Complexation and Protein Binding

COMPLEXATION AND
PROTEIN BINDING
1. Introduction
2. Types of Complexes
3. Metal-Ion Coordinate Complexes
4. Cyclodextrin Complexes
5. Ion-Exchange Resins
6. Protein-Ligand Interaction
7. Plasma Protein Binding

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Learning Objectives
• Understand the significance of complexation in pharmaceutical products

• Describe the mechanism of complexation

• Appreciate the clinical importance of complexation

INTRODUCTION

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 COMPLEXATION

Complexation. Characterize the covalent or non covalent interactions between two or

more compounds that are capable of independent existence

The ligand is a molecule that interacts with another molecule, the substrate to form a
complex

Drug molecules can form complexes with other small molecules or with macromolecules
such as proteins.

Ligand: molecules or ions surrounding the central metal atom (usually small organic
molecules)

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 COMPLEXATION
EXAMPLES OF DRUG COMPLEXES

Theophylline complexation with

↑ aqueous solubility
ethylenediamine to form aminophylline
Inclusion complexes of labile drugs with
↑ stability
cyclodextrins
Optimization of delivery
Ion-exchange resins
systems

Affect drug distribution Protein binding

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 COMPLEXATION
EXAMPLES OF DRUG COMPLEXES

↓ aqueous solubility Tetracycline complex with counterions

Coadministration of some drugs* with
↓ absorption
antacids in GI tract
Complexation of certain drugs with
↑ excretion
hydrophilic compound
Alter pharmacologic
Inhibit interactions with receptors
activity

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 COMPLEXATION
FORCES
Complexation of a ligand with a substrate molecule can occur as a result of coordinate
covalent bonding or one or more of the following noncovalent interactions
1. van der Waals forces
2. Dipolar forces
3. Electrostatic forces
4. Hydrogen bonding
5. Charge transfer
6. Hydrophobic interaction

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TYPES OF
COMPLEXES

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 TYPES OF COMPLEXES

Classified depending on the type of interaction involved in complexations, ligand-

substrate interactions are classified as:

1. Coordination Complexes
2. Molecular Complexes

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 TYPES OF COMPLEXES
COORDINATION COMPLEXES
Consists of a complex ion, typically a transition metal ion, with one or more attached
ligands or counterions, which are either anions or cations that will result in an electrically
neutral complex

Lewis base-acid reaction: interaction between the metal ion and the ligand
• Ligand [base]: donates a pair of electrons
• Metal ion [acid]: receives a pair of electrons from the ligand
This reaction forms a covalent bond.

𝐴𝑔+ + 2 : 𝑁𝐻3 → [𝐴𝑔(𝑁𝐻3 )2 ]+

Ag+ is the central metal ion interacting with NH3 to form the silver-ammonia coordinate
complex
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 TYPES OF COMPLEXES
COORDINATION COMPLEXES
Coordination Number. Maximum number of a toms or groups that can combine in the
coordination sphere with the central metal atom
K3[Fe(CN)6] CN = 6
Six cyano groups are complexed with the central iron atom.

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 TYPES OF COMPLEXES
COORDINATION COMPLEXES
What are the central metal ion, the ligand, and the coordination number in the
[Cu(H2O)6]2+ complex ion?

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 TYPES OF COMPLEXES
COORDINATION COMPLEXES
What are the central metal ion, the ligand, and the coordination number in the
[Cu(H2O)6]2+ complex ion?

Central metal ion: Cu2+

Ligands: water molecules (H2O)
Since there are six water molecules bound to Cu2+, the coordination number is 6.

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 LIGANDS
MONODENTATE LIGANDS
“one tooth”
Ligand binding to the center through only one atom

EXAMPLES: Cl- (chloro), H2O (aqua), OH- (hydroxo),

NH4+ (ammine)

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 TYPES OF COMPLEXES
BIDENTATE LIGANDS
Have two donor atoms which allow them to bind to a central metal atom or ion at two
points

EXAMPLES: H2NCH2CH2NH2/ethylenediamine (en), C2O42-/oxalate (ox)

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 TYPES OF COMPLEXES
POLYDENTATE LIGANDS
Range in the number of atoms used to bond to a central metal
atom or ion

EXAMPLE: ethylenediaminetetraacetic acid/EDTA (has six donor

atoms with electron pairs that can be used to bond to a central
metal atom or ion

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 TYPES OF COMPLEXES
CHELATION
A process in which a polydentate ligand bonds to a metal ion, forming a ring

The complex produced is called a chelate (claw) , and the polydentate ligand is referred to
as a chelating agent

THE CHELATE EFFECT. Enhanced affinity of chelating ligands for a metal ion compared to
the affinity of a collection of similar nonchelating (monodentate) ligands for the same
metal

