Pharmacology 3A: Principles of Pharmacology and Nuclear Receptors

University of Glasgow

College of Medical, Veterinary & Life Sciences

Pharmacology 3A (Session 2024-25) Block 1 : Principles of Pharmacology Nuclear Receptors Prof. Simon Kennedy simon.kennedy@glasgow.ac.ukILOS ... At the end of this class, you will be able to:

1. Understand what nuclear receptors are;
2. Describe how nuclear receptors are activated and the effects this can produce;
3. Describe some drugs which have nuclear receptors as their target and what they are used for.

Key Reference for Nuclear Receptors

Very nice and simple overview on NRS: Volle D.H. (2016) Nuclear receptors as pharmacological targets, where are we now ?. Cell. Mol. Life Sci. 73: 3777- 3780. Link: https://pubmed.ncbi.nlm.nih.gov/27506618/ Also: https://pubmed.ncbi.nlm.nih.gov/33676761/

History of Nuclear Receptors

• 1905 - Ernest Starling coined the word hormone
• 1926 - Edward Calvin Kendall and Tadeus Reichstein isolated and determined the structures of cortisone and thyroxine
• 1929 - Adolf Butenandt and Edward Adelbert Doisy - independently isolated and determined the structure of oestrogen
• 1958 - Elwood Jensen - isolated the oestrogen receptor
• 1970s- It was clear that receptors for steroid hormones such as oestrogen and the glucocorticoids were present in the cytoplasm of cells and translocated into the nucleus after binding with their steroid partner
• 1980s - Cloning of the oestrogen, glucocorticoid, and thyroid hormone receptors by Pierre Chambon, Ronald Evans, and Björn Vennström respectively
• 2004 - Pierre Chambon, Ronald Evans, and Elwood Jensen were awarded the Albert Lasker Award for Basic Medical Research, an award that frequently precedes a Nobel Prize in Medicine

Nuclear Receptors: Key Points

They are proteins found WITHIN cells and influence events over a long time frame. Examples of ligands are steroid and thyroid hormones. Once activated these receptors work with other proteins to regulate the gene expression, thus controlling such things as development, growth, homeostasis and metabolism. They can bind directly to DNA and regulate adjacent genes. Generally nuclear receptors are not constitutively active- ligand binding is required to activate gene regulation. As with RTKs, ligand binding activates a conformational change- causing either up- or down-regulation of gene expression. As they bind to DNA they play a key role in embryonic development and adult homeostasis.

Nuclear Receptors: Structure

N-terminal domain Hinge region C-terminal domain NH2 A-B C D E F -COOH DBD LBD Molecular mass is between 50,000 and 100,000 daltons.

• (A-B) N-terminal regulatory domain- domain is highly variable in sequence between various nuclear receptors.
• (C) DNA-binding domain (DBD)- highly conserved domain. Binds to DNA using specific sequences called hormone response elements (HRE).
• (D) Hinge region: a flexible domain that connects the DBD with the LBD. Influences trafficking and subcellular distribution.
• (E) Ligand binding domain (LBD)- moderately conserved in sequence but highly conserved in structure. Binds activator and repressor proteins.
• (F) C-terminal domain: Highly variable in sequence between various nuclear receptors.

Nuclear Receptor Structure Diagram

a N-terminal domain Hinge region C-terminal domain NH2 A-B C D E F COOH DBD LBD b Cytoplasm Ligands Nucleus Corepressors Coactivators Dimers Transcription target gene a XRE Hinge region allows conformational change upon ligand binding.

Nuclear Receptors: Types

Ligands for nuclear receptors such as hormones are small and lipophilic and so they can diffuse through the cell membrane and bind to nuclear receptors either in: The cytosol (type I NR) OR The nucleus (type II NR) of the cell.

Nuclear Receptors: Type I

Class I- largely receptors for steroid hormones- including the glucocorticoid and mineralocorticoid receptors (GR and MR), oestrogen, progesterone and androgen receptors (ER, PR and AR). Activation often causes a negative feedback effect to control biological events. In the absence of their ligand, these NRs are predominantly located in the cytoplasm, complexed with heat shock proteins and anchored to the cell cytoskeleton. When ligand binds, homodimers are formed which translocate to the nucleus. They can either transactivate or transrepress genes by binding to 'positive' or 'negative' hormone response elements.

