This document discusses protein-protein interactions (PPIs). It begins by stating that PPIs are important for many biological processes and are the basis of protein folding, assembly, and interactions. It then discusses that PPIs can be classified based on their interaction surfaces (homo- or hetero-oligomeric), stability (obligate or non-obligate), and persistence (transient or permanent). Transient interactions form signaling pathways while permanent interactions form stable complexes. PPIs modify enzyme kinetics, allow for substrate channeling, and construct new functional properties. Detection methods for PPIs include yeast two-hybrid, co-immunoprecipitation, and protein interaction databases.
2. CONTENTS:
Protein Binding
Function of Protein Binding
DRUG INTERACTIONS
Outcomes Of Drug Interactions
Mechanisms Of Drug Interactions
1)Pharmacokinetic
2)Pharmacodynamic
Protein – Drug Binding Interactions
Mechanisms Of Protein Drug Binding
Factors Affecting Protein Drug Binding
2
3. Protein-Protein Interaction Detection and
Methods
The significant properties of PPI
TYPES OF PROTEIN–PROTEIN
INTERACTIONS
Classification of PPI Detection Methods
Yeast two-hybrid
Co-immuno-percipitation
Applications
Protein interactions database
SIGNIFICANCE OF PROTEIN/TISSUE
BINDING OF DRUGS
Conclusions
REFERENCES 3
4. PROTEIN BINDING
At therapeutic concentrations in plasma,
many drugs exist mainly in bound form.
The most important plasma protein in relation
to drug binding is albumin. Albumin binds
many acidic drugs and a smaller number of
basic drugs
– Other plasma proteins include ß-
globulin, a-acid glycoprotein and Lipoproteins
4
5. FUNCTION OF PROTEIN BINDING
Serum albumins are important in regulating
blood volume by maintaining the osmotic
pressure of the blood compartment
– They also serve as carriers for molecules of
low water solubility this way isolating their
hydrophobic nature.
ß-globulins are a subgroup of globulin proteins
produced by the liver or immune system
– Mostly involved with transport.
5
6. α-acid glycoprotein is an alpha-globulin
and act as a carrier of basic and neutrally
charged lipophilic compounds
The amount of a drug that is bound to
protein depends on three factors:
– the concentration of the drug
– its affinity for the binding sites
– the concentration of protein
6
7. As an approximation, the binding reaction
can be regarded as a simple association
of the drug molecules with a finite
population of binding sites, analogous to
drug–receptor binding:
𝐷 + 𝑆 ⇌ 𝐷𝑆
free drug Binding site complex
The usual concentration of albumin in
plasma is about 0.6 mmol/l (4 g/100 mL).
With two sites per albumin molecule, the
drug-binding capacity of plasma albumin
would therefore be about 1.2 mmol/L.
7
8. – For most drugs, the total plasma
concentration required for a clinical effect
is much less than 1.2 mmol/l, so with usual
therapeutic doses the binding sites are far
from saturated, and the concentration
bound [DS] varies nearly in direct
proportion to the free concentration [D].
– Under these conditions the fraction
bound: [DS]/([D] + [DS]) is independent of
the drug concentration.
8
9. DRUG INTERACTIONS
Drug interaction is defined as the
pharmacological activity of one drug is
altered by the concomitant use of another
drug or by the presence of some other
substance.
OR
It is the modification of the effect of one drug
(the object drug ) by the prior concomitant
administration of another (precipitant drug).
9
10. The Drug whose Activity is effected by such
an interaction is called as a “Object drug.”
The agent which precipitates such an
interaction is referred to as the “Precipitant”.
Concomitant use of several drug in presence
of another drug is often necessory for
achiving a set of goal or in the case when the
patient is suffering from more than one
disease.
In these cases chance of drug interaction
could increase.
10
11. OUTCOMES OF DRUG INTERACTIONS
1) Loss of therapeutic effect
2) Toxicity
3) Unexpected increase in pharmacological
activity
4) Beneficial effects
e.g. additive & potentiating (intended) or
antagonism (unintended).
5) Chemical or physical interaction
e.g. I.V incompatibility in fluid or syringes
mixture
11
12. Mechanisms Of Drug Interactions
1)Pharmacokinetic
2)Pharmacodynamic
Pharmacokinetics involve the effect of a
drug on another drug kinetic that includes
absorption, distribution , metabolism and
excretion.
