DRUG DESIGN (19BI334)

Regulation – R19

Lab manual for the Academic Year (2022-23)

Ist semester

DEPARTMENT OF BIOTECHNOLOGY

Vadlamudi, 522 213, AP, India

Name of the Faculty: Dr. Abraham Peele

Department of Biotechnology

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 Department of Biotechnology

CERTIFICATE

This is to certify that Mr./Ms.______________________________bearing Registration

number____________________ is a student of B.Tech ____ Semester___ has successfully
completed ________ experiments in Lab during the academic year 2022-2023

Faculty In charge Head of the Department

External Examiner

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 INDEX
Name of the Lab:

S. No Date Title of the Experiment Marks & Sign

Page
No.

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 Experiment 1

Homology Modelling of a protein

Aim: To perform Homology modelling of a protein.

Description: Homology modeling, also known as comparative modeling of protein, refers to

constructing an atomic-resolution model of the "target" protein from its amino acid sequence and an
experimental three-dimensional structure of a related homologous protein. FASTA format is a text-
based format for representing either nucleotide sequences or peptide sequences, in which base pairs or
amino acids are represented using single-letter codes. A sequence in FASTA format begins with a
single-line description, followed by lines of sequence data. The SWISS-MODEL Repository is a
database of annotated 3D protein structure models generated by the SWISS-MODEL homology-
modelling pipeline.

Procedure:

 Get FASTA sequence and or valid UniProtKB AC: Go to UniProtKB

(https://www.uniprot.org// and search for cytidylate kinase
 Select the protein of species ‘Mycobacterium tuberculosis’ highlighted with the red box.
Here we get the UniProtKB AC ID highlighted with the blue box, P9WPA9)
 When we access this entry, we can download the concerned FASTA sequence
 Create account in SWISS-MODEL (https://swissmodel.expasy.org/) Start Modelling, Paste
the FASTA sequence or paste the code P9WPA9 in the search box and get the target
sequence
 Here, you can directly go for the option of ‘build model’ but as we will explore each step
separately thus ‘Search for template’.

Result:

S. No Component Max. Marks Marks Secured

1 Preparedness 2

2 Viva-voce 2

3 Experiment 3

4 Analysis & Record 3

Total 10

5
 Experiment 2

Evaluate the 3D structure of a protein

Aim: To Evaluate the 3D structure of a protein

Description: The level of protein structure at which an entire polypeptide chain has folded into a
three-dimensional structure is known as 3D structure of proteins. The SWISS-MODEL Repository is
a database of annotated 3D protein structure models generated by the SWISS-MODEL homology-
modelling pipeline. Saves server is used for structure validation scores of proteins. The PDB provides
access to 3D structure data for large biological molecules (proteins, DNA, and RNA).

Procedure:

 Model a protein structure using the swiss modeller and download the results in PDB format
 Go to SAVES version 6.0
 Upload your file in PDB format only
 Click on run programs
 The page redirects to multiple evaluating programs
ERRAT,VERIFY,PROVE,WHATCHECK,PROCHECK
 Click on start for each program to get respective results
 After evaluation click on results for details of each program evaluation.

Result:

S. No Component Max. Marks Marks Secured

1 Preparedness 2

2 Viva-voce 2

3 Experiment 3

4 Analysis & Record 3

Total 10

6
 Experiment 3
Active site/cavity in a receptor
Aim: To predict of active site/cavity in receptor

Description: Computer Atlas of Surface Topography of Proteins aims to provide comprehensive and
detailed quantitative characterization of topographic features of protein. UCSF Chimera is a program
for the interactive visualization and analysis of molecular structures and related data, including
density maps, trajectories, and sequence alignments. PyMol, a cross-platform molecular graphics tool,
has been widely used for three-dimensional (3D) visualization of proteins, nucleic acids, small
molecules, electron densities, surfaces, and trajectories. The PDB provides access to 3D structure data
for large biological molecules (proteins, DNA, and RNA).

