Molecular Pharmacology

• Molecular classification and receptor family expansion can be seen through gene cloning.

  • Identify and sequence the cDNA of receptor
  • Predicts the amino acid sequence of the receptor
  • Repeat for all receptor sub-types and compare amino acids to give molecular classification
  • Leads to identification of distinct sub-types

  
• Constricting responses:

  Noradrenaline > adrenaline > isoprenaline

  • Alpha receptor
• Dilating responses:

  Isoprenaline > adrenaline > noradrenaline

  • Beta receptor
• Differential pharmacology (structure function) and receptor associated signalling allows for sub-classification base on relative sensitivities to agonists/antagonists.
• cDNA screening: cDNA synthesised from mRNA or miRNA (catalysed by reverse transcriptase), then used to express a specific protein by heterologous expression.

  
• Homology: similarity due to shared ancestry between a pair of structures or genes in different taxa.
• Other methods of sequencing include:

  • Expressed sequence tags (ESTs)
  • Data mining
  • Genome sequencing (from organisms such as C. elegans, yeasts, drosophila etc)
• cDNA sequences allow for prediction of protein structures, which leads to sequence comparison, established function and protein prediction. This leads to diverse receptors being identified as relatives of one family.

  
• Superfamily > family > sub-family > sub-type
• Families of receptors:

  1. Adrenoceptors and rhodopsin
  2. Secretin
  3. Glutamates
  4. TAS2/Frizzled
  5. Adhesion

  
• The alpha 2 adrenoceptor is an inhibitor of neurotransmitters (adrenaline/noradrenaline).

  • A single neurotransmitter can activate many receptors
  • The signal transduced depends on the receptor and downstream signalling

  
• Biological function of receptors can be altered depending on the location e.g., beta 1 receptors in the heart to produce tachycardia.

  • Beta 2 receptors allow for vasodilation for the treatment of asthma and activation of premature labour for abortion
• Receptor signalling occurs from the stimulation of a receptor to transduce a signal inside the cell.

  • GPCRs - not a G-protein but a receptor that interacts with a G-protein
  • Ligand gated ion channels
  • Steroids - no cell surface receptor required and can bind to intracellular organelles
  • Receptor tyrosine kinases - leads to activation of phosphorylation of tyrosine residues on both the receptor and other proteins
• GCPRs:

  • Primary structure: multifaceted family
  • Secondary structure: all receptors predicted to be 7 TM spanning
  • Tertiary structure: are similar but different depending on the receptor
  • Quaternary structure: GCPR operates as dimers of individual subunits

  
• Ligand binding site is within the TM domain for family 1a. The third intracellular loop is in contact with the G-protein.

  • Binding is restricted to the N-terminal domain for larger ligands
  • In family 2 this is the same however the N-terminal is then cleaved and held by intramolecular forces to create the active site
  • Family 3 are dimers and use bi-lobed extracellular domain and have G-protein contact with loop 2

  
• TM1 and TM7 are close, with the structure being a pore-shape. There is space within the GPCR which can hold a hydrophilic molecule (dependent on primary structure).

  
• The receptor at rest will be activated by an agonist to become activated. This causes a conformational change, producing a G-protein binding site.

  • Specifically TM5 and TM6
  • Movement of the helix creates an opening around loop 3 to allow for G-protein contact
  • Poor activation does not allow for binding
  • Conformational change is stabilised with the agonist

  
• The nicotinic ACh receptor family is a group of ligand-gated ion channels.

  Primary: a number of related genes encoding sub-units that make up distinct members of the family

  Secondary: Each subunit exhibits a common transmembrane topology

  Tertiary: models supported by structural information for whole receptor/domains

  Quaternary: exists as an oligomeric receptor made of 5 subunits (pentameric)

  
• Most 5-HT receptors are GCPR, but some are ligand-gated ion channels.
• Basic transduction mechanism (nAChR):

  • Bind against agonist from outside of receptor
  • Conformational change or gate of ion channel (gating)
  • Selects specific ions to influx through the ion channel and pass through the membrane
  • Operate on a millisecond time scale

  
• Subunits of ligand-gates ion channels have interfaces between each other. Between these is where the ligand binding domain is located.

  • The final C terminus sits on the outside of the membrane, where ligand binding may occur on the Cys-loop exposed extracellularly
  • The TM region is formed of helices, whereas the extracellular region is made of pleated sheets

  
• The Cys-loop is specific for nicotinic receptors - this is separated by 13 amino acids and a common structural motif in the superfamily.
• Classical ACh receptors are heterooligomeric.