Pharmacodynamics are related to the
pharmacological activity of the interacting
drugs
E.g., synergism , antagonism, altered cellular
transport effect on the receptor site.
12
13. PHARMACOKINETIC INTERACTION
1) Altered GIT absorption:
•Altered pH
•Altered bacterial flora
• formation of drug chelates or complexes
• drug induced mucosal damage
• altered GIT motility
13
14. a) Altered pH:
The non-ionized form of a drug is more
lipid soluble and more readily absorbed
from GIT than the ionized form does.
Eg: antacids Decrease the tablet
dissolution of Ketoconazole (acidic).
Therefore, these drugs must be
separated by at least 2h in the time of
administration of both.
14
15. b) Altered intestinal bacterial flora:
Eg;
40% or more of the administered digoxin
dose is metabolised by the intestinal flora.
Antibiotics kill a large number of the
normal flora of the intestine.
Increase digoxin concentration and
increase its toxicity.
15
16. c) Complexation or chelation:
Eg;
Antacid (aluminum or magnesium hydroxide)
Decrease absorption of ciprofloxacin by 85% due to chelation.
d) Drug-induced mucosal damage:
Eg;
Antineoplastic agents (cyclophosphamide, vincristine,
procarbazine)
Inhibit absorption of several drugs
eg., digoxin
16
17. e) Altered motility:
Metoclopramide (antiemitic)
Increase absorption of cyclosporine
due to the increase of stomach empting time
and Increase the toxicity of cyclosporine.
f) Displaced protein binding:
It depends on the affinity of the drug to
plasma protein. The most likely bound drugs
is capable to displace others. The free drug is
increased by displacement by another drug
with higher affinity. 17
18. Phenytoin is a highly bound to plasma protein
(90%), Tolbutamide (96%), and warfarin (99%)
and Drugs that displace these agents are Aspirin,
Sulfonamides, phenylbutazone.
g) Altered metabolism:
The effect of one drug on the metabolism of the
other is well documented. The liver is the major
site of drug metabolism but other organs can also
do e.g., WBC, skin, lung, and GIT.
CYP450 family is the major metabolizing
enzyme in phase I (oxidation process). Therefore,
the effect of drugs on the rate of metabolism of
others can involve the following examples.
Eg. Enzyme induction
18
19. A drug may induce the enzyme that is
responsible for the metabolism of another
drug or even itself
Eg.
1) Carbamazepine (antiepileptic drug)
increases its own Metabolism.
2) Phenytoin increases hepatic metabolism
of theophylline leading to decrease its level
Reduces its action
19
20. PHARMACODYNAMIC INTERACTION
It means alteration of the dug action without
change in its serum concentration by
pharmacokinetic factors.
Propranolol + verapamil Synergistic or
additive effect.
Receptor interaction:
•Competitive
•Non-competitive
20
21. Sensitivity of receptor:
•Number of receptor
•Affinity of receptor
Alter neurotransmitter release /
drug transportation
Alter water/electrolyte balance
21
22. PROTEIN – DRUG BINDING INTERACTIONS
The interacting molecules are generally the
macromolecules such as protein, DNA or
adipose. The protein are particularly
responsible for such an interaction.
The phenomenon of complex formation of
drug with protein is called as protein binding
of drug.
As a protein bound drug is neither
metabolized nor excreted hence it is
pharmacologically inactive due to its
pharmacokinetic and Pharmacodynamic
inertness. 22
23. Protein + drug ⇌ Protein-drug complex
Protein binding may be divided into:
1. Intracellular binding
2. Extracellular binding
23
24. MECHANISMS OF PROTEIN DRUG BINDING
Binding of drugs to proteins is generally of reversible
&irreversible.
Reversible generally involves weak chemical bond such as:
1. Hydrogen bonds
2. Hydrophobic bonds
3. Ionic bonds
4. Van der waal’s forces.
• Irreversible drug binding, though rare, arises as a result of covalent
binding and is often a reason for the carcinogenicity or tissue
toxicity of the drug.
24
26. 1. BINDING OF DRUG TO BLOOD COMPONENTS
A. Plasma protein-drug binding:-
The binding of drugs to plasma proteins
is reversible.
