Insilico drug designing

Dinesh Gupta
Structural and Computational Biology Group
ICGEB
Modern drug discovery process
Target
identification
Target
validation
Lead
identification
Lead
optimization
Preclinical
phase
Drug
discovery
2-5 years
Drug discovery is an expensive process involving high R & D cost and
extensive clinical testing
A typical development time is estimated to be 10-15 years.

6-9 years
Drug discovery technologies
Target identification
Genomics, gene expression profiling and proteomics
Target Validation
Gene knock-out, inhibition assay
Lead Identification
High throughput screening, fragment based screening, combinatorial
libraries
Lead Optimization
Medicinal chemistry driven optimization, X-ray crystallography, QSAR,
ADME profiling (bioavailability)
Pre Clinical Phase
Pharmacodynamics (PD), Pharmacokinetics (PK), ADME, and toxicity
testing through animals
Clinical Phase
Human trials

Identify and validate target
Clone gene encoding target
Rational Approach to Drug Discovery
Express target
Synthesize modified lead compounds
Crystal structures/MM of target and target/inhibitor complexes
Preclinical trials
Identify lead compounds
Toxicity & pharmacokinetic studies
Bioinformatics tools in DD

Comparison of Sequences: Identify targets
Homology modelling: active site prediction
Systems Biology: Identify targets
Databases: Manage information
In silico screening (Ligand based, receptor
based): Iterative steps of Molecular
docking.
Pharmacogenomic databases: assist
safety related issues
Published by AAAS
J. Drews Science 287, 1960 -1964 (2000)
Currently used drug targets
This information is used by bioinformaticians to narrow the search in the groups
Insilico methods in Drug Discovery
Molecular docking
Virtual High through put screening.
QSAR (Quantitative structure-activity relationship)
Pharmacophore mapping
Fragment based screening
Molecular Docking
R L
Docking is the computational determination of binding
affinity between molecules (protein structure and ligand).
Given a protein and a ligand find out the binding free
energy of the complex formed by docking them.

L
R
Molecular Docking: classification
Docking or Computer aided drug designing can be
broadly classified
Receptor based methods- make use of the structure of the target
protein.
Ligand based methods- based on the known inhibitors
Receptor based methods
Uses the 3D structure of the target receptor to search for
the potential candidate compounds that can modulate
the target function.
These involve molecular docking of each compound in
the chemical database into the binding site of the target
and predicting the electrostatic fit between them.
The compounds are ranked using an appropriate scoring
function such that the scores correlate with the binding
affinity.
Receptor based method has been successfully applied
in many targets
Ligand based strategy
In the absence of the structural information of the target,
ligand based method make use of the information
provided by known inhibitors for the target receptor.
Structures similar to the known inhibitors are identified
from chemical databases by variety of methods,
Some of the methods widely used are similarity and
substructure searching, pharmacophore matching or 3D
shape matching.
Numerous successful applications of ligand based
methods have been reported
Ligand based strategy
Search for similar compounds
database known actives
structures found
Binding free energy
Binding free energy is calculated as the sum of the
following energies
- Electrostatic Energy
- Vander waals Energy
- Internal Energy change due to flexible deformations
- Translational and rotational energy
Lesser the binding free energy of a complex the more
stable it is
Basic binding mechanism
Complementarities between the ligand and the
binding site:
Steric complementarities, i.e. the shape of the
ligand is mirrored in the shape of the binding site.
Physicochemical complementarities
Components of molecular docking
A) Search algorithm
To find the best conformation of the ligand
and the protein system.
Rigid and flexible docking
B) Scoring function
Rank the ligands according to the interaction energy.
Based on the energy force-field function.



Success with vHTS

Dihydrofolate reductase inhibitor (1992)
HIV-protease (1992)
Phospholypase A2 (1994)
Thrombine (1996)
Carbonic anhydrase inhibitors(2002)

Virtual High Throughput Screening
Less expensive than High Throughput Screening
Faster than conventional screening
Scanning a large number of potential drug like
molecules in very less time.
HTS itself is a trial and error approach but can be
better complemented by virtual screening.


QSAR
QSAR is statistical approach that attempts to relate
physical and chemical properties of molecules to their
biological activities.
Various descriptors like molecular weight, number of
rotatable bonds LogP etc. are commonly used.
Many QSAR approaches are in practice based on the
data dimensions.
It ranges from 1D QSAR to 6D QSAR.

