A Comparative Study of Binding of Different Drugs on gp 120 Insight From Molecular Dynamics Simulation Study

HIV-I cellular infection triggered by CD4 receptor protein and viral envelop glycoprotein gp120 binding event. CD4:gp120 surface is directed by the contact points of a hydrophobic gp120 cavity capped by Phe43CD4 and ionic bonds residues Arg59CD4 and Asp368gp120. The binding sites originated by gp120 and CD4 interaction leads to the entry of HIV-I into the host membrane, where, gp120 and a CD4 binding site becomes the main mark for plenty of drug uncovering program. Here, we took the crystal structure of small-molecule of gp120 in a complex that concurrently pursues both of the hotspots of gp120 binding sites. All ligands in our study are small molecules that are able to obstruct the protein-protein interactions between CD4 and gp120. This study aims at the thermodynamical insights of the ligand binding in CD4 binding sites using Molecular Dynamics Simulations Study and calculation of binding free energy. The physical of binding of drugs distinctly indicates a hydrophobic and electrostatics interaction motivated binding of ligands which explicitly mark CD4 binding sites.


INTRODUCTION
By the end of 2017, over 70 million people were infected by HIV-1/AIDS, and about 36.9 million people are still septic by HIV-1 [1][2][3] .HIV-1(Virus type-I) changes in cells, as an originator gp160, and afterwards, split to go-41 and mature gp120.The HIV-1 Env (Envelope protein ), target host cells on viral entry into the cells [4][5][6] ., that mechanized as a trimer of gp120-gp41.Receptor CD4 and its co-receptor CCR5/CXCR4 binds with Env-gp120, in an indirect virus-cell membrane fusion.Viral Env protein undergoes big conformational alteration during its entry process.CD4 induces conformational changes into gp120 upon its binding, thus facilitating subsequent interaction through the CCR5 or CXR4 co-receptors.Gp120 binds together with receptor CD4 and co-receptor CCR5/CXCR4, therefore, triggering an extreme conformational change in gp41 which permits viral and cell membranes fusions.HIV pathological process is aided by a number of attachment effect whose initiation is carried out by the viral envelope glycoproteins.These glycoproteins are organized into trimeric spikes which are present on the surface of the virion 7 .Every individual trimeric envelope confounds consist of three gp41 transmembrane proteins and three gp120 envelope glycoproteins 8 .Each of these processes, in fact, the overall step informs new potent mark for drug discovery [9][10][11] .Apparently, initial entry step of the virion into CD4 cells is an important yet difficult approach for the prevention of HIV-1 pathological process and AIDS.Indeed, there have not been thorough studies on designing viral entry drugs that mark this entry process 12 .At the CD4−gp120 interaction point, residue Phe43 of CD4 is situated on the CDR2-like loop.This residue binds inside the hydrophobic cavity of gp120, this cavity known as "Phe43 cavity", whereas Arg59 of CD4 is situated on a neighbour β-strand and creates electrostatic interaction with Asp368 of gp120 [13][14] .Debnath et.al., recognized two drugs of CD4−gp120 namely NBD-556 and NBD-557 15 by preceding transmission of small-molecule inhibitors of viral fusion.Dual Hotspot HIV-1 Entry Inhibitors which engage both the sites i.e. hydrophobic Phe43 cavity and electrostatic interaction with Asp368 of gp120 with Arg59 of CD4, are used for inhibition 16 .
Although important residues are active in protein-ligand complexes, as suggested by present structural information, they do not furnish physical knowledge for the prominence in interaction pair of each residue.Hence, these become mandatory to clarify the physical assistance of phenomenon at the level of atomic resolution, so as to double-check the physical assistance of all residue near the attraction site participating in the stabilization of the complex.
Here we performed computational methods to investigate the interaction between gp120 and ligands using MD simulation methods.Our findings promote us to validate the thermodynamic result.In addition, this analysis shows that the method used is MMGBSA method and is capable to imitate the experimental validation of the result of binding free energies.
Once the system attained a 300K temperature and 1atm pressure, the equilibrium dynamics was carried for 4ns, with the previously described parameters.Afterwards, production dynamics was started and continued up to 200 ns for the protein-ligand system.The coordinate construction in the trajectories of production dynamics was gathered at an interval of 10ps.Ptraj module of Amber14 was employed to carry out all the analysis of trajectories while for visualization of structure VMD 1.6.725,Chimera-1.526module was used for the image purpose.