The extent or order of binding of drug to
plasma proteins is:
Albumin > α 1 -Acid glycoproteins >
Lipoproteins > Globulins.
26
27. 1. BINDING OF DRUG TO HUMAN SERUM
ALBUMIN
It is the most abundant plasma protein
(59%), having M.W. of 65,000 with large drug
binding capacity.
Both endogenous compounds such as fatty
acid, bilirubin as well as drug binds to HSA.
Four different sites on HSA for drug binding.
27
28. Site I: Warfarin & Azapropazone binding
site. Ex. Non-Steroidal Anti-Inflammatory
Drugs, Sulphonamides
Site II: Diazepam binding site. Ex.
benzodiazepines, medium chain fatty
acids, ibuprofen etc.
Site III: Digitoxin binding site.
Site IV: Tamoxifen binding site.
28
29. 2. Binding of drug to α1-Acid
glycoprotein: (Orosomucoid):
It has a M.W. 44,000 and plasma conc.
range of 0.04 to 0.1 g%. It binds to no. of
basic drugs like imipramine, lidocaine,
propranolol, quinidine.
3. Binding of drug to Lipoproteins:
Binding by: Hydrophobic Bonds, Non-
competitive.
Mol wt: 2-34 Lacks Dalton.
Lipid core composed of:
Inside: triglyceride & cholesterol esters.
Outside: Apoprotein. 29
31. B. BINDING OF DRUG TO BLOOD CELLS
In blood 40% of blood cells of which major
component is RBC (95%). The RBC is 500
times in diameter as the albumin. The rate &
extent of entry into RBC is more for lipophilic
drugs.
The RBC comprises of 3 components.
a) Haemoglobin: It has a M.W. of 64,500 Dal.
Drugs like Phenytoin, pentobarbital bind to
haemoglobin.
31
32. b) Carbonic anhydrase: Carbonic anhydrase
inhibitors drugs are bind to it like
acetazolamide & chlorthalidone.
c) Cell membrane: Imipramine &
chlorpromazine are reported to bind with the
RBC membrane.
32
33. 2. BINDING OF DRUG TO EXTRAVASCULAR
TISSUE PROTEIN
Importance:
1. It increases apparent volume of distribution of
drug.
2. localization of a drug at a specific site in body.
Factor affecting: lipophilicity, structural feature
of drug, perfusion rate, pH differences.
Binding order: Liver › Kidney › Lung › Muscles
33
35. FACTORS AFFECTING PROTEIN DRUG BINDING
1. Drug-related factors:
a. Physicochemical characteristics of the drug:-
Protein binding is directly related to the
lipophilicity of drug. An increase in lipophilicity
increases the extent of binding.
b. Concentration of drug in the body:-
Alteration in the concentration of drug
substance as well as the protein molecules or
surfaces subsequently brings alteration in the
protein binding process.
35
36. c. Affinity of a drug for a particular binding
component:-
This factor entirely depends upon the
degree of attraction or affinity the protein
molecule or tissues have towards drug
moieties.
For Digoxin has more affinity for cardiac
muscles proteins as compared to that of
proteins of skeletal muscles or those in the
plasma like HSA.
36
37. 2. PROTEIN/ TISSUE RELATED FACTORS
a. Physicochemical characteristics of protein
or binding agent:
Lipoproteins & adipose tissue tend to bind
lipophilic drug by dissolving them in their
lipid core.
The physiological pH determines the
presence of active anionic & cationic groups
on the albumin to bind a variety of drug.
37
38. b. Concentration of protein or binding
component:
Among the plasma protein , binding
predominantly occurs with albumin, as it is
present in high concentration in
comparision to other plasma protein.
The amount of several proteins and tissue
components available for binding, changes
during disease state.
38
39. 3. DRUG INTERACTIONS
a. Competition between drugs for the binding
sites[ Displacement interactions]:-
D2
D1+P D2+P
D1: Displaced drug D2: Displacer
drug
Ex. Administration of phenylbutazone to a
patient on Warfarin therapy results in
Hemorrhagic reaction.
39
40. b. Competition between drug & normal body
constituents:-
The free fatty acids are known to interact with
a no. of drugs that binds primarily to HSA. the
free fatty acid level increase in physiological,
pathological condition.
c. Allosteric changes in protein molecule:-
The process involves alteration of the protein
structure by the drug or it’s metabolite
thereby modifying its binding capacity.
