MOLECULAR DRUG TARGETS

LEARNING OUTCOMES

At the end of this session student shall be able to:

• List different types of druggable targets

• Describe forces involved in drug-receptor interactions

• Describe theories of drug receptor interaction

• Discuss the methods used to evaluate drug-receptor interaction

• Discuss the process of drug signal transduction

MOLECULAR DRUG TARGETS

• Molecular drug targets are cellular proteins that selectively bind the drug and initiate the drug’s effect

• A drug target has two characteristics:

– Recognition capacity

– Amplification capacity: ability to initiate a response

Drug targets include: Enzymes, Receptors, Ion channels and Transporters

There are about 8,000 targets in human proteins

Existing drugs interact only with 324 targets (266 human and 58 pathogens)

TERMINOLOGIES

Ligand: a molecule (drug) that binds to a target.

Pharmacophore: a fragment in the drug that enables it to bind to the receptor

Binding domain: a region on a target where a ligand binds

Signal transduction: the mechanism by which a message carried by the ligand is translated into a tissue response

TYPES OF LIGANDS Target Types of ligands Description

Enzyme

activator bind to enzymes and increase their enzymatic activity

competitive inhibitors Bind at the active site and inhibit enzyme activity

non-competitive inhibitors Bind at allosteric site and inhibit enzyme activity

Ion channel

openers bind to ion channels and allosterically open the ion channel

inhibitors (blockers) bind to ion channels and physically block the pore or cause an allosteric change that closes the pore

Transporter inhibitors bind to transporters and cause allosteric changes that

prevent proper functioning of the transporters

Receptor

agonist attach to binding site and give a similar response to that of the endogenous ligand

antagonist prevent the binding of the endogenous ligand and thus abolish the response

inverse agonists prevent the binding of agonists , elicit a response inverse to that of agonists

• Equilibrium between a drug, a receptor and a drug-receptor complex is:

• The biological activity of a drug depends on the stability of the D-R complex i.e. its affinity to the receptor

• The D-R complex can be stable if:

– the interaction between the drug and receptor is stronger than interaction between drug and receptor with solvent molecules.

– the enthalpy of interaction can compensate the entropic loss for both receptor and the drug.

• Thus, the Driving force for drug-receptor interaction is low energy state of drug-receptor complex

DRUG-RECEPTOR INTERACTIONS

The stability is measured as: 𝐾𝑑 =𝐷 [𝑅]

[𝐷−𝑅 𝑐𝑜𝑚𝑝𝑙𝑒𝑥]

the smaller the larger the more stable the cmplex

FORCES INVOLVED IN D-R COMPLEX

• Bonding between a drug and a receptor occurs if there is total decrease in free energy (i.e. G is negative)

G° = -RTlnKeq Keq = binding equilibrium constant

• Small change in energy have a major effect on drug-receptor equilibrium – E.g. decrease in G° of ~ 5.5 kcal/mol changes

binding equilibrium from 1% in drug-receptor complex to 99% in drug-receptor complex

FORCES INVOLVED IN D-R COMPLEX

Interactions in drug-receptor complex may involve covalent bond or non-covalent forces such as :

Hydrophobic interactions

Ion-dipole and dipole-dipole

Hydrogen bonding

Van-der-waals interactions

Charge-transfer

Ionic bonds

Covalent interactions

It is an irreversible link between the drug and the receptor

It is the strongest bond G from -40 to -110 kcal/mol

but is seldom found in drug action except with enzyme and DNA

Ionic interactions • Ionic bonding: are usually reversible and

their strength depends on the distance btw the two ions (G° ≈ -5 kcal/mol)

Arg, Lys and His can provide cations

Asp and Glu can provide anions

O

NH

O

O

NH

NH2

H2Npivagabine

Ion-dipole and dipole-dipole interactions

• dipole–dipole interaction: a dipole in a drug is attracted by a dipole in the receptor

N

N

N

N

O

H3 C

CH3

C

N

O O-

HO

ion-dipole

dipole-dipole

zaleplon

• ion–dipole interaction: a dipole in a drug is attracted by an ion in the receptor

G° ≈ -1 to -7 kcal/mol

Hydrogen bonds • Are a type of dipole-dipole interaction

between H on X-H and O, N or F

– (X is an electronegative atom)

G° ≈ -3 to -5 kcal/mol

intramolecular

intermolecular

O

O

O

H

H :OH

Charge transfer complexes Are forces btw an electron donor and an electron acceptor groups

– donors are usually π-electron-rich species

– Acceptors contain electron deficient π-orbitals

G° ≈ -1 to -7 kcal/mol

Chlorothalonil fungicide

+

CN

CN

Cl

Cl

Cl

Cl

OHacceptor donor

Hydrophobic interactions It occurs when non-polar sections of molecules are forced

together by a lack of water solubility

increased entropy of water decrease the free energy and stabilize the drug-receptor complex.

G° ≈ -0.7 kcal/mol (per CH2/CH2 interaction)

Hydrophobic Interaction

NH2

O

O

butamben

butamben - topical anesthetic

van der Waals forces

• Occurs due to the formation of transient dipoles within a structure

• G° ≈ -0.5 kcal/mol per CH2/CH2 interaction

charge transferor hydrogen bond

hydrophobic

dipole-dipoleor hydrogen bond

hydrophobic

ionic or ion-dipole

hydrogen bond

hydrophobic

:N

N

CH2CH2 N

H

CH2CH3

CH2CH3

H

OCH3CH2 CH2CH2O

+

dibucaine

hydrophobic

Dibucaine - local anesthetic

Drug receptor interaction - example

Means of measuring drug-receptor interactions

• Drug receptor interaction is measured by comparing a dose-response curve of endogenous ligand and the drug molecule. – If a molecule produce the same maximal response as the

ligand is called full agonist – If it show no response to the receptor but block the effect

of natural ligand depending on concentration of the ligand is a competitive antagonist

– If the effect of the antagonist is independent of the concentration of the ligand is called non-competitive antagonist.