Calculation of Absolute Binding Free Energies and Per-Residue Calculation
For the calculations of thermodynamic parameters and free energy of binding, MM-GBSA method was used.The principles of these methods are all well constituted and have been taken up elsewhere [27][28][29] .MMGBSA method used because it has been favourably applied for the analogous system, in this study but of various class in past studies [30][31][32][33][34][35] .The specific parameters employed in our approach are described here.The binding free energy (ΔG mmgbsa ) of the complex was calculated by using the following: Where ΔE MM is the total molecular mechanics energy of the molecular system in the gas phase, including the van der Waals (ΔE vdw ) and electrostatic (ΔE ele ) interaction energies.ΔG sol and ΔG ele , sol are electrostatic and non polar contributions to desolvation upon ligand binding, respectively, and -TΔS is the entropy contribution arising from changes in the degrees of freedom of the solute molecules, which we reconsidered here to obtain ΔG bind ; therefore, our values reported for the MMGBSA calculations can be called absolute binding free energies.In order to get the crucial residue study, the absolute binding free energies were determined in terms of the contributions of each individual residues by using free-energy-per-residue decomposition theory.

RESULT
Binding Free Energy Calculation Binding free energy of ligand with the receptor was calculated by using snapshots collected from trajectories during the last 40 ns time of Molecular Dynamics trajectories when the RMSD converges Fig. 2(a )and the finding are listed in Table 1.Binding free energy for all complex is shown in Fig. 2(b).According to this result the binding free energy (ΔG bind ) of the gp120-0LM, gp120-0LL, gp120-0LK, gp120-0LJ complexes are−8.46,−6.23, −12.67 and −8.39 kcal mol −1 , respectively, these results agree with the experimental findings.This finding discloses that the binding abilities of the third inhibitor, 0LK, are stronger than the other 3 inhibitors 0LL,0LK and 0LJ.Moreover, the change in entropy (− TΔS), induced by the inhibitor bindings yield a good correlation with enthalpy interaction(ΔHot).As observed from Table 1, the van der Waals interaction energy term (ΔE vdW ), non-polar solvation energy term (ΔG np ) give satisfactory involvement with inhibitor binding.Although the inhibitor bindings are favoured by the electrostatic interaction (ΔE ele ), this favourable factor is completely regulated by stronger unfavourable polar solvation energy term (ΔG gb ).
In contrast to the gp120-0LK complex, the van der Waals energy term and non-polar solvation energy term of the complex gp120-0LM binding are decreased by 13.83, and 1.42 kcalmol −1 , respectively.Similarly, in gp120-0LL too, the binding leads to the reduction of van der Waals energy and non-polar solvation energy by 6.91 and 0.68 kcalmol −1 respectively.Similarly, in gp120-0LJ the binding also goes to the reduction in van der Waals energy and non-polar solvation energy by 3.60 and 0.09 kcalmol −1 respectively.In a nutshell, this reduction in the van der Waals interaction may be considered as the main source of weaker binding affinities of inhibitors to 0LK, 0LL, and 0LJ in contrast to 0LK.The energetic contributions reveal that the association between gp120 and the four different ligands are chiefly governed by nonpolar energy (ΔE nonpolar ), with which the van der Waals energy (ΔE vdw ) contribute greatly.The gas-phase electrostatic energy (ΔE ele ) of the complexes are found to be favourable.For the first complex, gp120-0LM, electrostatic energy are very low and it is quite evident that it does not make strong Hbond with inhibitor.Scrutiny of the entropic contributions (TΔS) demonstrates that the formed complexes are distinguished by unfavourable entropy values due to a reduction in the degrees of freedom.The lowest entropy change (TΔS) was observed in the arrangement of the first complex gp120-0LM with ligand 0LM and the value of entropy change (TΔS) improved in terms of chain length thus disclosing that the ligand size plays a significant part in entropic part ( Fig. 1).The Colour map for all the residues of the receptor is shown in Fig. 3. Here, acidic residues like aspartic acid and glutamic acid are shown in red, hydrophobic residues (Ala, Val, Ile, Leu, Tyr, Phe, Trp, Met, Cys, Pro) in white, basic residues like histidine, lysine, arginine in blue, polar residues (Ser, Thr, Gln, Asn) in green and other residues like glycine in gray.

