Drug Mech: Basic Principles 2

• · Following binding of drug molecules to receptors, the occupancy-response coupling process is
• accomplished by a number of different molecular
• mechanisms (or transmembrane signaling pathways). Each one of these mechanisms has evolved to transduce many different signals.

• · Protein families that are involved in these molecular signaling pathways include receptors
• on the cell surface and within the cell, as well as enzymes, signal transducer proteins, and other molecules that generate, amplify, coordinate, and terminate postreceptor signaling by chemical second messengers in the cytoplasm.

· Five basic mechanisms of transmembrane signaling are well established in pharmacology. These five mechanisms account for the transduction of many of the most important drug/chemical signals that are of interest in pharmacotherapy. Each one of these signaling mechanisms uses a different strategy to transduce the chemical signal across the lipid bilayer of the plasma membrane. These basic mechanisms include:

• 1. Intracellular Receptors for Lipid-Soluble Agents
• 2. Ligand-Regulated Transmembrane Enzymes (Including Receptor Tyrosine Kinases)
• 3. Cytokine Receptors
• 4. Ligand-Gated Ion Channels
• 5. G Proteins and Second Messengers

• In this particular mechanism, a lipid-soluble ligand
• (or drug) crosses the plasma membrane and acts on an intracellular receptor (which may be an enzyme or a regulator of gene transcription).

• Examples of ligands that exert their effects via this pathway include:
• 1) nitric oxide
• 2) corticosteroids
• 3) sex hormones
• 4) thyroid hormone
• 5) vitamin D

The receptors in this pathway are transmembrane proteins consisting of an extracellular ligand-binding domain and a cytoplasmic (intracellular) enzyme domain (which may be a protein tyrosine kinase, a serine kinase, or a guanylyl cyclase).

The two domains (intracellular and extracellular) are connected by a hydrophobic segment of the polypeptide that crosses the lipid bilayer of the plasma membrane.

The ligand binds to the extracellular domain of the transmembrane receptor protein, thereby activating (allosterically regulating) the enzymatic activity of its intracellular domain.

• Examples of ligands that exert their effects via this
• pathway include:
• 1) insulin *classic example
• 2) epidermal growth factor (EGF)
• 3) platelet-derived growth factor (PDGF)
• 4) transforming growth factor-b (TGF-b)
• 5) many other trophic hormones

The Receptor Tyrosine Kinase signaling pathway begins with ligand binding to the receptor’s extracellular domain which results in a conformational change. The change in the receptor’s conformation causes receptor molecules to bind to one another, including the tyrosine kinase domains. The tyrosine kinase domains become enzymatically active and, as a result, phosphorylate one another as well as additional downstream signaling proteins.

Activated receptor tyrosine kinases phosphorylate tyrosine residues on different target signaling proteins and allow a single activated receptor to modulate several biochemical processes. For example, each one of the growth factor ligands initiates a complex array of cellular events in its specific target cells ranging from altered membrane permeability to changes in gene expression.

Specific inhibitors of growth factor-activated receptor tyrosine kinases are effective therapeutic agents in treating cancer (where over-expression of growth factor receptors and excessive growth factor signaling occur).

The mechanism for the cytokine receptors is very similar to the mechanism of receptor tyrosine kinases. However, in the case of the cytokine receptor, the protein tyrosine kinase activity is not intrinsic to the receptor molecule; instead, separate protein tyrosine kinase, from the Janus-kinase (JAK) family, binds noncovalently to the receptor.



In this particular pathway, the ligand binds to the extracellular domain of a transmembrane receptor protein that is already bound to a protein tyrosine kinase (JAK).

As a result of receptor-ligand binding, the protein tyrosine kinase is activated.

The activated tyrosine kinase phosphorylates tyrosine residues on the receptor.

This is followed by binding of the receptor to another set of proteins, the STATs (Signal Transducers and Activators of Transcription).

Following phosphorylation of the STATs by the bound protein tyrosine kinases, they (the STATs) dimerize, dissociate from the receptor, and travel to the nucleus, where they regulate the expression of specific genes.

• Examples of ligands that bind to and activate cytokine receptors include:
• 1) growth hormone
• 2) interferons
• 3) other regulators of growth and differentiation

The enzyme in Receptor Tyrosine Kinases (RTKs) are part of the intracellular receptor, whereas in Cytokine receptors, the protein tyrosine kinases are NOT intrinsic to the receptor molecule.

In this particular class of receptors, a ligand-gated transmembrane ion channel is induced to open or close by the binding of a ligand.