Procedure:
 Go to CASTp server(Computer Atlas of Surface Topography of protein)
 CASTp server takes protein structures in PDB format
 Go to search bar and enter the 4 letter PDB id or job id or submit the own protein structures
to request customized computation
 It takes probe radius as input radius of 1.4 Å is used, which is the standard value for
computing solvent accessible surface area. For customized computation request, users can
specify any probe radius desired.
 The CASTp server identifies all surface pockets, interior cavities and cross channels in a
protein structure and provides detailed delineation of all atoms participating in their
formation.
 The CASTp server also provides imprints of topographic features. These results can be
directly downloaded from CASTp server, which can be visualized using either the UCSF
Chimera or our PyMol plugin, CASTPyMOL.

Result:

S. No Component Max. Marks Marks Secured

1 Preparedness 2

2 Viva-voce 2

3 Experiment 3

4 Analysis & Record 3

Total 10

7
 Experiment 4
Building small molecules

Aim: To build small molecules

Description: PubChem sketcher is a chemical structure sketching tool based exclusively on CGI
server processing, client-side JavaScript functions, and image sequence streaming. Mol2 file will get
the coordinates and the protein information (residues, PDB atom names and so on). For PDB files,
only the ATOM information is read, thus no bond information. Open Babel is computer software, a
chemical expert system mainly used to interconvert chemical file formats.
Procedure:
 Go to PubChem sketcher from chrome
 The sketcher contains three visible sub-windows sections a drawing area(right),a mode
control pad(left), and a status line(top)
 Draw the required structure in the drawing area
 Export the file to required format if required add hydrogen and press done
 Convert the file format imported from the PubChem sketcher into .mol2 format in Open
Babel
 Open Avogadro software go to file and open the molecule
 Go to extensions > Optimize geometry and save the molecule structure.
Result:

S. No Component Max. Marks Marks Secured

1 Preparedness 2

2 Viva-voce 2

3 Experiment 3

4 Analysis & Record 3

Total 10

8
 Experiment 5

ADME prediction

Aim: To predict ADME parameters/properties.

Description: A SWISSADME is a free web tool to evaluate pharmacokinetics, drug-likeness and

medicinal chemistry friendliness of small molecules. SMILES (Simplified Molecular Input Line
Entry System) is a chemical notation that allows a user to represent a chemical structure in a way that
can be used by the computer. Chemical Entities of Biological Interest (ChEBI) is a freely available
dictionary of molecular entities focused on 'small' chemical compounds. The Drug Bank database is a
comprehensive, freely accessible, online database containing information on drugs and drug targets.
IUPAC nomenclature is based on naming a molecule's longest chain of carbons connected by single
bonds, whether in a continuous chain or in a ring.

Procedure:

 Open SWISSADME from chrome

 One or multiple molecules must be entered in the SMILES list field to be submitted to Swiss
ADME calculations
 As an input draw a new chemical structure in the main field of the sketcher or by clicking
import button to retrieve an existing molecule from a file or by a name current version
recognizes a name of drug or related molecule from Drug bank and ChEBI as well as IUPAC
nomenclature.
 When the list of input is ready for submission, start SwissADME calculations by clicking on
the “Run” button
 The button is red and active only if the SMILES list is not empty. All outputs are loaded in
the same page as the input.

Result:

S. No Component Max. Marks Marks Secured

1 Preparedness 2

2 Viva-voce 2

3 Experiment 3

4 Analysis & Record 3

Total 10

9
 Experiment 6

Protein – Ligand Docking

Aim: To perform protein-ligand docking

Description: Protein–ligand docking is a molecular modelling technique. The goal of protein–ligand

docking is to predict the position and orientation of a ligand when it is bound to a protein receptor or
enzyme. The PDB provides access to 3D structure data for large biological molecules (proteins, DNA,
and RNA).Auto Dock is molecular modeling simulation software. It is especially effective for
protein-ligand docking. Auto Dock 4 is available under the GNU General Public License. Reads and
writes Auto Dock PDBQT (Protein Data Bank, Partial Charge (Q), & Atom Type (T)) format. Note
that the torsion tree is by default. Auto Grid prepares a 3D grid representation of the non-covalent
interaction energies that various user-specified ligand atom types will experience around a user-
specified target macromolecule.

