Drug-Receptor interactions

• [IAS73] Drug Receptor Interactions overview
• Involves the interactions between drugs and receptors (targets)

Learning Outcomes Covered

• Identifying drug target examples
• Describing events preceding and following G-protein-coupled receptor activation
• Outlining the regulatory process of G-protein-coupled receptors
• Recognizing different types of receptors, including non-receptor drug targets
• Explaining drug-target interactions via receptor theory
• Deriving the fractional receptor occupancy equation
• Describing drug-target interaction characteristics in a two-state receptor model

Drug & Receptor Interactions Overview

• Part 1 focuses on G-protein-coupled receptors (GPCR), guanine nucleotide exchange, effector protein activation, receptor regulation, second messenger system, and other effects
• Part 2 covers drug targets other than GPCRs, including enzyme-linked receptors, intracellular receptors, ligand-gated ion channels, and voltage-gated ion channels
• Part 3 involves receptor theory, binding/unbinding from target, assessing dissociation constant (KD), and the two-state receptor model

G-Protein-Coupled Receptor (GPCR) as Drug Target

• The extracellular domain binds signalling molecules
• The intracellular domain interacts with trimeric G-proteins
• Quality of drug binding is affected by the properties of the transmembrane and extracellular domain
• GDP/GTP-binding protein is affected by the intracellular domain, where the alpha unit attaches to GDP
• Drug (agonist) binding activates GPCR

GPCR Activation Cycle

• An agonist binds to the extracellular domain of the receptor, causing a conformational change and receptor activation (R → R*)
• The intracellular domain acts as GEF (guanine exchange factor), activating the trimeric G protein and replacing GDP with GTP
• Activated G protein regulates activity of effector protein/ion channel (E), leading to either indirect (second messenger) or direct effector responses
• Signalling terminates via agonist unbinding, G protein inactivation (hydrolysis of GTP by α unit), and a reduction in the concentration of the second messenger

GPCR Signalling Pathway

• Based on the second messenger system
• Gas stimulates adenylyl cyclase to increase cAMP formation
  • α subunit of Gs protein activates adenylyl cyclase, converting ATP to cAMP
  • cAMP activates protein kinase A (PKA), which phosphorylates target proteins
• Gai inhibits adenylyl cyclase, decreasing cAMP formation
• Gq activates phospholipase C, increasing IP3 and DAG production
  • α subunit of Gq protein activates phospholipase C (PLC)
  • PLC cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) into DAG + inositol 1,4,5-triphosphate (IP3)
  • DAG activates PKC, which phosphorylates serine/threonine residues of protein targets
  • IP3 causes Ca2+ release

Signal Amplification and GPCR Signalling

• One agonist molecule binds to receptors, activating G proteins
• Adenylyl cyclase and phospholipase C amplify signals, leading to increased production of cAMP, IP3, and DAG
• Protein kinases, like PKA and PKC, amplify the signal further
• Effectors, including enzymes, transport proteins, contractile proteins, and ion channels, produce a large response
• Involves elements of Gai/o which inhibit AC
• Other elements (Gβy) directly act on ion channels
  • Can increase K+ efflux and reduce Ca2+ influx

Post-Activation GPCR Regulation

• Normal GPCR activation occurs initially
• GPCR is then phosphorylated upon interaction with agonists
• Phosphorylated GPCRs provide binding sites for β-arrestin
• β-arrestin trafficks the receptor to a specialized pit, leading to GPCR internalization
• GPCR is pulled downwards, off the cell membrane
• Unbinding of agonist, dephosphorylation of GPCR, and recycle it back to the cell membrane restarts the activation cycle
• Prolonged agonist binding can leads to lysosomal degradation of GPCR, receptor down-regulation, and a smaller response

Other Receptors as Drug Targets

• These receptors may contain drug-binding sites, e.g, voltage-gated ion channels use binding sites in pore-forming/allosteric regions
• Enzymes, a type of drug target, use active/allosteric sites

Enzymes as Drug Targets

• Receptor tyrosine kinases (RTK) involve receptor activation and trans/auto-phosphorylation
• Non-receptor tyrosine kinases (NRTK) recruit inactive NRTK, converting it to an active form
• Tyrosine phosphatase removes phosphate
• Serine/threonine kinase adds phosphate
• Guanylate cyclase converts GTP to cGMP

Intracellular (Nuclear) Receptors As Drug Targets

• Steroid hormones activate receptors, causing heat shock protein (hsp) displacement, receptor dimerization, and translocation to the nucleus
• Receptors, usually located in cytosol, leads to modulation of gene transcription and protein expression upon binding to hormone-response elements on DNA

Ion Channel as Drug Target

• Ligand-gated ion channels (LGIC) are where the receptor is an ion channel
• Channel opens when agonist binds to the receptor
• Examples include nicotinic acetylcholine receptor
• Orthosteric sites are typical agonist/antagonist binding sites
• Allosteric binding sites allow binding of allosteric/channel modulators and channel blockers
• Drug binding alters channel opening duration and probability
• Site within channel pore for ion flow
• Voltage-gated ion channels
  • Example shown is a subtype of voltage-gated Ca2+ channel with multiple binding sites for drugs
  • Other voltage-gated ion (e.g., Na+, K+) channels also contain multiple binding sites

Drug-Receptor/Target Interactions

• Mathematical expressions are used to describe drug-target interactions and ligands
• Involves target/receptor binding and unbinding reaction

Law of Mass Action

• The rate of concentration & concentration of product
• Free ligand (L) binds to free receptor (R) to form L-R complex
• k+1 & k-1 are forward & backward rate constants
• L-R complex dissociates to give free L & free R
• k+1 × [L] × [R] is proportional to [LR]
• k-1 × [LR] is proportional to [L] & [R] individually, where [L] = [R]
• Receptor (R) is a general drug target
• Ligand (L) is a drug
• Ligand-Receptor Complex (LR) is complex of the two

Dissociation Constant (KD)

• Kd indicates how tightly a ligand unbinds from the receptor
• KD = ratio of backward reaction / forward reaction
• At equilibrium, ratios are = 1
• k+1 < k-1; reaction is faster
• KD is big when k+1 > k-1; backward rxn is faster

Fractional Receptor Occupancy

• [Drug] binds to a fraction of the receptor
• Concentration of receptors that are bound (i.e., occupied) by ligand
• Concentration of all receptors in total (e.g., in a cell or tissue)
• Rt = sum of all receptors = ligand-bound receptors (LR) + free (unbound) receptors (R)
• When [L] increases, fractional receptor occupancy increases
• When KD decreases, fractional receptor occupancy does not change
• When KD = [L], fractional receptor occupancy = 1/2

Two-State Receptor Model

• The drug target switches state to reach equilibrium
  • State 1: Inactive resting state (R/occupation), no drug binding, no response (usually) is governed by affinity
  • State 2: Activated state (R*) response is present, agonist binding/efficacy
• Drug can bind to either or both states
• Describes when some drug receptor can be activated without agonist binding
• Even at baseline, equilibrium shifts to R*
• Receptor exists as R* state and does not depend on agonist activation -Agonist binding induces more conformational changes of AR*