DRUG DESIGN: ISONIAZID ANALOGUES
FOR TARGETING
ENOYL ACP REDUCTASE OF
MYCOBACTERIUM TUBERCULOSIS.
Under guidance of
[Link], ([Link],PhD),
( Professor, Department of Biotechnology)
By
[Link] NIVEDITHA(1220109111)
ANKITA DAS(1220109104)
BACKGROUND:-
Tuberculosis:-
Tuberculosis, which results from an
infection with Mycobacterium tuberculosis,
can usually be treated with a combination
of first-line drugs taken for several months.
1st LINE DRUGS-ACTION:-
DRUG ISONIAZID:-
Isoniazid (Laniazid, Nydrazid),
also known
as isonicotinylhydrazine (INH),
It is the first-line
antituberculosis medication in
prevention and treatment.
TARGET :- ENOYL-ACYL
CARRIER PROTEIN REDUCTASE
Multidrug-Resistant Tuberculosis (MDR TB)
MDR TB occurs when a [Link] strain is resistant to isoniazid and
rifampin, two of the most powerful first-line drugs.
To cure MDR TB, healthcare providers must turn to a combination of
second-line drugs.
Second line drugs may have more side effects, the treatment may last
much longer, and the cost may be up to 100 times more than first-line
therapy.
MDR TB strains can also grow resistant to second-line drugs, further
complicating treatment
2nd LINE DRUGS-ACTION:-
Extensively Drug-Resistant
Tuberculosis (XDR TB):-
XDR TB Occurs When A Mycobacterium Tuberculosis Strain Is
Resistant To
Isoniazid And Rifampin, Two Of The
Most Powerful First-line Drugs,
As Well As
Key Drugs Of The Second Line Regimen Like
Fluoroquinolones,p-amino salicylic acid etc.
XDR TB Strains May Also Be Resistant To Additional Drugs,
Greatly Complicating Therapy.
XDR-TB : DIMINISHED TREATMENT
OPTIONS:-
New Tuberculosis (TB) Drugs Under Development:-
STATEMENT OF
THE PROBLEM:-
Tuberculosis (TB) is one of the leading causes of death due to a
single infectious organism in the world.
In spite of the fact that TB is treatable; TB is predicted to an
increase of an alarming rate every year.
Since in the last 40 years there has not been a new drug for TB
introduced to the market, there is an unmet need to discover new
synthetic lead molecules.
Thus this has prompted research on new drug candidates and targets,
as well as elucidating the mechanism of drug resistance
OBJECTIVES:-
OBJECTIVES
To compile the available information on isoniazid
analogues from literature and various other databases
and to organize the information in a database for
further studies.
QSAR Studies carried out to predict MIC of
compounds whose MIC’s are not available from
experimental [Link] analogue predicted to have
the lowest value of MIC is reported.
Attempt to design some new structural analogues of
Isoniazid and predict their kd values and MIC values
DATABASES/TOOLS /
SOFTWARES USED:-
LIST OF BIOLOGICAL
DATABASES USED:-
1)The BINDING DATABASE:-
([Link]
6) CHEMBIOGRID:- 2)The RCSB Protein Data Bank:-
([Link] ( [Link] )
[Link] )
5)CHEMSPIDER:- (
3)PUBCHEM:- (
[Link]
[Link] [Link]
)
4)OSDD(OPEN SOURCE
DRUG DISCOVERY) :-(
[Link])
TOOLS/SOFTWARES USED:-
1)
2)
MOLINSP
nChI:-
IRATION:
8)Quantitati
ve Structure
Activity
Relationship
(QSAR):
Definition:-
QSAR includes all statistical methods where
Biological activities like MIC,IC50 etc are related with
physicochemical properties(Hansch analysis),
or
fields(3D QSAR).