Procedure:

 Click File in the top left corner of AutoDockTools GUI File > Preferences> In StartupDirectory
For ex. “. C:\Users\stemskillslab\Documents\SARSCOV23CL\”.Andclick Set and then Dismiss
 File > Read Molecule > in .pdb format
 Choose one chain select other chains and go to edit and delete > selected atoms
 Go to edit and delete water
 Add hydrogen using Edit > Hydrogen > Add add polar Hydrogen only
 Add charges using Edit > charge
 Assign AD4 type atoms from edit > Atoms
 Save the protein in .pdbqt format
 Go to ligand and input > open the ligand
 Load ligand Ligand > Torsion Tree > Detect Root… Ligand > Torsion Tree > Choose
Torsions…
 Ligand > Output > Save as PDBQT… > ligand.pdbqt
 Now we will prepare our protein for docking Grid > Macro molecule > Choose …
 Select macro molecule and click on select molecule
 Set grid box Grid > Grid Box… In present case, we will set Grid Box around bound ligand. Make
sure, ligand is occupied from all side. Just the box using x, y, z centre.
 Increase the box length by sliding x- , y-, z- dimension:
 Grid Options > File > Close saving current
 Preparing the Auto Grid Parameter File
 Grid > Set Map Types > Choose Ligand
 Choose Ligand> [open ligand]and then click Select Ligand Grid > Output > Save GPF
 Run the program Run > Run Auto Grid
 Using Browse window, select Program Filename autogrid4.exe and parameter file ligand.gpf And
then Launch the program
 Docking > Macromolecule > Set Rigid Filename… Select macromolecule.pdbqt
 Docking > Ligand > Choose... Selectligand.pdbqt
 Keep all options default and Accept it Docking > Search Parameters > Genetic Algorithm...Keep
all options default and Accept it
 Docking> Docking Parameters… Accept default parameters. And then close it

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 Docking > Output > Lamarckian GA (4.2)...
 Type-in receptor.dpf and save it. This will create docking parameter file. Finally, run docking using
following command Run > Run Auto Dock
 Using Browse window, select Program Filename autodock4.exe and parameter file receptor.dpf
 Steps for analysing docking results Analyse > Dockings > Open…
 Select receptor.dlg and click open.
 Analyse > Macro molecule > Choose: Select macro molecule receptor Analyse > Dockings >
Show Interactions
 Analyse > Conformations > Play, ranked by Energy.

Result:

S. No Component Max. Marks Marks Secured

1 Preparedness 2

2 Viva-voce 2

3 Experiment 3

4 Analysis & Record 3

Total 10

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 Experiment 7

Protein-Protein Docking

Aim: To perform protein-protein docking

Description: Protein-protein docking is the prediction of the structure of the complex, given the
structures of the individual proteins. Swarm Dock is a flexible docking method which uses a
population-based memetic algorithm to optimize parameters characterizing the orientation, position,
and conformations of protein subunits. The ClusPro server (https://cluspro.org) is a widely used tool
for protein-protein docking. The server provides a simple home page for basic use, requiring only two
files in Protein Data Bank format. ... Six different energy functions can be used depending on the type
of proteins. The PDB provides access to 3D structure data for large biological molecules (proteins,
DNA, and RNA). PyMol, a cross-platform molecular graphics tool, has been widely used for three-
dimensional (3D) visualization of proteins, nucleic acids, small molecules, electron densities,
surfaces, and trajectories. The PDB provides access to 3D structure data for large biological
molecules (proteins, DNA, and RNA).

Procedure:

