BIOLOGICAL TARGETS FOR SMALL

MOLECULE DRUGS AND THEIR MODE

OF ACTION

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 CELL MEMBRANE (PLASMA MEMBRANE)
outside the cell there are many protiens
ciruclating enzyme are outside the cell and most enzymes are in the cell

Phospholipid
Bilayer

• Outside of cell bounded by cell membrane

• Phospholipid bilayer with embedded proteins
• Proteins may be ion channels, transporters or receptors
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 Of the 4 major classes of macromolecules, drug
targets are usually proteins located in the cell
membrane or in the cytoplasm or in the nucleus

Proteins
Receptors – cell membrane – signal transductions
Enzymes – generally intracellular - biotransformations

Lipids
Cell membrane lipids

Nucleic acids
DNA – especially older anticancer agents
RNA – especially ribosomes antibiotics- target the bacterial ribosomes

Carbohydrates
Cell surface carbohydrates
Antigens and antibodies ©1
 DRUG TARGETS

• Drug targets are large molecules - macromolecules

• Drugs are generally much smaller than their targets
• Drugs interact with their targets by binding to specific binding sites
• Binding sites are typically pockets on the surface of macromolecules –
“active site” or “binding site” of enzymes or receptors
• Binding interactions involve non-covalent intermolecular bonds
• Most drugs are in equilibrium between being bound and in solution

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 DRUGS BIND IN THE ACTIVE OR BINDING SITE
Active or Binding
site

Drug Binding
groups

Intermolecular
bonds

Binding site

Binding Drug
site
diff points of interactions
Drug

Macromolecular target Macromolecular target

Unbound drug Bound drug

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 INTERMOLECULAR BONDING FORCES

Electrostatic or ionic bond

• Strongest of the intermolecular bonds (20-40 kJ mol-1)
• Takes place between groups of opposite charge
• Generally occurs between a protonated amine and a deprotonated acid
• At pH7 amines will be protonated (pKa 10) and acids deprotonated (pKa 4)
• Ionic bonds are the most important initial interactions as a drug approaches the
binding site
• Note the acid can be on the drug, amine in the protein OR vice-versa

O
Drug Drug NH3 O
O H3N Target Target
O

both ways
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 Higher PKa= More basic Lower PKA more Acidic

Which amino acids provide the acidic and basic side chains?

pKa 10.4
pKa 4.1 Protonated at
Deprotonated at Asp (D) Physiological pH
Physiological pH Lys (K)

pKa ~12
pKa 4.5 Protonated at
Deprotonated at Arg (R) Physiological pH
Physiological pH Glu (E) H O
+
N OH
pKa 6.3
NH2 His (H) Deprotonated and
N
protonated forms at
H
physiological pH
(Histidine shown in protonated form)

• Two acidic and three basic side chains on amino acids

• Acidic side chains have similar pKa values, generally deprotonated
• Three basic side chains vary in pKa
• Arginine always protonated; lysine generally protonated;
histidine exists in both neutral and protonated forms
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 Formulated as
Hydrochloride Salt

Side chain is
ionized and
Rimantidine
negatively charged
(racemic mixture)
The protonated amine is interacting with the deprotonated acid
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In this example the protonated amine is on the drug

F F

N
N
S N

+
H3N CH3

reaction is here

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In this example the deprotonated acid is on the drug

1
R

+
Side chain of Lysine 27
N
H H
– H –
O O

O O Note in this example two

S N S carboxylates are interacting
N N
with a single protonated amine

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 INTERMOLECULAR BONDING FORCES
Hydrogen bonds

• Vary in strength
• Weaker than electrostatic interactions
• A hydrogen bond takes place between an electron deficient hydrogen
and an electron rich heteroatom (N or O)
• The electron deficient hydrogen is usually attached to a heteroatom (O
or N)
• The electron deficient hydrogen is called a hydrogen bond donor
• The electron rich heteroatom is called a hydrogen bond acceptor
• Again can be either way around, like the ionic bond

- + -
- + - Drug Y H X
X H Y Target Target
Drug
HBD HBA HBA HBD

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 Hydrogen bonds
• Examples of strong hydrogen bond acceptors
- carboxylate ion, phosphate ion negatively charged

