INTRODUCTION

Docking:-

Molecular docking is one of the most used techniques in structure-based drug design
because it has the ability to predict the binding conformation of ligands to the specific
binding site of the receptor. It is a key tool in structural molecular biology and
computer-based drug design.

Aim of docking:-

The aim of docking is to achieve an optimized conformation for both receptor and
ligand & the relative orientation between protein and ligand such that the free energy
in the overall system is minimized.

Types of docking:-

 Rigid Docking (Lock andKey):-In rigid docking, the internal geometry of both
thereceptorand ligand is treated asrigid and the interaction is like “LOCK and
KEY” type.

 Flexible Docking (Inducedfit):-An enumeration on the rotations of one of the

molecule(usually smaller one) is performed. In every rotation, the energy is
calculated; later the most optimum pose isselected and the interaction is as like
“INDUCED FIT” type.

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Types of interactions between protein and ligand in Docking:-

 Electrostatic forces:-The forces are due to thechargesresiding in thematter.

 Electrodynamics forces: - The most widely known electrodynamics force is
the VanderWaalsinteraction.
 Steric forces:-These are caused by entropy. For example, in cases where
entropy is limited, there may be forces to minimize the free energy of
thesystem.
Solvent-related forces: - These are due to the structural changes of the solvent.
These structural changes are generatedwhen ions, colloids, proteins etc, are
added into the structure of the solvent.Themost commonly are hydrogen bond
and hydrophobic interactions.

FIG – 1 :-3D STRUCTURE OF PROTEIN – LIGAND DOCKING

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Stepsin MolecularDocking:-

 STEP-1:-Preparation of receptor :-The 3D structure of the receptor should be

considered, which can be downloaded from protein data bank or “PDB” ; later
the available structure should be processed.

 STEP-2:-Identification of the active site of receptor :- The active site within

the receptor is located and all sorts of energy minimization is done.

 STEP-3:-Ligand preparation: Ligands can be obtained from various databases

like PubChem, or can be sketched using tools Chemsketch .While selecting
the ligand, the “LIPINSKI’S RULE OF 5” should be applied.

 STEP-4:-Docking: - The ligand is docked onto the receptor and the

interactions are checked. The scoring function generates score depending on

which the best fit ligand is selected .

FIG 2 :- STRUCTURE OFPROTEIN – LIGAND DOCKING

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 EXPERIMENTAL WORK

Docking steps :-

1. Preparation of Receptor [β Amyloid Protein (2 BEG)]:-

Receptors are generally the protein molecules which are available in protein data bank
(PDB). The protein code (2BEG) for amyloid β protein was put on search bar in
PDB.The 3D structure along with various information about the protein was seen and
they were downloaded from protein data bank.3D structure of Alzheimer amyloid β
(1-42) fibril protein (2BEG) has been chosen for docking as a receptor molecule.

AMYLOID β PROTEIN (Aβ) :-

Amyloid β is a protein which is formed after sequential cleavage of the amyloid
precursor protein (APP), a transmembrane glycoprotein of undetermined
function.APP can be cleaved by the proteolytic enzymes α-, β- and γ-secretase. Aβ
protein is generated by successive action of the β and γ secretase.

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 FIG 3:- PATHWAY OF AMYLOID β FIBRIL FORMATION

Amyloid Precursor Protein ----- α-, β- and γ -Secretase  β- Amyloid Protein

Amyloid beta (Aβ) denotes peptides of 36–43 amino acids that are crucially involved
in Alzheimer's disease as the main component of the amyloid plaques formation in the
brains of Alzheimer patients.The most common isoforms are Aβ40 and Aβ42; the
longer form is typically produced by cleavage that occurs in the endoplasmic
reticulum, while the shorter form is produced by cleavage in the trans-Golgi network.
Amyloid β protein has a globular N-terminal domain and a flexible and disordered C-
terminal prion-forming domain.