 Go to chrome and open ClusPro2.0

 After signing up for an account and logging into the server
 The first thing you will want to do is choose a job name. If you don't choose a name for your
job, it will default to the job id.
 In each docking job, you will have a receptor and ligand. We will walk through choosing the
receptor. You have two options for choosing a receptor. You can choose a structure present in
the PDB or upload your own PDB by clicking on Upload PDB.
 You can optionally choose what chains to use in docking by specifying them below where the
pdb file was chosen. The chains should be single letters and white-space separated. If no
chains are specified, all chains in the file will be used.
 As an example input, we will consider the first case in the Protein-Protein Docking
Benchmark 3. Our receptor will be chain A of 1QQU and ligand will be Chain B of 1BA7.
Since 1QQU's only chain is chain A, we do not need to specify the chain.
 We will then be taken to view the queue. We can track the progress of our job from there
along with any jobs ahead of yours
 Depending on the queue on the supercomputer and the size of your protein, docking will
usually complete within a few hours. You will be notified via e-mail when your job
completes. You can then find your job in your results.
 Clicking on the id of your job will bring you to the results for that job. Here, you can view
and download the results of your docking. If you have extra information on the nature of your
complex, you can view the results for coefficient sets favouring electrostatics or
hydrophobicity. Clicking on the number above a picture will download that model as a pdb
file for your viewing. You can also download all the displayed models or all models for all
coefficients.
 By default, the server only displays the top 10 models. You can change the number of models
displayed if you don't find a satisfactory complex among the top 10.
 To analyse these results, we download the first model and view it in Pymol.

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Result:

S. No Component Max. Marks Marks Secured

1 Preparedness 2

2 Viva-voce 2

3 Experiment 3

4 Analysis & Record 3

Total 10

13
 Experiment 8

Combinatorial library Generation

Aim: To perform combinatorial library generation using RDKit.

Description: As a medicinal chemistry try to mimic the core elements of a wide range of natural
products such as nucleic acids, amino acids, carbohydrates, vitamins and alkaloids, heterocycles have
become a standard structure unit in drug discovery. These structures allow modulating important drug
properties such as potency and selectivity through bio steric replacements, lipophilicity, polarity and
aqueous solubility. This example is based on the synthetic approach to obtain bis heterocycles, linking
5 membered heterocycles building blocks containing one or more two heteroatoms (at least one
Nitrogen, Sulfur, Oxygen) to a set of azide containing building blocks through the formation of a 1, 4-
dsubstituted 1, 2, 3-triazole using click chemistry. A comprehensive introduction and installation
instructions can be found in the online documentation from the RDKit homepage
https://rdkit.org/docs/index.html.

 A strategy to build bis heterocycles

>>>import pandas as pd
>>>import rdkit as rk
>>>from rdkit import chem
>>>from rdkit.chem import Allchem
>>> from rdkit.chem.rdMolDescriptors import CalcNumHeteroatoms
#Read building blocks using a supplier.
>>>supp = Chem.SDMolSupplier (‘Sigma_bb.sdf’)
>>>for mol in supp:
>>>if mol is not None: mol.GetNumAtoms ()
#create a list of molecules
>>>mols=[x for x in supp]
>>>len (mols) #Number of building blocks

Procedure:

 Build or identify a library of commercially available building blocks used for this example
were taken from the sigma Aldrich(building blocks) catalog obtained from the ZINC DB,
consisting of 124,368 building blocks.
 Identifying the characteristics of building blocks for the strategy to be followed. Minor
components and duplicate compounds are removed, building blocks were removed, and
building blocks were selected to comply with the Congreve’s rule of three.
 The curated database can be found in Additional file 1:”sigma_bb.sdf”. The building blocks
were read in python using a supplier. Then compounds were filtered for the presence of
appropriate functional groups : a 5-membered heterocyclic ring with one(N,C,S) or 2
heteroatoms(N,S,O or at least one N)and a nucleophilic substituent (-OH,-SH) a terminal
alkyne 3-Bromo or chloro substituted and an azide.
 Setting up coupling reactions to generate the library of bis heterocycles, the reactions and
their corresponding SMIRKS were defined according to a synthetic approach reported by
Shafi.et.al. These reactions were used in the code to enumerate compounds that were
eventually exported in csv format.

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Result:

S. No Component Max. Marks Marks Secured

1 Preparedness 2

2 Viva-voce 2

3 Experiment 3

4 Analysis & Record 3

Total 10

15
 Experiment 9
Pharmacophore Modelling

Aim: To perform pharmacophore modelling.

