docking of FTH2. The solvent-accessible surface area for Holo- DeOxyHbS exhibited dissimilar oscillations when superimposed on the radius of gyration of Apo-DeOxyHbS-FTH2 over a time frame of 500 ps. The Apo-DeOxyHbS-FTH2 increase in Radius of gyration during simulation correlated well with its increase in solvent-accessible surface area.

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0 0 50 100 150 200 250 300 350 400 450 500 -100

-200

-300

Energy (kJ/mol) Energy -400

-500 DeOxyHbS DeOxyHbS-FTH2 -600 Time (ps)

Figure 9: van der Waal and electrostatic internal energy plot for Apo-DeOxyHbS-FTH2 (Gray) and Holo-DeOxyHbS (Holo)

The van der Waal (upper graph) and electrostatic internal energy (lower graph) were calculated using the charmm force field generated from CHARMM GUI, analysis of generated trajectory was performed using NAMD energy tool in VMD (Humphrey et al., 1996). Comparatively, from the observed molecular dynamics simulation trajectory over a time frame of 500 ps there was a decline in van der Waals while the electrostatic interaction for Holo- DeOxyHbS was subsequently lower than those of Apo-simulation; however, the sum of van der Waal interactions was significant enough to impact on the potential energy at the bound region for Apo-simulation. The variations in van der Waal energy has been attributed to the distortion of the deoxyhemoglobin dimer-dimer interaction, and implicated in deoxyhemoglobin polymerization (Galamba and Pipolo, 2018) therefore, although the van der Waals interactions are much weaker than electrostatic interactions their sum significantly impacts on the potential energy in the bound region (figure 9). The calculated mean of the P.E for the Apo and Holo simulations was –217.863kJ/mol and -234.423kJ/mol respectively (figure 10). Hence, decrease in the potential energy indicates that FTH2 was able to induce a reduction in the potential energy of the residues in the bound region of DeOxyHbS as earlier postulated from our in silico docking analysis due to significant impact of van der Waal interactions at the bound region.

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0 0 50 100 150 200 250 300 350 400 450 500 -100

-200

-300

-400

-500 Energy (kJ/mol) Energy -600 DeOxyHbS -700 DeOxyHbS-FTH2 -800 Time (ps)

Figure 10: Potential Energy plot of Apo-DeOxyHbS-FTH2 (Gray) and Holo-DeOxyHbS (Black)

Our result shows clear-cut evidence of the effect of Van der Waal energy on sickle erythrocyte polymerization in vitro because Deoxyhemoglobin is constrained by salt bridges (Perutz, 1970); interactions with these salts bridges can consequently trigger a delay in polymerization and subsequent breaking of the Heme-Heme interaction energy that constrains Deoxyhemoglobin (Perutz, 1970)

Conclusion:

Extracts of Ficus thonningii a known antisickling plant have been evaluated for its antisickling potential in vitro, here we report that an isolate from F. thonnngii, 3, 4-dihyroxyl benzoic acid has shown to be a potential antisickling agent in silico. Molecular docking and Molecular dynamics simulations studies were used to evaluate its interactions with DeOxyHbS, the results from the Molecular docking, RMSD, Rg, SASA, electrostatic energy, van der Waal, and potentials energy analysis suggest that the isolate may be a potential anti-sickling agent.

References:

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