• Examples of moderate hydrogen bond acceptors

- amide oxygen, ketone, ester, ether, alcohol

• Examples of poor hydrogen bond acceptors

- fluorine, aromatic ring, amide nitrogen, aromatic amine

• Example of good hydrogen bond donors

- Protonated amine, amide NH

Water molecules (structural not solvent) can often be involved in H-bonding

Often a water molecule can link different functional groups separated by distance

one h+ acceptor
2 H+ donors

Oxygen is an H-bond acceptor

Hydrogen is an H-bond donor

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 INTERMOLECULAR BONDING FORCES
Hydrogen bonds

• The interaction involves orbital overlap and is directional

• Optimum orientation is where the X-H bond points directly to the lone pair on
Y such that the angle between X, H and Y is 180o

X H Y X H Y
Hybridised 1s Hybridised
orbital orbital orbital
HBD HBA

• The hydrogen bond distance is around 0.3 nM between the heavy atoms
• Hydrogen bonding is crucial in DNA base pairing
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Amide backbones of proteins often acts as H-bond donors/acceptors

CH3

N
4 3 H 2
R R N R
NH
H
N O
HN

1 NH
O R HO

Glycine 351 Serine 516

H-bond donor H-bond acceptor

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Often a water molecule can participate in an extended H-bond network

2 H-bonds with amide backbone

O
NH
2 NH
R 3
N R
O

….
H

….
H O

Cl N N H3C
5
R
H
NH
1 Cl O
R
NH
H H
CH3

H3C
P
O ….H O ….H N
+
R
4

Structural water engaging in 3 H-bonds

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 Is this very strong interaction a salt bridge or a double H-bond?

1
R
Single +ve charge
delocalised across 3 N’s
+
H N
H

H
H N

H O Single -ve charge

delocalised across 2 O’s
O

2
R
S

• Doesn’t matter, probably better called a salt bridge (electrostatic interaction)

but the H atoms point into the O atoms

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 3. Hydrophobic (van der Waals) interactions

Can occur between…..

- alkyl groups and alkyl groups
- alkyl groups and aryl groups
- aryl groups and aryl groups
- aryl groups and heteroaryl groups (drug)
- or even aryl groups and amide groups!

• Ionic interactions and H-bonds are easy to define in eg X-ray structures

bigger the SA
• Hydrophobic interactions are weak, non-specific and surface area dependent bigger the interaction
• Van der Waals theory, induced dipole theory, desolvation theory ……. ? All very
complicated ……
• “Non-polar stuff likes to bind to other non-polar stuff”
• Drug must be physically adjacent to the binding region for interactions to occur
• The contribution of van der Waals/hydrophobic interactions can be crucial to drug binding,
often a bigger contribution to binding than ionic interactions depending of SA

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The aryl-aryl interaction involves orbital overlap (π-stacking)

Initially thought the ideal geometry was symmetrical

Now understood the ideal geometry is slightly offset

this is the optimum geometery for it

slightly offset

See below for a discussion

J. Am. Chem. Soc. 1990, 112, 14,

[Link]

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π-stacking can help stabilize the tertiary structure of proteins

Amino acids involved in π-stacking

π-stacking between the indole of tryptophan

and the phenyl group of phenyl alanine

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 π-stacking between a protein and a drug in the binding site

• The hydroxyphenyl of the tyrosine is π-stacking against the quinolone

moiety of the drug

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 Sometimes nearly all of the binding energy can come from hydrophobic interactions

–
O

CH3

• X-ray of long chain fatty acid in an enzyme shows the extent of the lipophilic interactions
• Three H-bonds to the carboxylate – the remainder of the binding is due to lipophilic interactions

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 Lipophilic side chains often form a lipophilic pocket to bind part of a drug

Valine
Leucine
lipophillic bit

Alanine

Lysine

• Note that the –(CH2)4- of the –(CH2)4NH3+ side chain of lysine

is contributing to the lipophilic pocket

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 Binding of a drug to a target results in a loss of entropy