Examples of natural ligands that regulate the flow of ions through plasma membrane channels, by binding to these channels, include:

• 1) the neurotransmitters acetylcholine,
• serotonin, g-aminobutyric acid (GABA)
• 2) the excitatory amino acids (e.g., glycine, aspartic acid, and glutamic acid).

Many important drugs act by either mimicking or blocking the actions of endogenous ligands that regulate ion channels.

In this particular signaling mechanism, the ligand binds to the extracellular domain of a transmembrane receptor protein (R).

The receptor in turn stimulates a GTP-binding signal transducer protein (G protein) located on the cytoplasmic face of the plasma membrane.

The G protein then activates an effector (E) (an enzyme or ion channel) that is responsible for modulating the production of an intracellular second messenger.



The family of G proteins contains a number of diverse subfamilies which mediate effects of receptors to effectors. Receptors coupled to G proteins belong to a family of proteins known as ‘Serpentine Receptors’ or ‘7-Transmembrane Receptors’; all serpentine receptors transduce signals across the plasma membrane in the same fashion.



A large number of extracellular ligands act by increasing the intracellular concentrations of second messengers, such as cAMP, calcium ion, or the phosphoinositides, via this G protein-coupled signaling pathway.

For example, for cAMP, the effector enzyme is adenylyl cyclase, a transmembrane protein that converts intracellular ATP to cAMP. The corresponding G protein, G_s, stimulates adenylyl cyclase after being activated by ligands that act via a specific receptor; examples of these ligands include catecholamines (b adrenoceptors), histamine (H₂ receptors), vasopressin (V₂ receptors), glucagon, FSH, LH, thyrotropin, parathyroid hormone, …etc.

G proteins use a molecular mechanism that involves binding and hydrolysis of GTP to amplify the transduced signal.

Amplification of the original signal (which is the result of binding of the ligand to its membrane receptor) is attributed to the fact that the active GTP-bound G protein remains in its active state for a relatively long time (tens of seconds). The duration of activation of adenylyl cyclase, for example, depends on the duration of activation of the G protein (not on the receptor’s affinity for binding to the ligand or the duration of that binding).

These pathways are highly efficient because of amplification.

One of the most important GPR examples is the beta-adrenergic receptor.

What matters is how long the G-protein is activated!

• Three well-studied intracellular Second Messenger Signaling Pathways are known:
• 1) cyclic adenosine monophosphate (cAMP)
• 2) calcium and phosphoinositides
• 3) the cyclic guanosine monophosphate second
• messenger pathways

• For example, cAMP mediates such hormonal responses as:
• 1) the breakdown of carbohydrates and triglycerides (stimulated by the b-adrenomimetic catecholamines)
• 2) conservation of water by the kidney (mediated by vasopressin)
• 3) calcium homeostasis (regulated by parathyroid hormone)
• 4) increased rate and contraction force of heart muscle (stimulated by the b-adrenomimetic catecholamines)
• 5) the production of adrenal and sex steroids (stimulated by corticotrophin or FSH)
• 6) relaxation of smooth muscle
• 7) numerous other endocrine and neural processes

cAMP exerts most of its effects by stimulating cAMP-dependent protein kinases. The specificity of cAMP’s regulatory effects is attributed to the distinct protein substrates (usually enzymes) of the kinases that are expressed in different cells.

• When the hormonal stimulus ends, the cAMP actions are terminated as follows:
• Phosphorylation of protein substrates is rapidly reversed by phosphatases and cAMP is degraded to 5’-AMP by phosphodiesterases (PDE).

• Second messenger signaling pathways may oppose one another (in terms of the biological response) in some cells, and complement one another in other cells (e.g., cAMP-mediated smooth muscle relaxation versus calcium-phosphoinositide mediated smooth muscle contraction).

The first messenger would be the ligand (also called the agonist or the drug) that binds to the receptor.

Recall how cAMP is degraded:

• When the hormonal stimulus ends, the cAMP actions are terminated as follows:
• Phosphorylation of protein substrates is rapidly reversed by phosphatases and cAMP is degraded to 5’-AMP by phosphodiesterases (PDE).

If a drug inhibits cAMP degredation to 5'-AMP, then cAMP can continue to effect, via its signal transduction pathway, a hormone stimulus. This effect could be considered a pharmacological effect.



Protein kinases are considered very important targets for drug discovery efforts in a number of diseases. Specific inhibitors of protein kinases have great potential as drugs, particularly in cancer chemotherapy (e.g., Imatinib, an inhibitor of a tyrosine kinase which is activated by growth factor signaling pathways, is used clinically for the treatment of chronic myelogenous leukemia).

The bottom line is: as long as the GPR is active, the effector is active!

• dephophorylation
• cAMP degraded by phosphodiasterase (PDE)