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 TYPES OF COMPLEXES
CHELATION
VALINOMYCIN
Natural antibiotic valinomycin (2,2’-bipyridine) selectively transports K+ ions across
bacterial cell membranes, killing the bacterium by dissipating its membrane potential

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 TYPES OF COMPLEXES
MOLECULAR COMPLEXES
Formed as a result of noncovalent interactions between ligand and substrate

The interactions can occur through oppositely charged ions (electrostatic forces), van der
Waals forces, charge transfer, hydrogen bonding, or hydrophobic effects

These complexes can occur between:

1. Small molecule and a small molecule (ethylenediamine + theophylline =
aminophylline)
2. Small molecule and a large molecule (protein binding of drugs, combination of iodine
and polyvinylpyrrolidone (Povidone) to form Povidone-Iodine)
3. Ion pairs (ion-exchange resins)
4. Self-association to form aggregates (surfactant micelles)
5. Inclusion complexes (cyclodextrin complexes)
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METAL-ION
COORDINATE
COMPLEXES

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 METAL-ION COORDINATE COMPLEXES

1. Iron complexes
2. Platinum complexes
3. Copper and cobalt complexes
4. Zinc complexes
5. Toxic heavy metal complexes

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 METAL-ION COORDINATE COMPLEXES
IRON COMPLEXES
The ability of metal ions to coordinate with and then release ligands in some processes
and to oxidize and reduce them in other processes makes them ideal for use in biological
systems.

Iron: the most common metal used in the body

a. Myoglobin
b. Hemoglobin

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 METAL-ION COORDINATE COMPLEXES
IRON COMPLEXES
MYOGLOBIN
Monomeric heme protein that is found mainly in
muscle tissue
Function: intracellular storage site for oxygen

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 METAL-ION COORDINATE COMPLEXES
IRON COMPLEXES
HEMOGLOBIN
Tetrameric heme protein [α(2):β(2)]
Found in erythrocytes
FUNCTION: binding oxygen in the lungs and transporting the bound oxygen through-out
the body where it is used in aerobic metabolic pathways

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 METAL-ION COORDINATE COMPLEXES
IRON COMPLEXES
MYOGLOBIN and HEMOGLOBIN
Both have heme
Carry oxygen
Carbon monoxide (CO) also binds coordinately to heme iron atoms in a manner similar to
that oxygen but the binding of CO to heme is 200 times stronger than that of oxygen
- Preferential binding of CO to heme iron is largely responsible for the asphyxiation that
results from carbon monoxide poisoning

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 METAL-ION COORDINATE COMPLEXES
IRON COMPLEXES
DEFEROXAMINE
Treatment for acute iron overdose
Chelation of ferric ion with deferoxamine results in a strong coordinate water-soluble
ferrioxamine complex that can be excreted through the kidneys

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 METAL-ION COORDINATE COMPLEXES
PLATINUM COMPLEXES
CARBOPLATIN and CISPLATIN
Platinum (II) complexes proven to be useful agents in the treatment of cancer
MOA: form identical types of adducts with DNA and have similar activities against ovarian
and lung tumors

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 METAL-ION COORDINATE COMPLEXES
COPPER COMPLEXES
Copper ion is present in a variety of important proteins and
enzyme, including hemocyanin, superoxide dismutase and
cytochrome oxidase

Typical state: Cu(I)

Complexes: colorless, tetrahedral complexes
EXAMPLE: hemocyanin (oxygen binding protein, colorless Cu(I)
complex that turns blue when bound to oxygen, indicating
transition to the Cu(II) state)

Cobalt ion is found in vitamin B12 (cyanocobalamin): crucial to the

normal function of the brain and the nervous system

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 METAL-ION COORDINATE COMPLEXES
ZINC COMPLEXES
Zinc is an important metal ion that is present in many proteins and confers structure and
stability.

The only metal ion found in crystalline insulin

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 METAL-ION COORDINATE COMPLEXES
TOXIC HEAVY METAL COMPLEXES
Lead and mercury toxicity is commonly treated by the administration of chelating agents
such as dicalcium salts of EDTA and 2,3-dimercaptopropanol (BAL/British Anti-Lewisite)

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CYCLODEXTRIN
COMPLEXES

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 CYCLODEXTRIN COMPLEXES
Cyclodextrins were isolated in 1891 by Villiers as degradation products of starch and were
characterized as cyclic oligosaccharides in 1904 by Schardinger.