The Gibbs free energy change of a binding event, ∆G = ∆H - T∆S

Delta S goes is -ve so makes
-Td(s) Positive
making delta G less negative
doesnt favour binding
• The free energy of binding (∆G, more negative, more binding) = the enthalpy of binding
(∆H, the more negative the more binding, resulting from intermolecular interactions like
H-bonds, lipophilic interactions etc) - T∆S (entropy)

• On binding of a drug to a protein, entropy is lost as the system is now less disordered,
so ∆S is negative

• The term -T∆S is therefore positive, so has the effect of making ∆G less negative

Entropy loss works against binding

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 Entropy penalties

ic50 conc of drug required to reduce activity of enzyme by 50%

lower IC50= more active

The extra ring is in solvent

• The lower the IC50, the greater the activity
• Compound (3) is constrained to the active conformation
• Smaller loss of entropy on binding compared to (2), more activity
in number 3
when its bound it unable to move around- removes the entropy lost
greater binding lower Ic50 mroe active ©1
 THE FOUR BIG ASPECTS OF DRUG BINDING

• IONIC BOND – between deprotonated –ve charged acid and

protonated +ve amine
• HYDROGEN BOND – between H-bond donor and H-bond
acceptor
• LIPOPHILIC INTERACTION – between lipophilic regions of
drug and protein target
• ENTROPY – reduce conformational freedom in drug for better
binding to the protein

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 PROTEINS (ENZYMES AND
RECEPTORS) AS DRUG TARGETS:
ENZYMES

HIV-1 protease plus inhibitor

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 Structure and function of enzymes
• Enzymes are proteins act as the body’s catalysts
• Speed up time for reaction to reach equilibrium
• Lower the activation energy of a reaction

Example:

LDH = Lactate dehydrogenase (enzyme)

NADH = Nicotinamide adenosine dinucleotide (reducing agent & cofactor)
Pyruvic acid = Substrate

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How do enzymes appear to make reactions go only one way
in biochemistry if they just make equilibrium happen faster?

• In principle A to E are all in equilibrium

• However if E is removed from the equilibrium ……
- insoluble macromolecule
can be removed if its this
- excreted from the cell
• Then the process will become linear as long as A is supplied

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 Enzymes are catalysts

Energy Energy
Transition state New
transition
Act. state
energy Act.
energy
Starting Starting
∆G ∆G
material material

Product Product

WITHOUT ENZYME WITH ENZYME

• Enzymes are proteins that catalyse biochemical reactions

• They speed up chemical reactions (106 – 1014 times faster than uncatalyzed!)
• They do this by lowering the activation energy
• They do NOT affect the position of equilibrium (∆G is the same in both cases)

KINETIC NOT THERMODYNAMIC EFFECT

its a kinetic effect
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 Structure and function of enzymes
Methods of enzyme catalysis

• Provides a place for the chemical reaction (the active site)

• Provides hydrophobic and polar binding opportunitiesfor substrates
• Bring reactants together in correct alignment for reaction
• May weaken bonds in the reactants
• Provide acid / base catalysis

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 The active site
• Hollow or cleft on the enzyme surface which generally
has both polar/non polar regions
• Accepts reactants (substrates and cofactors)
• Contains amino acids with appropriate side chains that ….
- bind reactants (substrates and cofactors)
- catalyse the reaction
Active site
Active site

ENZYME

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 Substrate binding
Bonding forces

• Ionic
• H-bonding
• van der Waals

Example: Binding of pyruvic acid in LDH

O O
H-Bond H
C O
H3C C O
Ionic
C O bond
O H3C C
H3N
O
Possible interactions vdw-interactions

H-Bond
van der Waals
Ionic ©1
 Catalysis mechanisms
Acid/base catalysis
Histidine pKa 7 +H
50% ionised NH NH
-H N
at physiological pH N
H
Non-ionised Ionised
Acts as a basic catalyst Acts as an acid catalyst
(proton 'sink') (proton source)

Nucleophilic residues

H H
H3N CO2 H3N CO2

L-Serine L-Cysteine
OH SH
very reactve

Key residues in eg serine and cysteine proteases

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 alcohol is the side chain of serine

aspartate pulls H+ off the histadine

histadine histadine is pulling a H+ from the serine
serine more nucloephilic able to attack the nitrogen
aspartate

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Try and work through this mechanism of a serine peptidase
cleaving a peptide bond
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In many diseases an overactive enzyme is the cause.
Many effective drugs are enzyme inhibitors.

