Title of the Project: Physicochemical studies of drug interaction with
biomolecules and cyclodextrins
Summary of the proposal
The present research proposal deals with the detailed interaction study
(Kinetic, mechanistic And thermodynamic) of Drugs with cyclodextrins (α, β
and γ) and biomolecules (such as, HSA, BSA, DNA). A thorough study of these
interactions is useful to gain basic information on the pharmacological action,
biotransformation, bio-distribution etc. of drug.
DRUG CYCLODEXTRIN INTERACTION
Cyclodextrins are hydrophilic cyclic oligosaccharides with a lipophilic
central cavity.The most common cyclodextrins are α, β and γ-cyclodextrin,
which contain six, seven and eight glucose units, respectively. The non-polar
cavity of CDs can form inclusion compounds with a variety of guest molecules,
the binding is governed by the molecular polarity and ability to closely fit
within the cavity [1]. The formation of these inclusion compounds has been
widely used to improve the solubility, bioavailability and chemical and
photochemical stability of pharmaceuticals [2-10]. They can also mask
undesirable side-effects, such as bad smell, irritation power and bitterness of the
drug can also be reduced or fully eliminated.[11, 12,13]. Cyclodextrin inclusion
complexes are of interest for scientific research, because they exist in aqueous
solution and can be used to study the hydrophobic interactions which are so
important in biological systems.
Drug protein interaction
A drug's efficiency may be affected by the degree to which it binds to the
proteins within blood plasma. The less bound a drug is, the more efficiently it
can traverse cell membranes or diffuse. Common blood proteins that drugs bind
to are human serum albumin, lipoprotein, glycoprotein, α, β‚ and γ globulins.
The interaction between proteins and various kinds of pharmaceuticals is
imperative for wide range of pharmacological, biological, and clinical
applications. Understanding the mechanism and related parameters of this kind
of interaction, such as number and location of binding sites and binding
constant, is crucial for gaining insights regarding the pharmacodynamics and
pharmacokinetics of a drug [14,15]. This includes providing information
concerning the influence of binding to proteins on the absorption, exertion,
distribution, and metabolic pathway of a drug. [16-35]. Therefore, to develop a
simple and reliable method to study the drug–protein interaction is of great
practical and theoretical importance.
Drug -DNA Interaction
The interaction of nucleic acids with drugs is one of the most important
parameters in drug discovery and development processes. DNA as carrier of
genetic information is a major target for drug interaction because of the ability
to interfere with transcription (gene expression and protein synthesis) and DNA
replication. Many anticancer, antibiotic, and antiviral drugs exert their primary
biological effects by reversibly interacting with nucleic acids. There is growing
interest in exploring the binding of small molecules with DNA for the rational
design and construction of new and more efficient drugs targeted to DNA as
well as in understanding how proteins recognize and bind to specific DNA
sequences [1]. In order to improve the clinical efficacy of existing drugs and
also to design new ones it is necessary to understand the molecular basis of
drug–DNA interactions in structural, thermodynamic, and kinetic detail. There
is a high demand for the development of improved techniques with rapidness
and low cost to elucidate the drug-DNA interactions in order to speed up drug
discovery and drug approval processes.
Objectives: Following objectives are intended to be achieved in proposed study
I.
II.
III.
IV.
V.
VI.
Interaction of drugs with HSA, BSA and glycoprotein with its
thermodynamic parameters.
Kinetic, mechanistic and thermodynamic study of interaction of drugs
with metal bound proteins.
Mechanistic and thermodynamic study of interaction of drugs DNA
and effect of counter ions such as Na+, Ca2+ and Mg2+ ions on these
interactions.
Thermodynamic of binding of drug with cyclodextrins to form
inclusion complexes.
Application of drug molecule in the molecular recognition of DNA
and protein.
Analytical application of above studies in the development of methods
for trace Detection of biomolecules in biological samples.