Ex. aspirin acetylates lysine fraction of albumin
thereby modifying its capacity to bind NSAIDs
like phenylbutazone.
40
41. 4. PATIENT-RELATED FACTORS
a. Age:
1. Neonates: Low albumin content: More
free drug.
2. Young infants: High dose of Digoxin due
to large renal clearance.
3. Elderly: Low albumin: So more free drug.
b. Intersubject variability:
Due to genetics & environmental factors. 41
44. INTRODUCTION
Protein-protein interactions (PPIs) handle a
wide range of biological processes, including
cell-to-cell interactions and metabolic and
developmental control .
Protein-protein interaction is becoming one
of the major objectives of system biology.
Noncovalent contacts between the residue
side chains are the basis for protein
folding,protein assembly, and PPI.
44
45. These contacts induce a variety of interactions and
associations among the proteins.
Based on their contrasting structural and functional
characteristics, PPIs can be classified in several
ways.
On the basis of their interaction surface, they may be
homo- or hetero oligomeric; as judged by their
stability, they may be obligate or non obligate; as
measured by their persistence, they may be transient
or permanent.
A given PPI may be a combination of these three
specific pairs.
The transient interactions would form signaling
pathways while permanent interactions will form a
stable protein complex.
Typically proteins hardly act as isolated species while
performing their functions in vivo
45
46. THE SIGNIFICANT PROPERTIES OF PPI
(i) modify the kinetic properties of enzymes
(ii) act as a general mechanism to allow for substrate
channeling
(iii) construct a new binding site for small effector
molecules
(iv) inactivate or suppress a protein
(v) change the specificity of a protein for its substrate
through interaction with different binding partners
(vi) serve a regulatory role in either upstream or
downstream level.
46
47. EXAMPLES OF PROTEIN–PROTEIN
INTERACTIONS
Signal transduction:
The activity of the cell is regulated by extracellular
signals
Transport across membranes:
A protein may be carrying another protein.
Cell metabolism:
In many biosynthetic processes enzymes interact with
each other to produce small compounds or other
macromolecules.
Muscle contraction :
Myosin filaments act as molecular motors and by
binding to actin enables filament sliding.
47
49. CLASSIFICATION OF PPI DETECTION METHODS
Protein-protein interaction detection methods are categorically
classified into three types, namely,
• in vitro
• in vivo, and
• in silico methods
The in vitro methods in PPI detection are
tandem affinity purification,
affinity chromatography,
Co-immuno-precipitation,
protein arrays,
protein fragment
complementation,
phage display,
X-ray crystallography, and
NMR spectroscopy.
49
50. In in vivo techniques, a given procedure is performed
on the whole living organism itself.
The in vivo methods in PPI detection are yeast two-
hybrid (Y2H, Y3H) and synthetic lethality.
The in silico methods in PPI detection are sequence-
based approaches,
structure-based approaches,
chromosome proximity, gene fusion
in silico 2 hybrid,
mirror tree,
phylogenetic tree, and
gene expression-based approaches.
50
51. YEAST TWO-HYBRID
Testing for physical interactions between two
proteins
first proven using Saccharomyces cerevisiae
as biological model by Fields and Song.
Bait – The protein fused to the DBD is
referred to as the ‘bait’ (yeast transcription
factor, like Gal4)
Prey- The protein fused to the AD
Reporter gene: LacZ reporter - Blue/White
Screening
51
52. Saccharomyces cerevisiae as biological
model by Fields and Song
One technique that can be used to study
protein-protein interactions is the "yeast two
hybrid" system.
transcription requires both the DNA-binding
domain (BD) and the activation domain (AD)
of a transcriptional activator (TA)
52
53. BASIC PRINCIPLE
If protein X and protein Y interact, then their
DNA-binding domain and activation domain
will combine to form a functional
transcriptional activator (TA). The TA will then
proceed to transcribe the reporter gene that
is paired with its promoter.