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 sit in acitve site and do nothing

In many diseases an overactive enzyme is the cause.

Many effective drugs are enzyme inhibitors.

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 Example of Competitive (reversible) inhibitor

• RNA-dependent RNA-polymerase is a “viral” enzyme

• It makes more viral RNA in the infected human cell
• This reversible inhibitor binds in the active site and blocks it
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Mechanism of hydrolytic cleavage of a peptide bond
by a protease enzyme using water
proton shift

Gem-diol intermediate

• The carbon of the amide is sp2 hybridized, planar geometry

• The carbon of the gem-diol is sp3 hybridized, tetrahedral geometry
• Groups bind to and stabilize the gem-diol LOWER the activation energy

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Medicinal Chemistry - Using mechanistic understanding to
design High Affinity Inhibitors that save lives

Note in this protease enzyme the gem-diol is

stabilized by an interaction with an aspartate
aspartate comes and stabilises the intermediate

In theory an sp3 alcohol of this type should bind with

higher affinity to the substrate
However this is an aminal – not a stable structure

Changing the N to C stabilizes the structure and is the

basis of many life-saving drugs of the HIV Protease
Inhibitors class
chemically stable ©1
 Example of a HIV-Protease Inhibitor

Position of amide carbonyl

in the peptide substrate Interactions of hydroxy group
with aspartates

• Examples include saquinavir, nelfinavir, atazanavir, indinavir etc

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 Allostery1
Allostery is where spatially separate binding sites in a protein
are functionally coupled so binding at one site influences binding
at the other.

• Binding at one site (allosteric site) changes the conformation of the

protein so that binding at the other site (effector site) is decreased
or increased.
• Biological function is to achieve greater control over
biochemical pathways

1Monod, J.; Jacob, F. Telenomic Mechanisms

in CellularMetabolism,
Cold Spring Harbor [Link]. Biol.
1961,26, 389−401
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 Types of Allostery in Enzymes

Ligand site
active site

Allosteric site

1. Allosteric inhibitors – reduce binding of enzyme substrate

2. Allosteric promoters – enhance binding of enzyme substrate

• Historically, allosteric ligands have been found by accident

• Structural biology/computational chemistry is enabling rational
discovery of new sites
(see J. Med. Chem 2019, 62, 6405) ©1
 Example of a non competitive allosteric inhibitor

NSC13345

• Cathepsin K is a cysteine protease overactive in arthritis

• The active site is in blue and the allosteric site is in red
• NSC13345 binds to the red region and the enzyme is inhibited
chnages the confirmation of the enzyme ©1
 PROTEINS (ENZYMES AND
RECEPTORS) AS DRUG TARGETS:
RECEPTORS

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Receptors and agonists – intercellular communication

• Proteins acting as a cell’s ‘letter boxes’ (message reception)

• Located mostly in the cell membrane, but some intracellular

• Receive “messages” from “chemical messengers” called

AGONISTS coming from other cells (hormones and
neurotransmitters)

• Transmit a message into the cell leading to a cellular effect

• Different receptors specific for different chemical messengers

• Each cell has a range of receptors in the cell membrane

making it responsive to different chemical messengers
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Chemical messengers that cause a biological response are called
………… “Agonists”

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Chemical messengers can be very small molecules through to small
proteins

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Receptors function as a kind of “on/off switch”

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 Structure and function of receptors
• Receptors contain a binding site (analogous to active site in an
enzyme) that the chemical messenger binds to
• Binding involves non-covalent intermolecular bonds
• Binding results in an “induced fit” of the receptor protein and a
conformational change in the receptor
• Conformational change in the receptor shape can result in a
conformational change or dissociation of a non-covalently
attached enzyme, resulting in enzyme activation
• Domino effect is known as Signal Transduction, leading to a
chemical signal being received inside the cell from the enzyme
• Chemical messenger does not enter the cell. It departs the
receptor unchanged and is not permanently bound
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 receptor is in contact non covalently to
an ezyme in the cell
agonist binds to the receptor and changes the
shape of the receptor and enzyme

opens active site

This is the commonest version – many variations are found

in nature
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 What is the difference between enzyme/substrate and
receptor/agonist interactions?