National and international status:
Research work done and in progress in Abroad; Following work has been done
in last decade by applying various techniques
Research
Technique
Refrence
Cyclodextrin Complexes of
Celecoxib: MolecularModeling,Characterization,
and Dissolution Studies
Differential scanning
calorimetry (DSC),
powder x-ray
diffractometry
(PXRD), and scanning
electron microscopy
(SEM)
M. N. Reddy et all.
2004
Complexation of Celecoxib
with b-Cyclodextrin:
Nuclear magnetic
resonance
spectroscopy (NMR),
differential scanning
calorimetry (DSC),
Xray
V.R. Sinha. 2005
Characterization of the
Interaction in Solution
and in Solid State
diffractometry (XRD),
scanning electron
microscopy (SEM),
infrared
spectroscopy
(IR), and polarimetry.
Physicochemical
characterization and
dissolution properties of
meloxicam–cyclodextrin
binary systems
Phase solubility
analysis, mass
spectrometry and 1H
nuclear magnetic
resonance (NMR)
studies, and in solid
state using differential
scanning calorimetry
(DSC), powder X-ray
diffractometry, and in
vitro dissolution
studies
N.B. Naidu 2004
Cyclodextrin complexes of
valdecoxib: properties
infrared spectroscopy,
differential
and anti-inflammatory
activity in rat
scanning calorimetry,
X-ray diffraction,
phase solubility
analysis, 1H
K. Rajendrakumara
2005
nuclear magnetic
resonance and circular
dichroism
spectroscopy
Physicochemical
Characterization and
Dissolution Properties of
Nimesulide-Cyclodextrin
Binary Systems
Taxol–DNA interactions:
fluorescence and CD studies
of DNA groove
phase solubility
analysis, mass specB. N. Nalluri 2003
trometry, and 1H
nuclear magnetic
resonance (1H-NMR)
spectroscopic studies,
differential scanning
calorimetry (DSC),
powder x-ray
diffractometry (XRD), scanning electron
micros-copy (SEM),
and in vitro
Fluorimetry and
circular dichroism
G. Krishn 1998
binding properties of taxol
Fluorescence Studies on the
Interaction of Furocoumarins
with DNA in the Dark
Fluoimetry
Interaction of furazolidone
with DNA
fluorimetry
M. Gupta
S.N. Chatterjee, 1975
Interaction between an 8UV-Vis and
methoxypyrimido[4 ,5 :4,5] fluorescence
thieno
spectrophotometry
(2,3-b)quinoline-4(3H)one
antitumour drug and
deoxyribonucleic acid
M GOPAL 1993
as well as by
hydrodynamic
methods
Thermodynamics of the
interaction of berberine with
DNA
spectrophotometry
G.S. Kumar 1993
Spectroscopic studies on
interaction of riboflavin and
other fluoroquinolones with
bovine serum albumin
Fluorimetry and
circular dichroism
Kamat et al., 2004 and
Kamat et al., 2005
Binding of Naproxen and
Amitriptyline to Bovine
Serum Albumin: Biophysical
Aspects
isothermal titration
calorimetry (ITC), in
combination
T. Banerjee 2006
Interaction between a potent
corticosteroid drug –
Dexamethasone
UV–vis absorption,
Fluoroscence and FTIR spectroscopy
with bovine serum albumin
and human serum albumin: A
fluorescence
quenching and fourier
transformation infrared
spectroscopy study
with fluorescence and
circular dichroism
spectroscopies
P.N. Naik 2010
Investigations on the
interactions of
aurintricarboxylic acid with
bovine serum
steady state and timeresolved fluorescence,
circular
M. Bardhan 2011
dichroism (CD), FT-IR
and fluorescence
anisotropy
albumin: Steady state/time
resolved spectroscopic and
docking studies
Study of the interaction of an
anticancer drug
UV-Vis and
fluorescence
spectrophotometry,
with human and bovine serum
circular divhroism
albumin: Spectroscopic
approach
P.B. Kandagal 2006
Research work done and in progress in India
Research
Techniques
Reference
Structure, Dynamics, and
Stability of βCyclodextrin Inclusion
Complexes of Aspartame
and Neotame
Mebendazole complexes
with various
cyclodextrins:
preparation
and physicochemical
characterization
1
J. R. Garbow. 2001
Mass spectrometry,
powder X-ray
diffractometry and
Fourier transform
infrared spectroscopy.