53
54. THE YEAST TWO-HYBRID ASSAY USES TWO
PLASMID CONSTRUCTS
The bait plasmid, which is the protein of interest fused to a
GAL4 binding domain, and the hunter plasmid, which is the
potential binding partner fused to a GAL4 activation domain
Selection genes encoding for amino acids, such as histidine,
leucine and tryptophan
54
55. PLASMID CONSTRUCTION
The 'bait' DNA is isolated and inserted into a plasmid
adjacent to the GAL4 BD DNA.
When this DNA is transcribed, the 'bait' protein will now
contain the GAL4 DNAbinding domain as well. The ‘Prey‘/
Hunter fusion protein contains the GAL4 AD.
55
56. TRANSFECTION
The 'bait' and 'hunter' plasmids are
introduced into yeast cells by transfection.
cells containing both plasmids are selected
for by growing cells on minimal media. Only
cells containing both plasmids have both
genes encoding for missing nutrients, and
consequently, are the only cells that will
survive.
56
57. The reporter gene most commonly used in the Gal4 system
is LacZ, an E. coli gene whose transcription causes cells to
turn blue4
LacZ gene is inserted in the yeast DNA immediately after
the Gal4 promoter
57
58. CO-IMMUNO-PERCIPITATION
Co-IP is a classic technology widely used for protein-protein interaction
identification and validation
New binding partners, binding affinities, the kinetics of binding and the
function of the target protein
58
59. THE ADVANTAGE OF THIS TECHNOLOGY
INCLUDES:
Both the bait and prey proteins are in their
native conformation in the co-IP assay
The interaction between the bait and prey
proteins happens in vivo with little to no
external influence
The limitation of this technology lies in,
Low affinity or transient interaction between
proteins may not be detected.
59
60. APPLICATIONS
Identify novel protein-protein interactions
Characterize interactions already known to
occur
protein domains
Conditions of interactions
manipulating protein-protein interactions in
an attempt to understand its biological
relevance
To know how mutation affects a protein's
interaction with other proteins
60
61. PROTEIN INTERACTIONS DATABASE
Protein interactions are collected together in
specialized biological databases
Databases can be subdivided into primary databases,
meta-databases, and prediction databases
Primary databases - published PPIs proven to exist
via small-scale largescale experimental methods. Eg:
DIP, Biomolecular Interaction Network, BIND,
BioGRID), HPRD
Meta-database – Primary and original data Eg: APID,
The Microbial, MPIDB, and PINA , and GPS-Prot etc.
Prediction Databases – predicted using several
techniques Eg: Human Protein–Protein Interaction
Prediction Database (PIPs), I2D, STRING, and
Unified Human Interactive (UniHI).
61
62. SIGNIFICANCE OF PROTEIN/TISSUE BINDING OF
DRUGS
Absorption
Systemic solubility of drugs
Distribution
Tissue binding, apparent volume of
distribution and drug storage
Elimination
Displacement interaction and toxicity
Diagnosis
Therapy and drug targeting 62
63. CONCLUSIONS
PPI methodologies have been developed in
yeast-methods are sometimes not suitable for
plant systems
All pharmacokinetic parameters can be
influenced by protein binding.
Bound drug cannot penetrate through blood
capillaries, so that the bound drug
pharmacologically inert.
Plasma–protein bound drug have longer
elimination half lives compare to the free drug.
Protein bound drug doesn’t cross BBB and
placental barrier.
63
64. REFERENCES
Brahmankar D.M. ,Jaiswal S.B. ,Biopharamaceutics
and pharmacokinetics ;A Treatise ,2nd ed. ,Vallabh
Prakashan ,p. 116-136.
International Journal of Proteomics-J. L.Hartman IV,
B. Garvik, and L.Hartwell, “Cell biology: principlesfor
the buffering of genetic variation,” Science, vol. 291,
no.5506, pp. 1001–1004, 2001.
H. Berman, K. Henrick, H. Nakamura, and J. L.
Markley, “The worldwide Protein Data Bank
(wwPDB): ensuring a single, uniform archive of PDB
data,” Nucleic Acids Research, vol. 35, no. 1, pp.
D301–D303, 2007
64
65. Paradkar A. ,Bakliwal S. ,Biopharamaceutics
and pharmacokinetics ,2nd ed. , Nirali
prakashan , p. 3.12-3.15.
Tripati K.D. Essential of Medical
pharmacology ,6th ed. , Jaypee brothers
Medical publisher Ltd. ,p. 20-23.
65