Pharmacophore mapping
It is a 3D description of a pharmacophore, developed by
specifying the nature of the key pharmacophoric features
and the 3D distance map among all the key features.
A Pharmacophore map can be generated by
superposition of active compounds to identify their
common features.
Based on the pharmacophore map either de novo design
or 3D database searching can be carried out.
Modeling and informatics in drug design
Increased application of structure based drug
designing is facilitated by:

Growth of targets number
Growth of 3D structures determination (PDB
database)
Growth of computing power
Growth of prediction quality of protein-
compound interactions


Summary: role of Bioinformatics?
Identification of homologs of functional
proteins (motif, protein families, domains)
Identification of targets by cross species
examination
Visualization of molecular models
Docking, vHTS
QSAR, Pharmacophore mapping

Example: use of Bioinformatics in
Drug discovery

Identification of novel drug targets
against human malaria
Malaria A global problem!
Malaria causes at least 500 million clinical cases and
more than one million deaths each year.
A child dies of malaria every 30 seconds.
Out of four Plasmodium species causing human malaria,
P.falciparum poses most serious threat: because of its
virulence, prevalence and drug resistance.
Malaria takes an economic toll - cutting economic growth
rates by as much as 1.3% in countries with high disease
rates.
There are four types of human malaria:
Plasmodium falciparum
Plasmodium vivax
Plasmodium malariae
Plasmodium ovale.






Approximately half of the world's population is at risk of malaria,
particularly those living in lower-income countries.
Today, there are 109 malaria affected countries in 4 regions

a) Chloroquine
b) Quinine
c) Artemether

d) Sodium artesunate
e) Dihydroartemisinin
f) Pyrimethamine
g) Sulfadoxine
h) Mefloquine

i) Halofantrine
j) Primaquine
k) Tafenoquine

l) Chlorproguanil
m) Dapsone
Chemical structures of drugs in widely used for treatment of Malaria


http://malaria.who.i
nt/docs/adpolicy_t
g2003.pdf

Problems with the existing drugs
Drug resistance is most common problem
Adverse effects (Shock and cardiac arrhythmias
caused by Chloroquine)
Poor patient compliance (Quinine tastes very
unpleasant, causes dizziness, nausea etc.)
High cost of production for some effective drugs
(Atovaquine).
Urgent need for identification of novel drug
targets which are effective and affordable.


Strategies for drug target identification in P.
falciparum
Parasite culture for functional assays are difficult and expensive.
Making computational approaches more relevant.
Malaria remains a neglected disease- very few stake holders!
Availability of the genomic data of P.falciparum and H.sapiens has
facilitated the effective application of comparative genomics.
Comparative genomics helps in the identification and exploitation of
different characteristic features in host and the parasite.
Identification of specific metabolic pathways in P.
falciparum and targeting the crucial proteins is an attractive approach
of target based drug discovery.
Comparison of proteomes helps in identifying
important indispensible parasite proteins
Out of 5334 predicted
proteins in P. falciparum,
60% didnt show any
similarity to known proteins.
Hence assigning a
physiological functional role
to these hypothetical
proteins using
bioinformatics approach still
remains a challenge.



A. gambiae
P. falciparum
H. sapiens
Predicted
proteome

Large set of proteins with no/low
similarity

Novel drug target identification in P.falciparum
BlastP
~40% identity threshold for
three-dimensional
modeling


Relational
Database of
homology
models

476 P.falciparum
proteins
Human
proteome
Putative drug
targets in
P.falciparum
Comparative genomics studies
Literature search for all these proteins
Check for physiological and biochemical
functions; etc ..
Proteasome
machinery (ClpQY
and ClpAP) in
P.falciparum

Targets identified by comparison of
proteins models
Identification of two proteasomal proteins
of prokaryotic origin, not present in hosts.
The protein degradation is an important
process in parasite development inside
host RBCs.
26S proteasome: eukaryotic type
19S regulatory + 20S proteolytic particle
Present only in Eukaryotes and archae
Degrades ubiquitinated proteins

> 20 different proteins involved
20S proteasome
ClpQY system: prokaryotic type
ClpY cap + ClpQ core particle
Present only in prokaryotes
No ubiquitination in prokaryote
Substrate specificity is not known
Only two proteins ClpQ & ClpY
Eukaryotic and prokaryotic proteasome machinery
ClpQ
ClpY
ClpY
Substrate protein
Peptides
ATP Dependent Protease Machinery
ClpQY (PfHslUV system)
The HslUV complex in prokaryotes is composed of an
HslV threonine protease and HslU ATP-dependent
protease, a chaperone of Clp/Hsp100 family.
HslV (ClpQ) subunits are arranged in form of two-stacked
hexameric rings and are capped by two HslU (ClpY)
hexamers at both ends.
HslU (ClpY) hexamer recognizes and unfold peptide
substrates with an ATP dependent process, and
translocates them into HslV for degradation.

