Drug-Receptor Binding & Signal Transduction PDF

Drug-Receptor Binding & Signal Transduction  Drug is any chemical that interact with a protein or another molecule to cause cellular response – Endogenous (made in the body) – Exogenous (delivered to the body) e.g. Poisons (substances with harmful effects, Toxins (poisons...

Drug-Receptor Binding & Signal Transduction  Drug is any chemical that interact with a protein or another molecule to cause cellular response – Endogenous (made in the body) – Exogenous (delivered to the body) e.g. Poisons (substances with harmful effects, Toxins (poisons of biological source) Receptor is a protein that interact with a molecule and cause a change in downstream signaling pathways  Nussinov et al 2013 Agonist + Receptor Activation Receptor Deactivation  Drug-receptor Binding Drugs binds to receptors via chemical bonds – Covalent (stable, atoms are sharing valence e-) – Hydrogen bonds – Electrostatic – Van der walls CO2 (Covalent bonds)  Parts of an Atom Protons (Subatomic particles with + charges): 8 Atomic Number 8 O 16 Atomic mass (P+N) Electrons (Subatomic particles with - charges): 8 Neutrons (Subatomic particles with neutral charges): 16 (Covalent bonds)  Parts of an Atom 11 17 Na 23 Cl 35 - -----------------------------  Drug-receptor Binding Agonist: Any drug that binds to a receptor and activates it Antagonist is any drug that binds to a receptor and inactivate it Crystal structures allow visualization of drug binding to receptors https://www.youtube.com/watch?v=Lh41xNU0Mgc  Drug-receptor Binding Drugs are weak acids or weak base as small changes in PH can change them between fat and water soluble When drugs are mixed with water they don’t completely dissociate into ionic form Mass Action PH7 PH >7 the drug can be completely dissolved PH< the drug will be protonated pKA is the PH at which the drug is balanced between the uncharged and the charged form  Blood-brain barrier (BBB) Brain is protected from many substances in the general circulation by the BBB. BBB is formed by tight junctions between the endothelial cells that make up the vessels of the central nervous system. Vessel is surrounded by cells called pericytes (which form a basement membrane) and astrocytes that also form a protective layer to keep drugs out of brain tissue. Lipophilic (fat loving) drugs can always just diffuse into the brain through membranes. But non-lipophilic drugs cannot get in, unless they have a transporter. Other tricks include binding the drug to a carrier that is transported across the BBB or giving a stimulus that transiently opens the BBB. The BBB Signal transduction mechanisms A. Ion channels B. G protein coupled receptors C. Enzyme-linked receptors D. Nuclear receptors  G-protein coupled receptors (GPCRs) G protein is a complex of an α, β, and γ subunit that binds GTP/GDP.  Fig. 1 Cartoons depicting the secondary structure and the location of agonist binding sites for different GPCRs. Brian K. Kobilka G protein coupled receptor structure and activation Biochimica et Biophysica Acta (BBA) - Biomembranes Volume 1768, Issue 4 2007 794 - 807 GPCR Cycle 1. Receptor and G-protein are inactive (A). 2. Ligand binds receptor recruiting G-protein (B). 3. GTP displaces GDP on the G- protein (B). 4. GTP-bound α subunit is active and dissociates from the βγ subunit (C). 5. Subunits perform their functions (C). 6. α subunit hydrolyzes GTP to GDP inactivating the subunit (D). 7. α subunit and βγ subunit rejoin (A). GPCR’s allow tremendous amplification. One agonist can bind to 1 GPCR, but each GPCR can activate many g-proteins. Each g-protein can phosphorylate many targets. Schrage et al 2015 GPCR Signaling Pathways  GPCR Signaling Pathways activation inhibition Sciavo et al 2001 Cholera toxin produces persistent activation of Gs proteins by blocking GTPase activity. Pertussis toxin prevents Gi proteins from coupling with receptors.  Second messengers Calcium- influx through channels or release from intracellular stores (endoplasmic reticulum, ER). Cyclic nucleotides- cAMP or cGMP are formed by adenylyl or guanylyl cyclase, respectively. Phospholipase C- breaks down phosphatidylinositol into IP3 and DAG. Arachidonic acid (AA) metabolites- phospholipase A2 cleaves AA out of the membrane. AA is then metabolized by cyclooxygenase (COX) or lipoxygenase enzymes into numerous bioactive substances.  