Drug Targets: Receptors and Channels

Drug Targets

• Cell surface receptors
  • G protein-coupled receptors (GPCRs)
  • Ion channels
    • Voltage-gated channels
    • Ligand-gated channels
  • Enzyme-linked receptors
    • Enzymes
    • Transporters
• Nuclear receptors

Targets of Current Marketed Drugs

• GPCRs (7TM1) account for 33% of small molecule drugs that target major families
• Ion channels represent 18%
• Kinases 16%
• Nuclear receptors 10%
• Other 3%
• Enzymes and transporters account for 30% of small molecule drugs that target major families

Ion Channels and GPCRs

• Ion channels
  • Neurotransmitter binds directly to ion channel protein
  • Channel opens immediately
  • Ions flow across membrane for a brief time
• G protein-coupled receptors (GPCRs)
  • Neurotransmitter binds G protein-coupled receptors
  • G protein becomes activated
  • G protein subunit moves to adjacent ion channel, causing short delay
  • The activated subunit also triggers second messenger systems (which are not shown in the diagram)
  • Channel opens, and ions flow across membrane for longer period

Class A Example: β-adrenergic Receptor

• Helices of membrane-spanning domains form binding pocket for norepinephrine
• Keeping molecule in place through molecular interactions

Major Classes of GPCRs

• Class A
  • Most numerous and diverse
  • ~45 kDa
  • Relatively short amino terminus
  • Small molecule binding site in lipophilic pocket
  • Binding within the 7 TM region
• Class B
  • Longer amino terminus, can sometimes bind ligands
  • Ligands predominantly bind to extracellular loops
  • Typical ligands are peptides and small proteins
• Class C
  • ~80 kDa
  • Very large amino terminus
  • Forms functional heterodimers
  • Agonists bind mostly in amino terminus
  • Binding sites can occur all over the receptor molecule

Class B Example: PAR-1 (Protease Activated Receptor)

• N-terminus of PAR-1 contains protease cleavage site
• Cleavage by thrombin results in a new N-terminus
• Sequence SFLLRN acts as tethered ligand
• Binds intramolecularly to heptahelical body of receptor
• Effects transmembrane signaling and G protein activation

Class C Example: Sweet and Umami Tastant Receptors

• Sweet taste receptor is heterodimer composed of two full-length GPCR monomers
• Long amino terminus forms "venus flytrap" binding pocket

Guanosine Tri- and Di-phosphates

• Nucleotides in G protein activation cycle
  • GTP is 3 phosphates
  • GDP has 2 phosphates

GPCRs Can Initiate a Signal Transduction Cascade

• G protein must be activated, GTP to GDP will inactivate nearby G proteins, but when attached to GTP it is activated.
• Ligand binds to receptor
• G protein releases GDP and binds GTP, activating G protein
• Subunits separate
• G protein subunits activate or inhibit target proteins
• Ga subunit hydrolyzes its bound GTP to GDP and becomes inactive
• Subunits recombine to form inactive G protein

Phospholipase C in an Enzyme

• PLC takes pieces from phospholipid searching for PIP2 to cleave A phosphate group, creating IP3 and DAG
• IP3 is a messenger for cell which is on surface of ER called IP3R, opening up the channel, CA2+ will exit into cytoplasm

IP3 Causes Release of Calcium

• IP3 binds IP3-receptor channels
• Calcium is released from ER and SR

IP3 Microinjection

• Microinjection of IP3 in neuroepithelioma cells evokes intracellular release of calcium and subsequent paracrine calcium signaling

Adenylyl Cyclase

• Adenylyl cyclase is an enzyme in the plasma membrane that converts cytosolic ATP to cAMP
• Takes ATP
• Creates cAMP
• PDE (phosphodiesterase) converts cAMP to AMP
• Inhibitors: Caffeine, Sildenafil, Theophylline

Down-regulation of Receptors

• Following agonist binding, GRKs are activated
• Phosphorylate agonist-bound receptor
• Phosphorylated C-terminus recruits β-arrestins
• β-arrestins bind and polymerize clathrin proteins, forming cages that stabilize the receptor complex

Up-regulation of Receptors

• Prolonged exposure to antagonist
• Chronically low levels of intrinsic neurotransmitter or hormone agonist
• Pathological factors impacting gene regulation and protein expression

Nuclear Receptors

• Nuclear receptors are transcription factors
  • Directly bind to DNA to regulate specific gene expression
• General structure
  • Ligand binding domain
  • DNA binding domain
• Examples of Agonists for Nuclear receptors
  • Steroid hormones
    • Estradiol
    • Testosterone
    • Cortisol
    • Aldosterone
  • Retinoic acid (metabolite of vitamin A)
  • Calcitriol (active form of vitamin D)
  • Triiodothyronine (thyroid hormone T3)

Nuclear Receptor Mechanism of Action - Class I

• Receptor located in the cytosol bound to Heat Shock Protein (HSP)
• Ligand diffuses across membrane, binds to receptor
• Ligand-Receptor (LR) complex dissociates from HSP
• LR complex translocates to nucleus
• LR complex binds Hormone Response Element (HRE) sequence on DNA
• LR complex recruits additional proteins to form a transcription initiation complex
• Transcription of DNA to RNA

Nuclear Receptor Mechanism of Action - Class II

• Unoccupied receptor already located in the nucleus bound to DNA
• Unoccupied receptor is bound to a corepressor protein
• Ligand binding to thyroid hormone receptor (TR) causes dissociation of corepressor and recruitment of coactivator protein, polymerase