FIG 4:- 3D STRUCTURE OF AMYLOID β PROTEIN

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 FIG 5:- AMINO ACID RESIDUE OF AMYLOID β PROTEIN

 The first 16 residues, Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu--

Val-His-His-Gln-Lys, are mostly hydrophillic with His13 and His14 acting as
a binding domain for Cu(II).
 Residues 12-23 function as the self-recognition region allowing for the
formation of dimers and/oroligomers.
 This region also serves as the binding site for cholesterol, apolipoproteinE and
amyloid beta-peptide binding region.
 The most reasonable determined structure consists of two helices; the first
helix (residues 8-25) is well defined and has an RMSD of 0.38 angstroms and
the second (residues 28-38) corresponds to the trans-membrane region of APP
and thus contains multiple small and hydrophobic amino acids.
 The two helices are connected by a kink (residues 26 and 27). The toxicity of
amyloid beta is due to the formation of aggregates and oligomers.

2. Identification of the active site of the receptor:-

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Curcumin interferes with the oligomer formation by destabilising Asp-23-Lys-28 salt
bridge of amyloid β protein, which forms the basis of fibril stability. Curcumin can
cross the blood brain barrier (BBB) when it is given through parenteral route. The
ferulic acid has a common phenolic structure that seems to be responsible for the
fibril destabilization .Residue 1-17 are in disordered form , residue 18-42 form a β-
strand which contains two parallel β-sheets, formed by residue 18-26 named as β1
and 31-42 named as β2 .

3. Preparation of ligands:-

Ligands are generally the small molecules which are specifically bound with the
receptor in proper orientation. The ligands are prepared by using various software
such as ChemDraw 3D,ChemSketch etc. Here the ligands of curcumin`s derivatives
were prepared by using ChemDraw 3D software and the bioactivity score and the
chemical characteristic features of the ligands (Lipinski`s rule) was checked by
using Mol inspiration website by coping the smiles of the ligand through
ChemSketch software. The ligands molecules that were fulfilled the requirements
(Lipinski`s rule) were saved inside the system.

STRUCTURE OF CURCUMIN

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 Ligands of curcumin`s derivative :-

MOLECULE R1 R2 R3 A B C R4 R5 R6

MOLECULE 1 -OCH3 -OH -OCH3 -C=O -CH2 -C=O -OCH3 -OH -OCH3

MOLECULE 2 -OCH3 -OH -Cl -C=O -CH2 -C=O -OCH3 -OH -Cl

MOLECULE 3 -OH -OH -H -C=O -CH2 -C=O -OH -OH -H

MOLECULE 4 -OCH3 -OH -H -CHOH -CH2 -CHOH -OCH3 -OH -H

MOLECULE 5 -OCH3 -OH -OCH3 -CHOH -CH2 -CHOH -OCH3 -OH -OCH3

MOLECULE 6 -OCH3 -OH -OCH3 -CHOH -C=O -CHOH -OCH3 -OH -OCH3

MOLECULE 7 - OCH3 -OH -H -C=O -CHOH -C=O -OCH3 -OH -H

MOLECULE 8 - OCH3 -OH -H -C=O -CHCH3 -C=O -OCH3 -OH -H

MOLECULE 9 - OCH3 - OCH3 -OCH3 -CHOH -CH2 -CHOH -OCH3 -OCH3 -OCH3

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MOLECULE -H - OCH3 -H -CHOH -CH2 -CHOH -H - OCH3 -H
10

MOLECULE - -OCH3 -H -CHOH -CH2 -CHOH -OCH3 - OCH3 -H

11 OCH3

MOLECULE -OCH3 -OCH3 -H -CHOH -C=O -CHOH -OCH3 - OCH3 -H

12

MOLECULE -H -OCH3 -H -CHOH -C=O -CHOH -H - OCH3 -H

13

MOLECULE - OCH3 -OH -H -CHOH -C=O -CHOH - OCH3 -OH -H

14

MOLECULE - OCH3 -OH -OCH3 -C=O -CHOH -C=O - OCH3 -OH - OCH3
15

MOLECULE - OCH3 -OH -OCH3 -C=O -CHCH3 -C=O - OCH3 -OH - OCH3
16

Ligands of imidazole derivative :-

The development of new moieties for inhibiting amyloidogenic pathway is

challenging and various small organic molecules like imidazole derivatives can also
be a promising analog for protein fibrillation like polyphenols, flavonoids etc.
Imidazole was selected as a small organic molecule as it is known to show anti-
inflammatory, anti-analgesic, anti-bacterial, anti-fungal, anti-cancer, anti-HIV agents
etc. Small groups like Imidazole forms main structure of amino acid histidine,
vitamin B12 and a DNA base structure. Various drugs like Cimetidine,
Metronidazole possess the structure of Imidazole.