Crystal structure of HslUV complex
in H. influenzae
PfClpQY complex model in
P. falciparum
ATP Dependent Protease
machineries ClpQY (PfHslUV
system)
The HslUV complex in prokaryotes is
composed of an HslV threonine
protease and ATP-dependent protease
HslU, a chaperone of clp/Hsp100
family.

HslV subunits are arranged in the form
of two-stacked hexameric rings and
are capped by two HslU hexamers at
both ends.

In an ATP dependent process, HslU
hexamer recognizes and unfold
peptide substrates and translocate
them into HslV for degradation.
MFIRNFVNIIGSQKSITKTIARNYFSDNSKLIIPRHGTTILCVRKNN
EVCLIGDGMVSQGTMIVKGNAKKIRRLKDNILMGFAGATADCFTLLDKFETKIDEYPNQL
LRSCVELAKLWRTDRYLRHLEAVLIVADKDILLEVTGNGDVLEPSGNVLGTGSGGPYAMA
AARALYDVENLSAKDIAYKAMNIAADMCCHTNNNFICETL
For full length & matured active protein
Length : 207 aa (170)
Pro domain : 37aa

Important motifs found:
TT at N terminal in mature protein
GSGG common chymotrypsin
protease signal.
Lys(28) and Arg(35) are two
conserved amino acids play some
role in the activity.
PfClpQ component
PK_ClpQ TTILCVRKNNEVCLIGDGMVSQGTMIVKGNAKKIRRLKDNILMGFAGATADCFTLLDKFE
PV_ClpQ TTILCVRKNNEVCLIGDGMVSQGTMIVKGNAKKIRRLKDNILMGFAGATADCFTLLDKFE
PF_ClpQ TTILCVRKNNEVCLIGDGMVSQGTMIVKGNAKKIRRLKDNILMGFAGATADCFTLLDKFE
PY_ClpQ TTILCVRKNNEVCLIGDGMVSQGTMIVKGNAKKIRRLKDNILMGFAGATADCFTLLDKFE
PB_ClpQ TTILCVRKNNEVCLIGDGMVSQGTMIVKGNAKKIRRLKDNILMGFAGATADCFTLLDKFE
************************************************************

PK_ClpQ TKIDEYPDQLLRSCVELAKLWRTDRYLRHLEAVLIVADKDVLLEVTGNGDVLEPSGNVLG
PV_ClpQ TKIDEYPDQLLRSCVELAKLWRTDRYLRHLEAVLIVADKDVLLEVTGNGDVLEPSGNVLG
PF_ClpQ TKIDEYPNQLLRSCVELAKLWRTDRYLRHLEAVLIVADKDILLEVTGNGDVLEPSGNVLG
PY_ClpQ TKIDEYPDQLLRSCVELAKLWRTDRYLRHLEAVLIVADKDTLLEVTGNGDVLEPSGNVLG
PB_ClpQ TKIDEYPDQLLRSCVELAKLWRTDRYLRHLEAVLIVADKDTLLEVTGNGDVLEPSGNVLG
*******:******************************** *******************

PK_ClpQ TGSGGPYAIAAARALYDVENLSAKDIAYKAMNIAADMCCHTNNNFICETL
PV_ClpQ TGSGGPYAIAAARALYDVENLSAKDIAYKAMNIAADMCCHTNNNFICETL
PF_ClpQ TGSGGPYAMAAARALYDVENLSAKDIAYKAMNIAADMCCHTNNNFICETL
PY_ClpQ TGSGGPYAMAAARALYDIENLSAKDIAYKAMNIAADMCCHTNHNFICETL
PB_ClpQ TGSGGPYAIAAARALYDIENLSAKDIAYKAMNIAADMCCHTNHNFICETL
********:********:************************:*******

