Molecular Dynamics of Sialic Acid Analogues and their Interaction with Influenza Hemagglutinin

Synthetic sialic acid analogues with multiple modifi cations at different positions(C-1/C-2/C-4/C-8/C-9) are investigated by molecular mechanics and molecular dynamics to determine their conformational preferences and structural stability to interact with their natural receptors. Sialic acids with multiple modifi cations are soaked in a periodic box of water as solvent. Molecular mechanics and a 2 nanosecond molecular dynamics are done using amber force fi elds with 30 picosecond equilibrium. Direct and water mediated hydrogen bonds existing in the sialic acid analogues, aiding for their structural stabilization are identifi ed in this study. The accessible conformations of side chain linkages of sialic acid analogues holding multiple substituents are determined from molecular dynamics trajectory at every 1ps interval. Transitions between different minimum energy regions in conformational maps are also noticed in C-1, C-2, C-4, C-8 and C-9 substituents. Docking studies were done to fi nd the binding mode of the sialic acid analogues with Infl uenza hemagglutinin. This fi nding provides stereo chemical explanation and conformational preference of sialic acid analogues which may be crucial for the design of sialic acid analogues as inhibitors for different sialic acid specifi c pathogenic proteins such as infl uenza toxins and neuraminidases.

The binding of a virus particle to the surface receptors of a host cell is mediated by viral proteins, which specifically recognize receptor determinants, such as peptides, lipids or carbohydrates [1] .The receptor determinants of Infl uenza virus are shown to be sialic acid residues, which constitute more than 20 naturally occurring derivatives of neuraminic acid collectively referred to as a family of sialic acids.These are found widely distributed in animal tissues and in bacteria, especially in glycoproteins and gangliosides.Influenza A and B bind to the most abundant sialic acid, N-acetyl neuraminic acid (Neu5Ac or NANA).The binding of infl uenza A virus is mediated by the major virus surface glycoprotein, hemagglutinin (HA) that binds to the terminal sialic acid residues as the fi rst step of viral infection and mediates both the initial attachment of virus to target cells and the subsequent fusion of the viral and cell membranes [2] .The region of the HA involved in receptor binding has been deduced from studies of mutant HAs with different binding specificities to involve a pocket of amino acids at its membrane distal surface [3] .The three-dimensional structure of infl uenza virus HAs complexed with cell receptor analogues show neuraminic acid bound to this pocket fundamentally through hydrogen bonds and van der Waals interactions [4] .
Based on this knowledge, it should, in principle be possible to fi nd a neuraminic acid analogue that mimics the cell receptor and thus preferentially binds to the virus, thereby blocking attachment.One approach in the design of high-affi nity inhibitors is to use Neu5Ac (or its 2α-O-methyl derivative) as a scaffold and to modify its functional groups in order to increase its affi nity for the HA.The confi guration of neuraminic acid places the carboxylate in the axial position is the alpha-anomer of neuraminic acid.
Present work was initiated with the modeling of the alpha-anomer of neuraminic acid and its derivatives having multiple substitutions at C-1, C-2, C-4, C-8 and C-9 positions.Molecular mechanics and molecular dynamics calculations were performed.The conformational behaviour of varying substituent holding side chains of neuraminic acid in aqueous environment

Research Research Papers Papers
were studied.The direct and water-mediated hydrogen bonds, which played a major role in the structural stability of neuraminic acid were also analyzed.Docking studies were done to study the binding mode of neuraminic acid derivatives into the binding pocket of Infl uenza HA.
Molecular mechanics calculations were carried out in the Pentium IV workstation using SANDER module of software AMBER10 [7] .The force fields AMBER ff03 and gaff (general amber force fi eld) were used.Water molecules are added from the solvent library of AMBER10 and care is given to maintain the number of water molecules to be same for all the neuraminic acid derivatives.
A periodic box enclosing the neuraminic acid analogues in solution is constructed to turn it into a periodic system for the simulation programs and periodic boundary conditions are applied on constant volume (Table 2).The non bonded pair list was updated for every 10 steps.The non-bonded cutoff was specifi ed as 8Å.For initial 100 cycles steepest descent method was used, then conjugate gradient is switched on.The convergence criterion for the energy gradient is less than 0.01kcal mole -1 .The energy minimized structures for all the neuraminic acid derivatives are intensively analyzed through graphics software VEGA ZZ to work out the direct and solvent mediated hydrogen bonds.
To understand the conformational dynamics of the neuraminic acid analogues in aqueous environment, molecular dynamics calculations were performed over a period of 30ps equilibration followed by a Docking studies were done for all the 18 sialic acid analogues using Schrodinger (maestro).The protein is prepared by optimizing and minimizing the structure using Protein Preparation Wizard.The grid is generated using Receptor Grid Generation by picking the reference ligand which is already present in the PDB structure.HTVS is performed by importing 18 minimized sialic acid analogues using GLIDE module of Schrodinger software [8,9] .Induced fit is carried out for the five top scoring sialic acid analogues using Schrodinger to predict the ligand-induced conformational changes in receptor active sites [10] .