The substrate is chemically changed by the enzyme, giving the product

- That is the job of the enzyme

The agonist is NOT chemically changed by the receptor, released unchanged

- The activated enzyme in the cell is performing all the chemical reactions

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 Messenger binding is non-covalent
vdw
interaction

H-bond
Binding site
H ionic
O Phe
bond
Ser
CO2

Asp

Receptor

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 Holding on and Letting Go

• The agonist is bound to the receptor for a short period of time

• This is known as the residence time
• The length of the residence time depends on how good the binding is
better binding the longer
• During the residence time, the activated enzyme might catalyse
thousands of reactions (signal transduction)
• Eventually the agonist is “let go” by the receptor and diffuses away
• The enzyme reverts to the inactive conformation ©1
 Properties of “agonists”
• Agonist binds reversibly to the binding site
• Similar intermolecular bonds formed as to natural messenger
• Induced fit alters the shape of the receptor in the same way as
the normal messenger
• Receptor is activated
• Agonists are often similar in structure to the natural
messenger

Agonist Agonist Agonist

Induced fit

RE RE
R

Signal transduction
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 Example of an Agonist – diabetes drug

Natural agonist

Synthetic drug agonist

• Activation of the receptor (GPR-40) releases insulin

• Natural agonists are long chain fatty acids
• Synthetic agonists are used to stimulate insulin secretion
• Agonist drugs can lead to overstimulation and side effects
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What do we mean by agonist and antagonist?

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 How to manipulate these processes to treat disease

• A compound that turns a receptor on is called an agonist

• The natural chemical messengers are agonists
• Unnatural chemicals that have the same effect are also agonists
• Unnatural chemicals that bind but do not turn on the receptor
are called antagonists
• Antagonists prevent agonists binding and prevent any response
• Many drugs are antagonists – they “tone down” an
overactive physiological process

How do antagonists achieve this effect at the molecular level?

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Antagonists bind to the receptor but do not induce the
correct conformational change
prevent binding 5-HT,
natural agonist

Risperidone Clozapine Ondansetron Spiperone

• The above drugs are all 5-HT antagonists

• Note the structural differences between themselves and to 5-HT
• They ALL bind to the 5-HT receptor WITHOUT inducing the
conformational change required for activation
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 Noncompetitive allosteric antagonists

Allosteric antagonist
Binding site for agonists
binding at a different site
and antagonists
changes the shape of the receptor
the agonist can not bind at its normal site anymore

RORγt nuclear receptor

Allosteric antagonists are able to bind at a different site on the

receptor, remote from the usual messenger binding site, and alter
the conformation of the receptor such that the agonist cannot bind
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 Example of a noncompetitive allosteric antagonist

SDF1

CXCR4 Receptor GSK812397

• GSK812397 is a noncompetitive antagonist of the CXCR4 receptor
• The natural agonist is a peptide called SDF1
• The HIV virus binds to the agonist site as part of the entry process
• GSK812397 binds to an allosteric site and prevents
prevents the HIV binding
binding to the agonist site
• GSK812397 protects human cells against HIV infection © 1
 Summary
• Drug targets are usually proteins (but can be RNA/DNA)
• Binding of a molecule to a protein involves non-covalent interactions
- hydrophobic interactions, H-bonds, ionic interactions
- conformation constraint increase binding through entropy effects
• Enzymes – the substrate molecule undergoes reaction
and the product is released
- an inhibitor molecule generally remains unchanged
• Receptors – no chemical reaction of the ligand
- Natural messenger is an agonist (turns receptor on)
- Unnatural agonists are sometimes useful as drugs
- Antagonists bind to the receptor but do not activate
- Many drugs are antagonists of receptors
• Allosteric drugs act remotely from the normal site on the protein
- and can activate or inhibit biological processes

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