Description: A pharmacophore model is the ensemble of common and steric and electronic features that are
necessary to ensure the optimal molecule interactions with a specific biological target and trigger (or block)
its biological response.
Procedure:
 Go to PharmaGist server.
 Input molecules: The server accepts up to 32 input molecules in mol2format.The user can upload the
molecules in a single mol2 file or in a zip of several ml2 files. Notice that through the PharmaGist
server treat the molecules as flexible, it may only change their torism angles. Bond length and bond
angle must be correct in the input files. Hydrogen atoms should also explicitly specified. An example
for Mol2 file can be found.
 No of output pharmacophores: The link with the no of candidate pharmacophores that will be shown
for each no of input molecules.
 Email Address: The link with the results of your request is sent to this address. Using this link you
can view the results. The results are kept on the server for at least a month, so they can be viewed
with the link later again. It also consist of advanced options.
 Key molecule: The user can send the first ligand in the input to serve as a key molecule. In this case
all the other ligands are aligned onto it and all the pharmacophore candidates includes this key
ligands. By default the algorithm iteratively selects each input ligand to serve as a key. This molecule
can be the ligand with the highest affinity to the receptor or the one with the lowest of rotatable
bonds.
 Minimum no of features in pharmacophores: The number of spatially distinct features in the reported
pharmacophore modelling candidate.
 Feature weighting options: There are four types currently supported by the algorithm. (i) Aromatic
ring. (ii) Hydrogen bond donor/acceptor. (iii) Charge (anion/cation). (iv)Hydrophobic.
 The user can modify weights of the features in the scoring function of the algorithm. For example: if
the user knows that the interaction with the target receptor is mostly hydrophobic, he/she is
encouraged to increase the weight assigned to a hydrophobic feature.
Result:

S. No Component Max. Marks Marks Secured

1 Preparedness 2

2 Viva-voce 2

3 Experiment 3

4 Analysis & Record 3

Total 10

16
 Experiment 10
Virtual Screening

Aim: To perform Virtual Screening

Description: PyRx is an open source software to perform virtual Screening. It is a combination of several
softwares such as Auto Dock Vina, Auto Dock 4.2, Mayari open Barbel etc... PyRx uses vina and Auto dock
4.2 as docking softwares.
Procedure:
 Go to PyRx Software.
 Download PyRx software – http://pyrx.sourceforge.io/downloads
 Loading molecules into PyRx workspace: Use upper left button to load your protein and ligand into
PyRx workspace.
 Converting .pdb files to .pdbqt files(vina and input file format)
 After successfully loading molecules into the workspace, convert them into Auto Dock input files
(pdbqt) files.
 Right click on ligands>Auto Dock>Make ligand.
 Right click on protein>Auto Dock>Make Macromolecules.
 After converting pdb files into auto dock input files you will see them under Auto Dock tab.
 Now the protein and ligand files are ready for Docking.
 Click on start here button under Vina execution mode.
 Select protein and ligand by simply clicking on them. You will see a window like below->click to
forward run vina.
 Selecting vina search space: In this step you will see a grid box in the 3D scenes. This grid box allows
you to select search space in the protein.
 To help locating the binding site we can use binding site amino acids. Click here to see the list of
amino acids->after selecting amino acids click on Toggle selection spheres button to see the selected
amino acids as pink spheres. Toggle selection spheres Full Screen->click molecules button under
Navigator panel, then click on + button located in front of protein tab.
 Make sure you select the grid box size big enough to allow the ligand to move freely in the search
space. Click forward button to start vina calculations->once the calculations are done, results will be
populated as a table with the binding affinity values. More negative the binding affinity better the
orientation of the ligand in the binding site.
 Results can be exported to other software programs like UCSF Chimera or PyMol for analysis. Click
on Edit>Preferences. You will see a pop up window.
 All your results will be saved in location specified as workspace.
 Analysing results with PyMol-> To open .pdbqt files in PyMol. Select all files in open pop up
window.
 Open protein .pdbqt files followed by ligand1- output .pdbqt to analyse the results.
 Viewing protein ligand Interactions: Select the ligand by using upper sequence bar and click on sele
A find > Polar contacts > to any atoms.

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Result:

S. No Component Max. Marks Marks Secured

1 Preparedness 2

2 Viva-voce 2

3 Experiment 3

4 Analysis & Record 3

Total 10

18