Contribution of hydrogen bonds in structural stability:
Figs. 1-3 showed the formation of water mediated hydrogen bonds and direct hydrogen bonds in neuraminic acid derivatives.It is well known from the previous fi ndings that water mediated hydrogen bonds formed within the molecule plays a major role in stabilization of the molecule [11] .The watermediated hydrogen bonds and direct hydrogen bonds are shown in Table 4. Comparison of water mediated hydrogen bonds with direct hydrogen bonds revealed that analogue 10 (5-N-acetyl-9-amino-9-deoxy neuraminic acid) has 4 water mediated hydrogen bonds and 4 direct hydrogen bonds.The nitrogen atom from the substituted NH 2 group contributed to both water mediated and direct hydrogen bond.It is also observed that analogue 6 (2α-O-methyl-4-Ocapriloyl-5-N-acetyl neuraminic acid) has three water  in each neuraminic acid derivatives are responsible for the minimum energy and its stabilization of the molecule.

Molecular dynamics of sialic acid analogues:
To study the conformational dynamics of the neuraminic acid derivatives, a 2ns molecular dynamics simulation was carried out.An in-depth analysis on the conformational features of all the 10 neuraminic acid analogues was done by collecting the frames for every 1ps.

Modeling of sialic acid analogues and Influenza hemagglutinin complexes:
High Throughput Virtual Screening was done for ten neuraminic acid analogues to find out the structures (ligands) most likely to bind to the Influenza hemagglutinin.The top fi ve ligands with best docking score and minimum energy are subjected to induced fi t docking.The glide score along with the glide energy is displayed in Table 5.The inter-molecular interactions between the top five ligands and the Influenza  hemagglutinin are noted and displayed in Table 6.Figs. 6 and 7 shows the neuraminic acid analogues 3 (benzyl 2α-O-methyl-5-N-acetyl-8,9-O-isopropylidene neuraminate) and 10 (5-N-acetyl-9-amino-9-deoxy neuraminic acid) at the active site of Influenza HA, respectively.The bound state conformations of the substituent holding side chains of sialic acid analogues are displayed in Table 7.

DISCUSSION
The current study reveals the probable conformational models for neuraminic acid derivatives with multiple substitutions at positions C-1/C-2/C-4/C-8/C-9 in aqueous environment.Water mediated hydrogen bonding interaction plays a dominant role in stabilizing the conformational structures of these neuraminic acid derivatives.The accessible conformations for neuraminic acid analogues with multiple substituents holding side chain linkages observed by the present MD study correlate well with those reported for similar linkages in various Neu5Ac-α2→8-Neu5Ac moiety present in all the di-and tri-sialogangliosides   by earlier studies [11,12] .Present MD results show a dynamic behaviour for χ 1 of analogue 10 (5-N-acetyl-9-amino-9-deoxy neuraminic acid) at the cost of 10 kcal/mol (Fig. 5c), which is a vivid indication that this molecule prefers -60° region.mediated hydrogen bonding schemes greatly involve in stabilizing the three dimensional conformational structures of these neuraminic acid analogues.This study also shows the dynamics trajectory and distribution plot for the substituent holding side chain linkages of the neuraminic acid analogues.The high affinity inhibitors modeled in this study saturate the hemagglutinin (HA) receptor [5] and can be used as potential antiinfl uenza drugs.

TABLE 4 : HYDROGEN BONDS IN EACH NEURAMINIC ACID ANALOGUE WITH MULTIPLE MODIFICATIONS
*is the atom from the substituent group.