  • The alpha subunit is the major binding domain in the muscle
  • Two alpha subunits means that the effector organ (muscle) if activated by two ACh molecules
  • This is activated by binding to gamma or delta subunits
  • Amino acids that make contact with ACh make hydrogen bonds, van der Waals and dipole interactions

  
• Solvation of an ion prevents crossing of the hydrophobic environment across the membrane. The channel allows for dehydration of the ion, passing through then partial dehydration once passed through the channel.

  
• When an agonist binds, the structural change shifts the position of M2 to allow the ion to enter. There are two conformations, open or closed.

  • Beta sheets twist when the agonist binds
  • TM helices are levered, shifting the M2 position and creating a bigger space
  • Rotation in an individual subunit coordinated across subunits of oligomeric protein increases the size of the ion channel

  
• Tyrosine-kinase linked receptors are activated, leading to downstream signalling.

  Primary: a number of different growth factor receptor gene families

  Secondary: single transmembrane spanning or membrane associated domain

  Tertiary: isolated ligand binding domain or kinase domains confirm likely structures

  Quaternary: clear dimerisation required during signal transduction as some exist as oligomers before ligand

  
• Basic transduction mechanism (TKs):

  • Activated by extracellular ligand
  • Conformational change drives receptor dimerisation
  • Dimerisation activated intrinsic tyrosine kinase or recruits non membrane associated kinase
• Steroid receptors respond to steroid molecules such as cholesterol. Cholesterol is the starting point for synthesis of mineralocorticoids (aldosterone), glucocorticoids (corticosterone), and sex steroids (e.g., oestrogen).
• Steroid molecules are lipid soluble, so diffuse into the cell and act on intracellular receptors.

  • The receptor is inactive before binding with the steroid
  • The heat-shock protein (HSP-90) keeps the receptor inactive and dissociated when the steroid binds
  • Dimerisation of the receptor occurs upon dissociation of HSP-90 and becomes activated, binding to DNA, and activates transcription

  
• Zn2+ is required as a cofactor in steroid receptors. The N terminus is defined in elements that are involved in gene transcription.

  • The zinc finger domain is classical of the receptor and aids in binding to DNA/dimerisation
  • The hinge region allows for movement
  • The steroid binding domain slightly overlaps with the nuclear localisation
  • HSP-90 is between the steroid binding site and nuclear localisation

  
• Pharmacogenomics: the study of genetic determinants and variability effecting the way in which a drug works.
• Pharmacogenomics is used to determine the outcome that reduces side effects and optimal efficacy. This has an impact on both pharmacokinetics and pharmacodynamics.

  • The biological processes that control these are based on genes - the protein interaction with any system and the effect of the drug
  • Common targets are receptors, ion channels, enzymes and the immune system

  
• If a drug is too quickly metabolised, there is no/little response. Increased drug response can be produced with slow metabolism, excessively high drug levels at usual dosage, as well as a higher risk of ADRs.
• Adverse drug reaction (ADR): a harmful, unintended result caused by taking medication.
• Succinylcholine (Suxamethonium) is used as a short lived drug.

  • 1 : 3000 have a genetically altered enzyme that has reduced activity
  • Only occurs in individuals carrying two copies of the modified gene (will have an adverse reaction)
  • This is by sustained NMJ muscle block

  
• The human genome varied between individuals every ~500 nucleotides e.g., 14 million SNPs across the 3.2 billion nucleotides.
• Single nucleotide polymorphism (SNP): a germline substitution of a single nucleotide at a specific position in the genome that is present in a sufficiently large fraction of considered population.
• A reference gene, with an intact intron, and an exon spliced out to encode a protein.

  • Single nucleotide variations have an effect depending on where it is in a codon
  • Small insertions can have a consequence
  • Larger changes may occur by insertions of additional genes
  • A deletion of a coding exon
  • Inversion of introns and exons
  • Duplication with an additional exon copy
  • Translocation of the order of the gene

  
• Functional consequences include parts of DNA that encode promoter elements.

  • A coding SNP gives a polymorphism that changes the sequence of RNA
  • Exonic deletion causes a whole section of protein to be missing
  • Gene duplication causes a doubling of protein output
  • Deletion/inactivation of transcription factor binding sites (no transcriptor = no protein)

  
• Genomic modulation of pharmacokinetic parameters int eh CYP450 family can cause modification of metabolism.