Homologs of PfClpQ protein in other Plasmodium spp
PfClpQ
1kyi
Conservation of catalytic residues
S125-G45-T1-K33
Homology modeling of PfClpQ
Structural alignment of PfClpQ and HslV
(H.influenzae)
E. coli
S. enterica
H. influenzae
X. campestris
W. pipientis
P. falciparum
T. brucei
T. cruzi
L. infantum
E. coli
S. enterica
H. influenzae
X. campestris
W. pipientis
P. falciparum
T. brucei
T. cruzi
L. infantum
E. coli
S. enterica
H. influenzae
X. campestris
W. pipientis
P. falciparum
T. brucei
T. cruzi
L. infantum
Homology Modeling of PfClpQ
Most of the conserved residues in different bacterial species
were either identical or similar in PfClpQ
Km =19.18 mM
Cbz-GGL-AMC
Lactacystin
Activity assay for PfClpQ protein
0
50
100
150
1h 2h 3h 4h 5h 6h
Time
Threonine protease like
Substrate:
Inhibitor:
Biochemical characterization of PfClpQ protein
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Substrate conc (mM)
Km = 58.22 mM Km =37.79 mM
Chymotrypsin like
Suc-LLVY-AMC
chymostatin
Peptidyl glutamyl hydrolase
Z-LLE-AMC
MG132
0
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30 60 90 120 150 180
Time in minutes
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1h 2h 3h 4h 5h 6h
Time
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Substrate conc (mM) Substrate conc (mM)
Fluorogenic
peptide
substrate
Fluorescence
Protease
Top 100 solutions
Out of top 40 only 10 compounds available for purchase
Drug-like compound
library (1,000,00)

Molecular
docking

Ligand docked into proteins
active site
Insilico identification of novel inhibitors against PfClpQ ,
a novel drug target of P.falciparum by high throughput
docking
PfclpQ
Phe46
Arg36
Val21
Gly49
Gly48
Ser22
Thr2
Thr50
ClpQ interaction with ligand identified by virtual screening
Crystal structure of
HslV complexed
with a vinyl sulfone
inhibitor
Compound Gold
Score
Flexx
score
Chemical Structure
1 52.54 -25.14
2 54.76 -17.37
3 54.66 -24.43
4 52.84 -24.47

A regulatory component of ClpQY system

Recognizes the substrate; unfolds the substrate; feeds it
into the degradation machine (ClpQ)

Belongs to AAA+ family of proteins


Identification of P. falciparum ClpY (PfClpY) gene
PfClpY
~1.3 kb
Contain all the three
ClpY domains- N, I and C
N-Domain
C-Domain
I-Domain
N
I C N
DOMAINS
Walker A
Walker B
ATPase domain
ClpY
ClpY
ClpQ
Variation in I domain:
plays role in recognition of
different substrate
Homology of PfClpY protein with homologs in other organisms
Targeting the ClpQY interaction
Crystal structure of HslUV in H. influenzae Modeled ClpQY interaction in P.falciparum
J Biomol Struct Dyn. 2009 Feb;26(4):473-9
EXTRACTING THE
MICROARRAY DATA FROM
NCBI GEO
NORMALIZATION IF NECESSARY
OTHERWISE PREPARING EXCEL
FILES FOR WGCNA ANALYSIS
EXCEL SHEET OF NORMALIZED DATA
AND GENE SIGNIFICANCE
ANALYSING THESE FILES IN R
LANGUAGE AND RUNNING THEM IN
ANOTHER R PACKAGE WGCNA
PRINCIPLE BEHIND CONSTRUCTING
NETWORK IS THAT THE GENES
WHICH ARE CO-EXPRESSED,
RELATED AND CAN BE CONNECTED
TO MAKE A NETWORK , USING
PEARSON CORRELATION
COEFFICIENT
VISUALIZATION OF
NETWORKS BY DIFFERENT
GRAPHS AND SOFTWARE IN R
PACKAGE
FINDING DIFFERENT HUB GENES AND
MODULES WHICH CAN BE USED AS
DRUG TARGET BY REFERING TO THESE
NETWORKS
IDENTIFICATION OF DRUG TARGETS USING INTERACTION NETWORKS
THESE NETWORKS CAN BE USED FOR FINDING THE DRUG
TARGETS
THESE CAN ALSO BE USED FOR ANNOTATION OF PROTEINS AND
GENES BY COMPARING THEM BY INTERACTOME STUDIES
THESE NETWORKS CAN BE USED FOR PATHWAY ANNOTATION
BETTER THAN OTHER STUDIES AS THEY ARE BASED ON THE
MICROARRAY DATA
Tools used:
Sequence analysis: Pairwise and multiple
sequence alignments, Pfam.
Molecular modelling: Modeller
Docking: Tripos FlexX, GOLD, Arguslab
PP network: R package and Visant

Molecular docking hands on
Download and install Arguslab in windows
Load a PDB file, practice Arguslab tools
Follow the tutorial at
http://www.arguslab.com/tutorials/tutorial_
docking_1.htm
Molecular Docking using Argus lab:
Ex : Benzamidine inhibitor docked into Beta Trypsin
Create a binding site from bound ligand
Setting docking
parameters
Analyzing docking results
Polypeptide builder.