Imidazole moieties were modified by using various electron withdrawing groups like
CF3, COOH, CHO and electron donating groups like CH3 groups.

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MOLECULE R1 STRUCTURE

MOLECULE -CF3
17

MOLECULE1 -COOH
8

MOLECULE1 -CH3
9

10
MOLECULE2 -SO4
0

MOLECULE -CHO
21

MOLECULE -SH
22

4. Docking :-

At first the receptor molecule (2BEG) was opened in AutoDock Vina software
and the water molecules was deleted which was associated with the protein and
the polar hydrogen atoms were added into the protein .After that the grid box
was made, having specific dimension [center, X=0.385;Y=0.250;Z= -8.949 ] and
then the modified protein along with grid box was saved as .pdbqt format inside
the operating system. Then the ligands were opened in Autodock tools and they
were saved as .pdbqt format in the operating system and after that the docking
was done by using comment prompt and the docking output was saved inside the
operating system.

After getting the docking output of each molecule, the receptor – ligand
interaction was checked by using Discovery Studio software and the forces
(electrostatic, van der walls forces, hydrogen bond etc.) which were involvedin

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 the receptor – ligand interaction, were determined by 2D picture of the
interaction. The Docking scores are obtained and the software generates various
out ligand file by arranging them in best suitable conformers. The
outligand.pdbqt files are read in the software to understand the exact location of
ligand and receptor binding.

FIG 6:- SEQUENCE OF AMYLOID β PROTEIN ( 1- 42 )

OBSERVATION

CURCUMIN`S DERIVATIVES AND PROTEIN INTERACTION:-

Sr. No. Molecule Docking score(KJ/Mole )

1. Curcumin -7.1

2. Molecule 3 -8.0

3. Molecule 8 -7.3

4. Molecule 4 -7.2

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 5. Molecule 9 -7.1

6. Molecule 10 -7.1

7. Molecule 2 -7.0

8. Molecule 7 -7.0

9. Molecule 13 -7.0

10. Molecule 14 -7.0

11. Molecule 12 -6.9

12. Molecule 1 -6.7

13. Molecule 5 -6.6

14. Molecule 15 -6.6

15. Molecule 6 -6.5

16. Molecule 11 -6.5

17. Molecule 16 -5.4

PROPERTIES RECEPTOR- LIGAND INTERACTION

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 LogP:1.69
Enzyme
inhibitor:.13
Mw:340.33
nON :6
nOHNH:4
MOL- 3 DOCKING SCORE:
(-8.0)

LogP:2.67
Enzyme
inhibitor:..40
Mw:372.42
nON :6
nOHNH:4
MOL- 4 DOCKING SCORE:
(-7.2)

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 LogP:2.78
Enzyme
inhibitor:.0.02
Mw:382.41
nON :6
MOL-8 nOHNH:2
DOCKING SCORE:
(-7.3)

IMIDAZOLE DERIVATIVES AND PROTEIN INTERACTION :-

Sr. No. MOLECULE DOCKING

SCORE(KJ/Mole)
1. MOLECULE 17 -7.5
2. MOLECULE 21 -6.0
3. MOLECULE 19 -5.9
4. MOLECULE 22 -5.3
5. MOLECULE 20 -4.5
6. MOLECULE 18 -3.9

PROPERTIES RECEPTOR- LIGAND INTERACTION

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 LogP:4.27
Enzyme
inhibitor:0.33
Mw:288.27
nON :2
nOHNH:1
MOL- DOCKING
17 SCORE: (-7.5 )