M. Lahiani-Skiba
(2007)
H NMR
HPLC and solubility
HPLC
study of the interaction
between pindolol and
cyclodextrins
Spectrofluorimetry
Study on the inclusion
interaction of methylated
β-cyclodextrins with
albendazole by
spectrofluorimetry and
its application
C. Gazpio,(2005)
G. Y. Zhao, (2008).
Physicochemical Study
of the Complexation of
Nortriptyline and Human
Serum Albumin
conductivity, ζ potential, static and
dynamic light scattering
methods, and UV-vis
spectroscopy
D. Leis, 2002
Interactions of Two
Amphiphilic Penicillins
with Myoglobin in
Aqueous Buffered
Solutions: A
Thermodynamic and
Spectroscopy Study
equilibrium dialysis, - ζ
potential, isothermal
titration calorimetry
(ITC) and UV-Vis
absorbance techniques.
P. Taboada 2004,
Interaction of
Tetrandrine with Human
Serum Albumin:
a Fluorescence
Quenching Study
Fluorescence quenching
spectra, synchronous
fluorescence spectra and
ultra-violet spectra.
C. WANG 2007
Coupling microdialysis
with flow-injection
chemiluminescence
detection for a protein–
drug interaction study
coupling system of online microdialysis
sampling with flowinjection
chemiluminescence
detection (FI-MD-CL)
H. Chena (2006)
Characterization of the
docking
binding of angiotensin II and molecular dynamics
receptor
simulation.
blockers to human serum
albumin using docking
and molecular dynamics
simulation
J. Li (2010)
Characterization of
antihistamine–human
serum protein
interactions by capillary
electrophoresis
ultrafiltration and
M.A. Mart´ınez-G´omez,
capillary electrophoresis. (2007)
Binding of
UV–vis absorption,
Z. CHI, 2010
Oxytetracycline to
Bovine Serum Albumin:
Spectroscopic and
Molecular Modeling
Investigations
Fluoroscence, Circular
dichroism, Molecular
modelling
Spectroscopic
investigation of the
interaction
between rifabutin and
bovine serum albumin
fluorescence
C. Wang,(2007)
spectroscopy and circular
dichroism (CD).
Spectroscopic
Investigations of the
Binding Interaction of a
New Indanedione
Derivative with Human
and Bovine Serum
Albumins
Fluorescence
spectroscopy
D. Stan , 2009,
Spectroscopic study on
the interaction of
celecoxib with human
carbonic anhydrase
II: Thermodynamic
characterization of the
binding process
UV–Vis, fluorescence
and circular dichroism
(CD) spectroscopy and
differential scanning
calorimetry (DSC).
M. Mehrabi , (2009)
Spectrofluorimetric
study on the interaction
between antimicrobial
drug
sulfamethazine and
bovine serum albumin
steady state and
synchronous
fluorescence
spectroscopy
A. D. Bani-Yaseen.
Interaction of the
anticancer drug
epirubicin with DNA
differential pulse
A. Erdem (2001)
voltammetry (DPV) and
cyclic voltammetry (CV)
at
carbon paste electrode
(CPE)
A Fluorescence
Spectroscopic Study of
the Interaction Between
Norfloxacin and DNA
Fluorescence
spectroscopic.
G.Song 2004
Interaction of anticancer
drug mitoxantrone with
DNA analyzed
by electrochemical and
spectroscopic methods
UV/Vis, fluorescence
and Raman techniques.