Cyclodextrins are donut-shaped molecules of D-glucopyranose

CYCLODEXTRIN TYPE NO. OF GLUCOSE RESIDUES
Alpha 6
Beta 7
Gamma 8

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 CYCLODEXTRIN COMPLEXES
Improvements in Properties of Selected Drug Compounds by Complexation with Cyclodextrins
1. Enhanced aqueous solubility
2. Improved stability
3. Enhanced absorption and bioavailability
4. Change from liquid to solid
5. Decreased volatility
6. Improved taste and odor
7. Decreased stomach irritation
8. Inhibit red blood cell lysis
9. Prevention of incompatibilities

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 ION-EXCHANGE RESINS
Ion-exchange. A method of complexation that is based on electrostatic interactions between the
surface-bound ions on a solid particle (or resin) and the oppositely charged ions in aqueous solution

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PROTEIN LIGAND
INTERACTION

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REVIEW: PROTEIN STRUCTURE
Levels of Protein Organization
1. Primary: [Assembly] amino acid sequence
2. Secondary: [Folding] sequence linked by H-
bonds
3. Tertiary: [Packing] attraction between alpha
helices and pleated sheets
4. Quaternary: [Interaction] protein with more
than one amino acid chain

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 PROTEIN LIGAND INTERACTION
Protein-ligand interaction is important in:
• Drug binding to its receptor for pharmacologic activity
• Enzyme-substrate interaction in catalysis
• Antibody-antigen recognition
• Interaction between drugs and proteins in plasma that affects the distribution profile of a drug in
the body

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 PROTEIN LIGAND INTERACTION
𝑃 + 𝐿 ↔ 𝑃𝐿
Many interactions (not all) between protein and low-molecular-weight compounds occur in a
reversible manner.
[P]: molar concentration of the protein
[L]: molar concentration of the ligand (or drug)
[PL]: molar concentration of the protein-ligand complex

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 PROTEIN LIGAND INTERACTION
𝐾𝑎 : association constant or the measure of the
affinity between the protein and the ligand

[𝑃𝐿]
𝐾𝑎 =
𝑃 [𝐿]

Unit: 1/M or liters per mole

𝐾𝑑 : dissociation constant is the reciprocal of the

association constant

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PLASMA PROTEIN
BINDING

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 PROTEIN LIGAND INTERACTION
Upon systemic administration of therapeutic agents, those agents encounter a massive
pool of plasma proteins, such as albumin and α1-acid glycoproteins, that can interact with
drug molecules
PLASMA PROTEINS
Different functions of proteins in plasma
1. maintenance of osmotic pressure between intracellular and extracellular fluids
(albumin)
2. coagulation of blood (clotting factors, thrombin, fibrinogen)
3. immune reaction (antibodies)
4. transport of endogenous and exogenous compounds
5. function as enzymes and hormones

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 PROTEIN LIGAND INTERACTION
IMPORTANT PROTEINS IN THE PLASMA IN DRUG BINDING
1. Albumin
2. α1-acid glycoprotein

MOST ABUNDANT AND MOST IMPORTANT: ALBUMIN

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 ALBUMIN
• acidic protein (isoelectric point of 4.8) with a
molecular weight of 69,000
• very soluble in water
• concentration of 30 to 50 mg/mL in normal human
plasma
• a very stable protein with 17 disulfide bonds in the
tertiary structure
• elimination half-life of 17-18 days
• primary function: serve as an osmotic agent for the
regulation of pressure differences between
intracellular and extracellular fluids
• additional function: important transport protein in
the plasma for both endogenous and exogenous
compounds

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 ALPHA1-ACID GLYCOPROTEIN
• globular protein with a molecular weight of 44,000 Daltons
• plasma concentration is very low
• preferentially binds with BASIC DRUGS (ex. imipramine, propranolol, lidocaine)

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 OTHER PROTEINS
LIPOPROTEINS
• high molecular weight complexes of proteins with lipids
• Classified according to their densities and separation in an ultracentrifuge (VLDL, LDL,
HDL)
• Responsible for the binding and transport of plasma lipids
• Also bind with drugs when albumin-binding sites are saturated

IMMUNOGLOBULINS

TRANSFERRIN

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 SIGNIFICANCE OF PLASMA PROTEIN BINDING
Highly protein bound drugs tend to remain mainly in the
systemic circulation as opposed to binding with adipose tissue
and have a relatively lower volume of distribution
therefore: longer biological half-life

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 SIGNIFICANCE OF PLASMA PROTEIN BINDING
FACTORS
Affinity and Capacity

AFFINITY
• association constant, measure of strength of the interaction between the protein and
the drug molecule

Since drug binding is reversible, a drug with higher affinity (or larger Ka) can displace
another drug from the same binding site

Displacement of bound drug can lead to significant toxicity as the free drug interacts with
active site to produce a pharmacologic response

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 SIGNIFICANCE OF PLASMA PROTEIN BINDING
WARFARIN
• Extensively bound to plasma proteins but with relatively weak affinity
• NSAIDs have higher affinity, therefore NSAIDs can displace warfarin from its binding site
and cause serious hemorrhagic complications

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 SIGNIFICANCE OF PLASMA PROTEIN BINDING
• Another factor is the changes in concentration of available proteins caused by disease,
age, malnutrition, trauma.

==> If the protein concentration decreases, the relative increase in free drug
concentration will cause significant toxicity at the same dose.

END CLASS. 50