Type I Nuclear Receptor Mechanism

a Steroid hormone Plasma membrane HSP complex Steroid receptor Nucleus Target-gene transcription Response element hormone 0 HSP changed cell function 0 NA dimer protein 1000 NR/hormone complex nuclear pore MRNA NR/HSP complex ribosome cytoplasm coactivator nuclear envelope mRNA RNA polymerase NR dimer nuclear DNA VAVAVAVAVAVAY HRE target gene cell membrane

Nuclear Receptors: Type II

Class II- Ligands for class II NRs are usually already present within the cell. Examples include the peroxisome proliferator-activated receptor (PPAR) that recognises fatty acids; the liver oxysterol receptor (LXR) that recognises and acts as a cholesterol sensor, the farnesoid (bile acid) receptor (FXR) also vitamin A and D receptors and thyroid receptors. Also includes receptors which recognise foreign substances- xenobiotics and drugs. A typical response might be to induce drug-metabolising enzymes such as CYP3A (which is responsible for metabolising about 60% of all prescription drugs) Normally operate as heterodimers together with the retinoid receptor (RXR) to mediate positive feedback effects (e.g. occupation of the receptor amplifies rather than inhibits a particular biological event).

Type II Nuclear Receptor Activation

changed cell function hormone cytoplasm protein COR Nucleus nuclear pore nuclear pore mRNA 2 ribosome nucleus 1 RNA polymerase 3 + T4 CoA coactivator corepressor TRE Gene DBD T Cytoplasm AVAVAVAVAVAVAY 4 HRE target gone HRE target gene nuclear envelope T or Gene expression corepressor O coactivator LBD RNA polymerase CXC CXC mRNA RXR TRZ T3 T3 T3 Thyroid receptor activation

Nuclear Receptors: Co-Reg Proteins

Once a nuclear receptor is bound to its HRE it can recruit a number of other proteins called TRANSCRIPTION COREGULATORS. These facilitate or inhibit the transcription of the associated target gene into mRNA. Nuclear receptors may bind specifically to a number of coregulator proteins, and thereby influence what happens within the cell. Coactivators Binding of an agonist eg Oestrogen to its nuclear receptors induces a conformation of the receptor that preferentially binds coactivator proteins which promote gene transcription. Corepressors Binding of an antagonist eg mifepristone at the progesterone receptor, induces a conformation of the receptor that preferentially binds corepressor proteins which repress gene transcription.

Co-Reg Proteins and Gene Expression

coactivator gene expression H12 agonist Estrogen Receptor coactivator H12 gene expression hormone Estrogen Receptor antagonist

Nuclear Receptors: Drug Targets

PPAR (Peroxisome proliferators activated receptor)

Types of PPAR Receptors: PPARa expressed in Liver, Kidney, Heart, Muscle, Adipose Tissue and others. PPAR ß expressed mainly in brain, adipose tissue and skin. PPAR y almost all tissues. PPARs heterodimerize with Liver X Receptors (LXR), Retinoid X Receptors (RXR) or Vitamin D receptors. Functions: Control of Cellular differentiation and development. Control of Metabolism (Carbohydrate, Lipid, Protein). Thought to be involved in the following diseases: Type 2 Diabetes, Atherosclerosis, Obesity and Hyperlipidaemia

PPAR Agonist Drugs

PPAR agonist drugs: Glitazones and Fibrates. PPARy agonists: Thiazolidinediones (Roziglitazone, Pioglitazone). Pharmacology: Agonist Receptor complex enhances transcription of responsive genes. Glucose entry to muscle and gluconeogenesis suppressed- reversal of insulin resistance PPARY, RKR - Ligands Fatty acids TZDs (Actos, Avandia) · Effects (adipose) Adipocyte differentiation Lipid storage Insulin sensitization Glucose (1) · Target genes 1GLUT4,CYP4B,ADRP, Adiponectin, Leptin, CD36 Į Resistin

MoA of Fibrates

MoA of fibrates: Effective in lowering serum triglycerides Second line behind statins and other drugs except where serum TGs are >10mmol/L Fibrate PPARx Activated PPARo/RXR 1 LDL particle size Į Triglycerides PPRE/target genes 1 HDL synthesis Į Inflammation 1 Reverse cholesterol transport Figure | Mechanism of action of fibrates. Abbreviations: HDL, high-density lipoprotein; LDL, low-density lipoprotein; PPARo, peroxisome proliferator-activated receptor-c; PPRE, peroxisome proliferator response elements; RXR, retinoid X receptor; 1 indicates increase; \ indicates decrease. From: Goldenberg et al 2008- Vascular Health and Risk Management. 4: 131-141

Glucocorticoids and Inflammation

Glucocorticoids and Inflammation: Annexin effects on neutrophils: ^L-selectin shedding glucocorticoid endothelial adherence ¥ endothelial transmigration membrane receptor Widely used in Asthma, UC, Crohns etc. Cell membrane COX-2 PLA2 Rapid (non-genomic) responses Immunophilin Anti-inflammatory Anti-pyretic Anti-hyperalgesic Immunophilin GR Hsp90 Annexin A1 cytoskeleton & dynein motor transport protein GR Hsp52 Hsp90 mRNA L ĮCOX-2 transcription dimer GR GR NNNNNNI GRE Nuclear transcription / repression of glucocorticoid-sensitive genes Topically in creams, eye drops etc. dyn protein synthesis Nucleus Delayed genomic response Inflammatory cytokines