LogP:3.17
GPCR ligand:-
0.01
TPSA:45.75
Kinase inhibitor:
0.30
Natoms:19
MOL- Enzyme
22 inhibitor:0.20
Mw:248.28
nON : 3
nOHNH: 1
Volume:226.67
DOCKING SCORE
(-5.3 )

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 LogP:3.36
GPCR ligand:-
0.08
TPSA:28.68
Kinase inhibitor:
MOL- 0.39
19 Natoms:18
Enzyme
inhibitor:0.30
Mw:234.30
nON : 2
nOHNH: 1
Volume:224.25
DOCKING SCORE
(-5.9 )

CONCLUSION

The curcumin derivative molecules were modified by changing R1, R2, R3, R4, R5,
R6 groups and A, B, C groups were also changed to see their bioactivity score. The
derivatives had passed the Lipinski rule of 5. Mostly EDG groups’ likes -OCH3, -Cl,-
OH were substituted or deleted.Thus change of groups in R3 & R6 showed increase
in activity.

Small molecules like Imidazole were also chosen and substituted to get a future
molecule to inhibit protein fibrillation. Molecule-17 gave a good docking score in the
range of 7.5. Substitutions in the ligand can be however modified as they could not
show interactions with Asp-23 but showed various electrostatic interactions with Val
B-40 and Phe A-19 which can suggest Imidazole like molecule to be a future
molecule but with better substitutions.

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 Molecule-3 shows good bioactivity score like curcumin which includes changes in
R1, R3,R4 and R6 respectively with docking score of equivalent to
standard.Interactions shown by Molecule-3 was with Leu C-17 (Vander walls
interaction) and Phe B-19 (showed electrostatic interaction)

Molecule-4 replacement of ketone groups by alcohol group was done and -7.2
docking score was achieved. Molecule -4 has vander walls interaction with Asp A-23
and Phe A-19 showed electrostatic interaction.

Molecule-8 showed a docking score -7.3 which involves a significant change of

curcumin with electron donating group and change in A,B and C leads to enhanced
activity .It showed electrostatic interaction with Phe A-19.The molecules mentioned
above can thus be a future promising molecule that can inhibit the salt bridge formed
by Asp 23 in 2BEG protein.

REFERENCES

1. Lührs T, Ritter C, Adrian M, Riek-Loher D, Bohrmann B, Döbeli H, Schubert D,

Riek R. 3D structure of Alzheimer's amyloid-β (1–42) fibrils. Proceedings of the
National Academy of Sciences. 2005 Nov 29;102(48):17342-7.

2. Schmidt M, Sachse C, Richter W, Xu C, Fändrich M, Grigorieff N. Comparison of

Alzheimer Aβ (1–40) and Aβ (1–42) amyloid fibrils reveals similar protofilament
structures. Proceedings of the National Academy of Sciences. 2009 Nov
24;106(47):19813-8.

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3. Mangione MR, Piccionello AP, Marino C, Ortore MG, Picone P, Vilasi S, Di Carlo
M, Buscemi S, Bulone D, San Biagio PL. Photo-inhibition of Aβ fibrillation mediated
by a newly designed fluorinated oxadiazole. RSC Advances. 2015;5(21):16540-8.

4. Ahsan MJ, Choupra A, Sharma RK, Jadav SS, Padmaja P, Hassan M, Al-Tamimi A,
Geesi MH, Bakht MA. Rationale Design, Synthesis, Cytotoxicity Evaluation, and
Molecular Docking Studies of 1, 3, 4-oxadiazole Analogues. Anti-Cancer Agents in
Medicinal Chemistry (Formerly Current Medicinal Chemistry-Anti-Cancer Agents).
2018 Jan 1;18(1):121-38.

5. Feng BY, Toyama BH, Wille H, Colby DW, Collins SR, May BC, Prusiner SB,
Weissman J, Shoichet BK. Small-molecule aggregates inhibit amyloid
polymerization. Nature Chemical Biology. 2008 Mar;4(3):197.

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