Residue-wise Decomposition Free Energy
Energy decomposition investigation allows us to explain the part of respective amino acid in deciding complex stabilization.The figure demonstrates the primal residues for bonding and the contributions of total free energy(ΔG total ).

Fig. 4. The binding free energy of protein-inhibitor complexes are evaluated using MM-GBSA methodology which is depicted from interaction map
These figures represent that all the complexes are stabilized mainly by hydrophobic and polar amino acid (Fig. 4).All ligands are surrounded by several hydrophobic and polar residues and detail interaction of this amino acid with all the four drugs is shown in figure( Fig. 5).It is self-evident that the ligands 0LM and 0LL interact with a maximum number of amino acids.In case of 0LM -Trp290, Met289, Asn288, and Glu242 play strongly and are principal amino acid responsible for strong binding.For 0LL -Asn288, and Ile338 provide the main interactions.Similar is the case with ligand 0LK in which Trp290, Met289, Asn288, Ile243 and Glu242 are crucial.Also, in regard to the ligand 0LZ, Trp290, Asn288, and Glu242 are the major contributors for favourable interactions.
In accordance with the total free energy contributions, residues Trp290, Asn288 and Glu242 of gp120 have the greatest impact in the binding energy which proposes that these amino acid play a critical part in ligand binding.In addition, the role of polar, non-polar, van der Waals and electrostatics energy for all the amino acid are given in (Table 2) for all the four complexes.From figure one can infer that the van der walls and electrostatics energy play an essential role in total binding energy for almost all hydrophobic and polar residues.Asn288 and Glu242 have maximum electrostatics contribution in total binding affinity.Still, hydrophobic residues contribute via van der walls interaction

DISCUSSION AND CONCLUSION
Here, the binding interaction between gp120 and four experimentally known ligands were evaluated with the help of conformational analysis and the binding free energy calculated over 200 ns dynamics using the MMGBSA methodology.The energetic analysis revealed a qualitative agreement of the theoretically calculated binding free energies with their experimentally reported values.The calculated results tell that the inhibitors produce stronger binding to 0LK as compared to 0LM,0LL, 0LZ.Also, the increase in van der Walls interaction of inhibitors with 0LK relative to the other inhibitors is the main factor responsible for stronger bindings of inhibitors.The fact that the reduction in van der waals energy may be a main cause of weaker binding of inhibitor 0LK than to other inhibitors However, decomposition free energy asserted not only that plausive free energies arise only from favourable interactions due to residues ASn288 and Glu242 but also these actions were amongst the most favourable role, proclaiming the essential contribution of these energy interactions is helpful in the ligand structure.The Entropic analysis demonstrated that all four complexes did undergo a conformational reduction which in turn played important role in bringing the MMGBSA results in more closer to the observational absolute binding free energies.Furthermore, energetic contributions to the binding are attributed by a large number of hydrophobic contacts.Asn288 makes a strong H-bond with all inhibitors and is significantly accountable for electrostatic interactions (Fig. 5).Moreover, Trp290 also provides a great energetic interaction via its hydrophobic side chain and is the chief contributor for the enhanced van der Waals interactions.
In the present study, the per-residue binding free energy decomposition tells us to acknowledge Asn288, Glu242, Trp290 Asp240 and Met289 as the crucial amino acid for the complex stabilization of the four ligands, which is also in good agreement with experimental values for the GP120 complexes.Val145, Thr147, Glu242, Ser247, Tyr256, Ile287, Met289, Gly292, Gly335, Asn337, and Ile338 as the chief amino acid for the complex stabilization of the four ligands.Since these residues are very close and near to the binding site, hence they become potentially crucial targets for advance drug uncovering projects as we can design new inhibitors which can act more efficiently with them and may be an excellent inhibitor than inhibitors named as 0LM, 0LL, 0LK and 0LJ respectively

Fig. 1 .
Fig. 1.Chemical structure of all the inhibitors named as a) 0LM, b) 0LL, c) 0LK, d) 0LJ respectively from top to bottom

Fig. 2 (Fig. 2 (
Fig. 2(a).The RMSD curve for Cα atoms of protein with respect to time tells conformational fluctuations arising during molecular dynamics simulation in 200ns of time scale