N. Lia,(2005)
Capillary electrophoresis
for studying drug–DNA
interactions.
Electrochemical studies
of the interaction of the
anticancer herbal drug
emodin with DNA
capillary electrophoresis
F. Araya,
(2007)
differential pulse
voltammetry (DPV) and
cyclic voltammetry
(CV) at the bare or DNA
modified GCE and
ultraviolet–visible (UV)
spectra
L. Wang, (2006)
Electrochemical studies
and spectroscopic
investigations on the
interaction
of an anticancer drug
with DNA and their
analytical applications.
Differential pulse
Shankara S. Kalanur
voltammetry
(2009),
(DPV) and spectroscopic
techniques viz., UV–vis
absorption, circular
dichroism and
fluorescence
Anticancer Drug-DNA
Interactions Measured
Using a Photoinduced
Electron-Transfer
Mechanism Based on
Luminescent Quantum
Dots
Electrochemistry of
interaction of 2-(2nitrophenyl)benzimidazole
derivatives with DNA
UV/vis absorption,
Fluorescence,
luminescence
spectrometer
J.Yuan, 2009,
differential pulse
voltammetry at glassy
carbon electrode
M.Catalán (2010)
Study of interactions
between DNA and
aflatoxin B1 using
electrochemical
and fluorescence
methods
differential pulse
voltammetry (DPV),
Fluorescenec
spectroscopy.
M.H. Banitaba 2011.
Work plan (including detailed methodology and time schedule
UV–vis, steady state and time-resolved fluorescence and circular
dichroism spectroscopic investigations would be used to to obtain a
multidisciplinary insight into the molecular and energetic details of the drug –
protein, drug-DNa and drug-Cyclodextrins. Understanding of molecular
recognition processes between drug and biological macromolecules requires a
complete characterization of the binding energetics and correlation of
thermodynamic data with the structural properties of the interacting molecules
.The fluorescence spectroscopy has been widely used to monitor the molecular
interaction because of its high sensitivity, reproducibility and relatively easy
use. Now a days Resonance Rayleigh scattering (RRS), second-order scattering
(SOS) and frequency doubling scattering (FDS) has been extensively applied
for the determination of macromolecule polysaccharides, organic and inorganic
molecule. Thus, studying on the RRS, absorption, fluorescence spectra and CD
of drug with cyclodextrins and biomolecule systems can not only provide much
information about the tendencies and conditions of the interaction, explain the
mechanism and the binding modes, but also can lay the foundation of
developing new highly sensitive methods for the determination of drug, and
recognition of biomolecules.
The experiment will be conducted in these stages.
Binding studies.
UV–vis absorption spectra, steady state and time-resolved fluorescence
spectra, resonance Rayleigh scattering spectra of biomolecules and
cyclodextrins at varying concentration of drug would be recorded. These data
will be employed to establish the kinetic quenching parameters, binding
parameters via Stern–Volmer and its modified form, double logarithm
regression curve and its modified form, Benesi–Hildebrand equation and
Scatchard’s equation. Therefore, valuable information’s such as binding
mechanism, binding constant, binding distance, Energy transfer efficiency and
binding sites can be obtained using fluorescence quenching study. Additionally,
the conformational changes of biomolecules occurred in presence of drug have
been analyzed by using circular dichroism (CD) and synchronous fluorescence
spectroscopy.
Thermodynamic Studies.