QSAR MODEL
CALIBRATION:-
PRINCIPLE PARTIAL LEAST MULTIPLE
COMPONENT SQUARES LINEAR
ANALYSIS (PLS) REGRESSION
(PCA) (MLR)
MODEL QUALITY ASSESSMENT:-
R2 = 1- RSS/TSS
= 1-∑(yi – yi,calculated)2 /∑(yi – yi,mean)2
Q2 =1 - PRESS/TSS
= 1 - ∑(yi – yi,predicted)2 /∑(yi – yi,mean)2
RMSD = √∑(yi – yi,calculated)2 / N
AAE = ∑ ∣ (yi – yi,calculated) ∣/ N
9)AUT
ODOC
K:-
AutoDock is a suite of automated docking tools.
It is designed to predict how small molecules, such as substrates or drug candidates, bind
to a receptor of known 3D structure.
Monte Carlo simulated annealing (SA) method , Genetic algorithm along with improved
Lamarckian Genetic Algorithm (LGA) is incorporated.
STEP I-
DATABASE CREATION:-
List of tables in database:-
#TABLE NUMBER NAME OF TABLE CONTENT [Link] ENTRIES
Analoguename,
logP,
1. [Link] Molecularweight, 161
HBD,HBA,
SASA.
Analogue name and
2. [Link] SMILES string. 161
Analogue name and
3. [Link] Structural InChi keys. 161
Analoguename,
Kd, 11
4. [Link] MIC, 78
IC50. 58
Analogue name and
5. [Link] references. 161
STEP II-
QSAR STUDIES:-
MIC LogP Molecular weight HBA
HBD SASA TPSA Polarizibility
Isoelectric point Molar Refractivity Pi Energy FRB
Weiner indices Randic indices Kd IC50
TEST SET#1-
Values were entered in following order:
◆ MIC ◆ LogP
◆ Molecularweight
◆ HBA ◆ HBD ◆ SASA ◆ TPSA
◆ Polarizibility ◆ Isoelectricpoint
◆ Molar refractivity ◆ Pi Energy
◆ FRB ◆ Weiner indices ◆ Randic indices.
O r i g i n a l Tr a i n i n g S e t - 7 8
Te s t S e t - 6 8
TEST SET#2-
Values were entered in following order:
◆ MIC ◆ LogP
◆ Molecularweight
◆ HBA ◆ HBD ◆ SASA ◆Kd ◆ TPSA
◆ Polarizibility ◆ Isoelectricpoint
◆ Molar refractivity ◆ Pi Energy
◆ FRB ◆ Weiner indices ◆ Randic indices.
O r i g i n a l Tr a i n i n g S e t - 11
Te s t S e t - 0 7
TEST SET#3-
Values were entered in following order:
◆ MIC ◆ LogP
◆ Molecularweight
◆ HBA ◆ HBD ◆ SASA ◆ IC50 ◆ TPSA
◆ Polarizibility ◆ Isoelectricpoint
◆ Molar refractivity ◆ Pi Energy
◆ FRB ◆ Weiner indices ◆ Randic indices.
O r i g i n a l Tr a i n i n g S e t - 5 8
Te s t S e t - 3 9
c)QSAR run:-
PRINCIPLE COMPONENTS PARTIAL LEAST SQUARES
REGRESSION(PCR) METHOD (PLS) METHOD
Settings for the run:- Settings for the run:-
▶Autoscaled data values(a) ▶Autoscaled data values(a)
▶ Minimal level output(m)
▶ Minimal level output(m)
▶ Principle component
regression(2) ▶ Partial least squares(3)
▶ Specific model(s) ▶All available descriptors
were used.
▶ Property coloumns to use
were specified
STEP III-
DESIGN OF NEW
ANALOGS & DOCKING
STUDIES
DESIGN OF NEW ANALOGS:-
The analogue with least value of MIC from above set
of data was selected and modification attempted to
design better isoniazid analogues with good binding
constant with the Enoyl ACP Reductase of [Link].