Effectors downstream of Ca++ Calmodulin- protein that binds Ca++ via 4 EF-hand domains. Activates calcium/calmodulin-dependent protein kinase (CaMK), calcineurin (phosphatase), MAPK, phosphodiesterase (PDE), histone deacetylases (HDACs). CaMK consists of an N-terminal catalytic domain, a regulatory domain, and an association domain. In the absence of Ca++, the regulatory domain inhibits the catalytic domain. Protein kinase C- phosphorylates many proteins MEK- mitogen activated protein kinase (MAPK)/extracellular signal-related kinase (ERK) kinase Phosphoinositide kinase 3 (PI3K)- phosphorylates PDK1 which phosphorylates protein kinase B (also known as Akt) Calcium Signaling  Cyclic Nucleotides Cyclic AMP or cAMP is produced by adenlyl cyclase (AC). GPCRs coupled to Gs stimulate and those coupled to Gi inhibit AC. Most ACs are activated by forskolin. cAMP is a ligand for ion channels and it also activates protein kinase A (PKA). PKA then phosphorylates cAMP response element binding protein (CREB). cGMP is generated by guanylyl cyclase (GC) which is most commonly activated by nitric oxide (NO) but also by membrane bound GCs. cGMP regulates ion channels, relaxes smooth muscle, and contributes to the mammalian visual transduction pathway. Activates protein kinase G (PKG). Nitrates are used as vasodilators because they activate GC, make cGMP and dilate vessels. Examples: nitroglycerin, sodium nitroprusside  Adenylyl Cyclase and cAMP Gs stimulates Gi inhibits  Breakdown of cAMP/cGMP Phosphodiesterases (PDEs) perform this function. There are at least 10 PDEs, PDEI-PDEX. Targets of drugs: – Trifluoperazine inhibits PDEI (but also adrenergic and dopaminergic receptors)- used as an antipsychotic. Also vinpocetine inhibits. – Milrinone, Enoximone, Amrinone (aka Amiodarone)- inhibit PDEIII- used for congestive heart failure. – Rolipram inhibits PDEIV- anti-inflammatory and may be effective as an antidepressant. – Ibudilast inhibits PDEIV- bronchodilator/vasodilator. Also Roflumilast for COPD – Sildenafil (Viagra), Tadalafil (Cialis), Vardenafil (Levitra)- inhibit PDEV- used for erectile disfunction. – Methylated xanthines e.g. theophylline, caffeine- nonspecific inhibitors of PDE (but also adenosine receptor antagonists)  Phospholipase C (PLC) PLC catalyzes the breakdown of phosphatidylinositol into IP3 and diacyl glycerol (DAG). Most commonly activated by Gq proteins, specifically the βγ subunits. IP3 binds to the IP3 receptor on the ER and opens a Ca++ channel. – Ca++ is pumped into the ER by the sarcoplasmic/endoplasmic reticulum Ca++ ATPase (SERCA) pump. Thapsigargin blocks this pump. DAG (along with Ca++) activates protein kinase C (PKC). Phosphatidylinositol (PI) Hydrolysis O’Day 2011  Arachidonic acid metabolites Arachidonic acid (AA) is an eicosanoid meaning it has 20 carbons AA is cleaved out of the membrane by phospholipase A2. PLA2 is cytosolic and activated by Ca++ as well as phosphorylation by MAPK. There are also secreted PLA2s, some of which are found in various venoms. AA is converted by 5-lipoxygenase (5-LO) into leukotrienes. 5-LO is activated by 5-LO activating protein (FLAP). AA is converted by cyclooxygenase 1 and 2 (COX1 and 2) into prostaglandins (PGE2, PGD2, PGF2), prostacyclin (PGI2), thromboxanes TXA2. COX1 is constitutive and expressed throughout the body e.g. the upper GI tract where it contributes to the production of mucus that lines the stomach. Aspirin, acetaminophen, NSAIDs (e.g. ibuprofen) inhibit COX1 as well as COX2 so produce GI irritation. COX2 is inducible during inflammation… This is inhibited by rofecoxib (Vioxx, withdrawn), celecoxib (Celebrex) which have lower incidence of GI side effects. Enzyme Linked Receptors Cytokine ligands- interleukins, interferons, tumor necrosis factor… Tyrosine kinase ligands- epidermal growth factor, nerve growth factor… Serine/threonine kinase receptors- transforming growth factor  Tyrosine Kinase Receptors Kinase cascade Dimerization and autophosphorylation are critical for signaling in TK receptors. Nuclear Receptors Ligands Ligands for nuclear receptors must be able to cross the plasma membrane. If they cross they can bind to the receptor which is intracellular.