Drug-receptor binding thermodynamics has proved to be a valid tool for
pharmacological and pharmaceutical characterization of molecular mechanisms
of receptor-recognition phenomena. The large number of membrane receptors
so far studied has led to the discovery of enthalpy-entropy compensation effects
in drug-receptor binding and discrimination between agonists and antagonists
by thermodynamic methods. Thermodynamic parameters relying on
temperatures were analyzed to characterize the acting forces between drug and
BSA. Basically, four types of interactions play vital roles in drug–receptor
binding, that is, hydrogen bonds, van der Waals forces, electrostatic forces, and
hydrophobic interactions. Thermodynamic parameters, free energy changes (G◦)
enthalpy changes (H◦) and entropy changes (S◦) of interactions are essential to
interpret the binding mode. For the purpose of clarifying interaction of drug
with biomolecules, the temperature dependence of binding constant was
resulted from binding studies carried out at three different temperatures.Thus,
variation in the binding constant as a function of temperature has been fitted
according to the van’t Hoff equation.
The significance of the thermodynamic approach is related to the more
complete information on drug-receptor interaction mechanisms obtainable by
full thermodynamic methods with respect to the simple affinity constants
measurements.
TIME SCHEDULE:
MONTHS
WORK PLAN
0-6
Placing of order for chemicals glassware’s and
instrument
7-13
UV–vis absorption spectra, steady state and timeresolved fluorescence spectra, resonance Rayleigh
scattering spectra of biomolecules at varying
concentration of different drug would be recorded
14-20
Binding study at different temperature, determination
of conformational change s by circular dichroism and
synchronus
fluorescence
spectroscopy.Thermodynamic study to evaluate type
of forces acting.
21-27
UV–vis absorption spectra, steady state and timeresolved fluorescence spectra, resonance Rayleigh
scattering spectra of cyclodextrins at varying
concentration of different drug would be recorded.
28-33
Complexation study at different temperature, and at
different pH. Calculation of inclusion constant,
stiochiometry, binding mechanism and Determination
of the thermodynamic properties of the complexes to
enrich the thermochemical database.
34-36
Statistical analysis of data and completion of report
Expected deliverables/outcome.
The interaction of drug with biomolecules and cyclodextrins can cause
quenching and enhancement of RRS intensity. Therefore,valuable
physicochemical parameters can be obtained from these quenched and
enhanced signals. Highly sensitive method may be developed for detection
of biomolecules or determination of drug utilizing these signals.
Significance of the expected outcome with respect to the state-of-the-art in
the field
The investigation and output will be valuable contribution to the chemical
literature. The success of proposal will also open up new perspective for
application of biosensors in analytical measurement and molecular recognition
of proteins and nucleic acid.. The main beneficiaries will be those working in
other universities, research institution or in pharmaceutical/biochemical
industry. The outcome will be helpful to gain basic information on the
pharmacological action, biotransformation, bio-distribution etc. of drug. These
studies may provide information of the structural features that determine the
therapeutic effectiveness of drug and become an important research field in life
science, chemistry and chemical science. The data may have a practical interest
for the development of new drugs.
11. Name, address of the institution and bio-data of the scientist-mentor
with whom the proposed R&D study will be executed.
12. Facilities in terms of laboratory, equipment, etc. likely to be made
available to the candidate by the host institution for pursuing the above
studies
1. UV/Visible spectrophotometers
2. Fluorescence Spectrometer
3. pH meters
4. Electrical Balance
5. Incubator
6. Deep Freezer
7. Deionizer/ Millipore water purification system
8. Conductivity meter
9. Distillation Plant
Infrastructure Facilities
1. Water and electricity
2. Standby power supply
3. Laboratory space and furniture
4. Air conditioned room for sophisticated instruments
5. Library facility
6. Telecommunication
10. Computational facilities with internet etc
Justification for the contingency and consumables:
Our proposal aims at the physicochemical studies of drug interaction with
biomolecules and cyclodextrins. Chemicals and biochemicals of high analytical
grade are required. Latest drugs (anticancerous) in market will be used. The
drug in pure form and biomolecules such as HSA, BSA, DNA are costly and
most of them are to be imported. In view of the high cost of chemicals and
glassware the budget under said heading is well justified.
14. Details of research funding received and applied for (mention reference
no., title, duration, cost, funding agency and brief achievements). NO
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