– If it produced less than the maximal response is called partial agonist

– If it showed opposite response is an inverse agonist

% M

usc

le C

on

trac

tio

n

Dose-response curve of Ach

W showed same response as Ach is agonist

Y showed less than maximal response is a partial agonist

X blocks the response independent on conc. of Ach is a non-competitive antagonist

X showed no response X blocked the response depending on conc. of Ach is a competitive antagonist

Z showed less maximal response opposite to Ach is a partial inverse agonist

Z showed full response but opposite to Ach is an inverse agonist

Stages in drug-receptor interactions • Drug receptor interaction involves two stages

1. Affinity: capacity of drug to bind to the receptor 2. Efficacy (): ability of drug to initiate a biological effect

• Affinity and efficacy are uncoupled: a compound can have great affinity but poor efficacy (and vice versa).

• Agonist and antagonist depends on the biological system.

– A compound can be an agonist for one receptor and an antagonist or inverse agonist for another receptor

Equal efficacies Different affinities Equal affinities Different efficacies

Drug-Receptor Theories

• Occupancy theory

• Rate theory

• Induced-Fit theory

• Activation-Aggregation theory

• Multistate model

Occupancy Theory

• In 1926

• Intensity of pharmacological effect is directly proportional to number of receptors occupied

• Does not rationalize why two drugs that can occupy the same receptor can act differently. (as agonists, antagonist or inverse agonists)

Rate Theory (1961)

In 1961

• Activation of receptors is proportional to the total number of encounters of a drug with its receptor per unit time.

• Does not rationalize why different types of compounds exhibit the characteristics they do.

Induced Fit Theory

In 1958

•

• Agonist induces conformational change – response

• Antagonist does not induce conformational change - no response

• Partial agonist induces partial conformational change - partial response

Activation-Aggregation Theory

In 1965-1967

• Receptor is always in a state of dynamic equilibrium between activated form (Ro) and inactive form (To).

𝑅𝑜 ⇌ 𝑇𝑜

• Agonists shift equilibrium to Ro

• Antagonists shift equilibrium to To

• Partial agonists bind to both Ro and To

• Binding sites in Ro and To may be different, accounting for structural differences in agonists vs. antagonists

biological response

no biological response

Two-state (Multi-state) Receptor Model

• R and R* are in equilibrium (equilibrium constant L), which defines the basal activity of the receptor.

In the multi-state model there is more than one R state to account for variable agonist and inverse agonist behavior for the same receptor type.

• Partial agonists bind preferentially to R*

• Partial inverse agonists bind preferentially to R

• Antagonists have equal affinities for both R and R* (no effect on basal activity)

• Full agonists bind only to R*

• Full inverse agonists bind only to R

G-PROTEIN-COUPLED RECEPTORS (GPCRs)

GPCRs has seven transmembrane –helices in a single chain of 350–1,200 residues,

The amino-terminal contains N-linked glycosylation sites.

Interaction with G-protein is through the third loop, and the C-terminal

Are integral plasma membrane proteins that transduce signals from extracellular ligands to signals in intracellular G-proteins (GTP binding proteins)

Types of GPCRs Receptors

About 860 genes of the human genome encode GPCRs.

More than 50% of GPCRs are activated by sensory stimuli (8 by light, 22 by taste compounds and 388 to 460 by odorant stimuli).

The endogenous ligands of GPCRs are small neurotransmitters, neuropeptides, peptide hormones, inflammatory mediators, lipids and ions

Types of GPCRs Receptors Endogenous ligand Example

Biogenic amines Acetylcholine, Adrenaline, Dopamine,

Histamine, Serotonin

Peptides/Proteins Adrenocorticotrophin (ACTH,

Adrenomedullin, Amylin, Angiotensin II,

Bradykinin, chemokines, Gastric

inhibitory peptide, Gastrin, Neuropeptide

Y/W/FF, Opioids

Amino acids Glutamate, GABA

Lipids Leukotriene, Lysophosphatidylcholine,

Platelet-activating factor, Prostacyclin,

Prostaglandin, Thromboxane A

Nucleotides/Nucleosides Adenosine, ADP, ATP, UDP, UTP

Proteases Thrombin, Trypsin

Ions Calcium

•G-protein consist is complex of three units GαGβγ.

•At rest Gα bound to GDP

•When a receptor is activated: 1. GDP is converted to GTP. 2. complex dissociates to active Gα-GTP and Gβγ 3. Hydrolysis of GTP leads to reassociation

G-protein signaling pathways

Note

• Signaling may take up to tens of seconds to be completed.

• in a few cases, such as vision using rhodopsin and transducin, the responses take only tens of milliseconds.

G-protein Pathway

Gαs activates adenylyl cyclases, increase cAMP, stimulates PKA

Gαi inhibit most adenylyl cyclases

Gαq activate PI3K increase IP3 and DAG that release Ca+2 and activate PKC, respectively

Gα12/13 enhance Rho kinase

Gαtransducin Release cGMP actiavte vision and taste systems

Gβγ inhibit opening of Cav channels stimulate PLC β and (PI3K).

G-protein signaling pathways