DOCKING STUDIES:-
Preparation of coordinate files using AutoDockTools
Calculation of atomic affinities using AutoGrid
Docking of ligands using AutoDock
Analysis of results using AutoDockTools
A - Preparation of coordinate files using AutoDockTools.
An extended PDB format, termed PDBQT, is used for
coordinate files, which includes atomic partial charges and
atom types.
Flexible residues are specified in the [Link] file.
SET OF FLEXIBLE RESIDUES(#) IN RECEPTOR
#95,94,142,147,149,165,191,192,194,199,122(11 RESIDUES)
Files generated:- ♦Ligand PDBQT Files ♦ Rigid
Receptor PDBQT Files ♦ Flexible Receptor PDBQT
Files
B - Calculation of atomic affinities using AutoGrid
RAPID ENERGY EVALUATION IS ACHIEVED BY PRECALCULATING
ATOMIC AFFINITY POTENTIALS FOR EACH ATOM TYPE IN THE
LIGAND MOLECULE BEING DOCKED.
.
THE GRID PARAMETERS ARE SET BASED ON RIGID AND FLEXIBLE
RESIDUES.
THE FILE SAVED WITH .gpf EXTENSION AND USED TO RUN
AUTOGRID
C - Docking of ligands using AutoDock
N
LD u
Oo m
Cc kAb
Ni ea uLn r m
g
p r a
Sm b E
oe e A
ft r e
R
Cr H
i
Ps o
tAa esR
fv r
eA
d a s
rMad uEto n i
cT
os
kE
=unfi. 5s
Rsd
10ellS 00ge=d 0.
.
D - Analysis of results using AutoDockTools
The p redicted values of
Binding constant (Kd) of the
set of ligands with Receptor
Enoyl ACP Reductase were
noted.
STEP I-
DATABASE CREATION:-
Table#[Link]:-
Table#[Link]:-
Table#[Link]:-
Table#[Link]:-
Table#[Link]
STEP II-
QSAR STUDIES:-
(A)TEST SET 1-QSAR table#1
for all descriptors except Kd
and IC50:-
a)Using PCR:-
R2 and Q2 corelation 0.3029 -0.4088
Average Absolute 1.4167 1.8596
Error
Root Mean Square 1.7790 2.5575
Deviation
b)Using PLS:-
R2 and Q2 corelation 0.2581 -1.2701
Average Absolute
Error 1.4747 2.0703
Root Mean Square
Deviation 1.8560 3.2465
(B)TEST SET 2-QSAR table #2
for all descriptors except
IC50(includes Kd):-
a)Using PCR:-
R2 and Q2 corelation 1.00 1.00
Average Absolute
Error 0.00 0.00
Root Mean Square
Deviation 0.00 0.00
b)Using PLS:-
R2 and Q2 corelation 0.9996 -0.4648
Average Absolute
Error 0.0414 1.9689
Root Mean Square
Deviation 0.0564 3.3356
(C)TEST SET 3-QSAR table#3 for
all descriptors except
Kd(includes IC50):-
a)Using PCR:-
R2 and Q2 corelation 0.3029 0.0298
Average Absolute
Error 1.5658 1.7820
Root Mean Square
Deviation 1.9423 2.2912
b)Using PLS:-
R2 and Q2 corelation 0.4151 -0.0658
Average Absolute
Error 1.3967 2.1385
Root Mean Square
Deviation 1.7751 2.9953
Predicted Value of MIC
of TEST SET 2:-
ANALOGUE WITH LOWEST MIC
PREDICTED:-
IUPAC PRED.
Name: MIC
COMPOUND# VALUE IMAGE
5-pentyl-
2-
1.5856
#59 phenoxy
mM
phenol
(5PP)
STEP III-
DESIGN OF NEW
ANALOGS & DOCKING
STUDIES
Display of the active site of the ACP
reductase with isoniazid:-
0RIGINAL MOLECULE:-
[Link] Experimental Kd
NAME ORIGINAL STRUCTURE
value value
5PP 257.15µM 5-11.8nM
#1-MODIFIED MOLECULE:-
Pred.
STRUCTURE [Link] ENERGY values
NAME
values (Kcal/mol)
MM#1 38.2mM -2.15
#2-MODIFIED MOLECULE:-
Pred.
[Link] ENERGY values
NAME STRUCTURE
values (Kcal/mol)
MM#2 458.23mM -0.48
#3-MODIFIED MOLECULE:-
Pred.
[Link] ENERGY values
NAME STRUCTURE
values (Kcal/mol)
MM#3
25.72mM -2.17
#4-MODIFIED MOLECULE:-
Pred.
[Link] ENERGY values
NAME MODIFIED STRUCTURE
values (Kcal/mol)
MM#4 246.32mM -0.83
#5-MODIFIED MOLECULE:-
Pred.
[Link] ENERGY values
NAME ORIGINAL STRUCTURE
values (Kcal/mol)
MM#5 5.55mM -3.08
#6-MODIFIED MOLECULE:-
Pred.
[Link] ENERGY values
NAME STRUCTURE
values (Kcal/mol)
MM#6 21.93mM -2.26
#7-MODIFIED MOLECULE:-
Pred.
[Link] ENERGY values
NAME STRUCTURE
values (Kcal/mol)
MM#7 907.01µM -4.15
STEP IV:-
QSAR STUDY TO PREDICT MIC VALUE OF MODIFIED MOLECULE TEST #6
and #7 : -
a)Using PCR:-
R2 and Q2 corelation 1.000 1.000
Average Absolute
Error 0.000 0.000
Root Mean Square
Deviation 0.000 0.000
MIC PREDICTED FOR TEST#6 = 1.7365 micromoles.
MIC PREDICTED FOR TEST#7 = 68.17 micromoles.
b)Using PLS:-
R2 and Q2 corelation 0.996 -0.4648
Average Absolute
Error 0.0414 1.9689
Root Mean Square
Deviation 0.0564 3.3356
MIC PREDICTED FOR TEST#6 =2.465 micromoles.
MIC PREDICTED FOR TEST#7 =85.77 micromoles.
The database constructed as part of
this project organizes information
regarding activity and molecular
descriptors of isoniazid analogues.
Quantitative structure activity
relationships(QSAR) studies were carried
out to predict the MIC values for isoniazid
analogues , if experimental MIC values
were not available.
The analogue predicted to have
the lowest MIC value i.e 5pp
was subjected to further
modification.
Docking studies were carried
out to predict the binding
constant of the designed
analogues.
Furthermore , the MIC values
of these designed analogues
were predicted by using
QSAR.
Thedatabase
The compound “5 pp”
is aimed at i.e.5 -
Some of-the
equipping
pentyl-2 the analogues
efforts on research
and development
may of novel
phenobe better
xypheno thanwas
l(5PP)
technologies and approaches
the
identified
commercial from
needed for the QSAR
drugs
global surveillance
STUDIES
and foin
control of
available r further
drug-resistant M.
the market.
optimizatio n.
tuberculosis.
In
futur
e
resea
rch
work
cross
react
ivity
prop
erties
of
these
comp
ound
s can
be
teste
d by
using
docki
ng
studi
es for
predi
cting
inter
actio
n
with
hum
an
enzy
mes
like
hum
an
dihy
drofo
late
redu
ctase
etc
.If
these
resul
ts are
prom
ising
precl
inical
and
clinic
al
trials
may
be
cond
ucted
.
By providing a We envision that this
comprehensive,single resource database will expand as
of Isoniazid analogues we hope additional analogues are
to accelerate and encourage
new analogue based drug identified in the coming
discoveries in TB that will have years and will serve as a
application in targeting drug- platform for diverse
resistant [Link] infections . analyses and projects.