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Receptors and Nucleic Acids

Prof. Alicia Catabay Department of Pharmaceutical Chemistry


1. Receptor superfamilies





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Ion channel receptors (Ligand gated ion channels)
• Receptors that control ion channels are part of a five-protein ion channel structure • They are glycoproteins which traverse the cell membrane • The fact that the receptor is part of the ion channel structure means that binding of a chemical messenger leads to a rapid response crucial to speed and efficiency of nerve transmission

Ion channel receptors (Ligand gated ion channels)
General structure
Binding site Messenger

Cell membrane

INDUCED FIT ‘GATING’ (ion channel opens)

Cell membrane

Five glycoprotein subunits traversing cell membrane

Cationic ion channels for K+, Na+, Ca2+ (e.g. nicotinic) = excitatory Anionic ion channels for Cl- (e.g. GABAA) = inhibitory


• Ion selectivity of different ion channels is dependent on amino acid lining the ion channel • Mutation of 1 amino acid changes a cationic selective ion channel to an anionic selective channel


4-TM B.2-TM (TM.transmembrane) ©1 .Families of receptors involved in the control of ion channels A.3-TM C.

5HT3 (serotonin) receptor 3.Nicotinic acid receptor 2.GABAA receptor Binding of a neurotransmitter to its binding site causes a conformational change in the receptor: opening up the central pore to allow ions to flow ©1 .Structure and function of 4-TM • Includes: 1. Glycine receptor 4.

Ion channel receptors (Ligand gated ion channels) Transverse view (nicotinic receptor) Binding sites Ion channel b a g a b d g a Cell membrane d a 2xa. g. d subunits Two ligand binding sites mainly on a-subunits ©1 . b.

2x b subunits Three ligand binding sites on a-subunits ©1 .Ion channel receptors (Ligand gated ion channels) Transverse view (glycine receptor) Binding sites Ion channel a a a b a a b b Cell membrane b a 3xa.

Ion channel receptors (Ligand gated ion channels) Structure of protein subunits (4-TM receptor subunits) Neurotransmitter binding region H2N Extracellular loop CO2H Cell membrane TM1 TM2 TM3 TM4 Intracellular loop Variable loop 4 Transmembrane (TM) regions (hydrophobic) ©1 .

Ion channel receptors (Ligand gated ion channels) 2.3 Detailed structure of ion channel TM4 TM1 TM3 TM4 TM1 TM3 TM4 TM2 TM1 TM2 TM3 TM3 Protein subunits TM1 TM2 TM3 TM1 TM4 TM2 TM2 TM4 Transmembrane regions Note: TM2 of each protein subunit ‘lines’ the central pore ©1 .

several knock on effects from initial binding process • Opening of locked gate nicotinic a receptor controlled ion channels ©1 .• Conformational change is quite complex.

Ion channel receptors (Ligand gated ion channels) Gating Neurotransmitter binds Induced fit at binding site ‘Domino effect’ Rotation of 2TM regions of each protein subunit Ion flow TM2 Cell membrane TM2 TM2 TM2 TM2 TM2 TM2 TM2 TM2 TM2 TM2 Transverse view of TM2 subunits TM2 Transverse view of TM2 subunits Closed Open ©1 .

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Ion channel receptors (Ligand gated ion channels) Gating • Fast response measured in msec • Ideal for transmission between nerves • Binding of messenger leads directly to ion flows across cell membrane • Ion flow = secondary effect (signal transduction) • Ion concentration within cell alters • Leads to variation in cell chemistry ©1 .

each has 3 transmembrane segments: TM1. TM2.hydrophobic segment embedded in the intracellular side of the membrane • Ligand binding site involve both N terminal chain and extracellular loop • Example: Calcium ion channel receptors ©1 .3-TM ion channel receptor • Ion channels controlled by these receptor also contain 5 protein subunits. and TM4 • TM2.

• These channels play important roles in neurotransmission. kinetic. and ion permeability properties. memory acquisition as well as acute and chronic disorders of the brain. • Most cells are responsive to glutamate which activates Calcium channels with different pharmacological.Calcium ion channels in the CNS • L-glutamate is the major excitatory neurotransmitter in the vertebrate central nervous system. ©1 .

General structure of the voltage-dependent calcium channels. ©1 .

Glutamate receptors ©1 .

Calcium Channel Blockers & the CNS: Ntype Omega-conotoxin MVIIa (SNX-111): A selective blocker for N-type calcium channels from the cone snail Conus magus ©1 .

2 TM Ion channels • A 5 protein subunit where each of the subunits contain 2 transmembrane segments • N.and C-terminal chains are both inside the cell. most protein are extracellular and include a hydrophobic region embeded in the outer surface of the cell membrance • Example: ATP is thought to control an ion channel of this type ©1 .

2-TM ion channel ©1 .

G-protein coupled receptor • Do not affect directly ion channels or enzymes • They activate signalling proteins called Gproteins which then initiate a signalling cascade involving a variety of enzymes • Also calle 7-TM receptors ©1 .

Single protein with 7 transmembrane regions Extracellular loops NH2 N-Terminal chain Transmembrane helix Membrane VII G protein binding region VI V IV III II I HO2C C-Terminal chain Variable intracellular loop Intracellular loops ©1 .G-protein-coupled receptors (7-TM receptors) Structure .

C-terminal intracellular ©1 .• GPCR are proteins embedded in the cell membrane and have region exposed to both the outside and inside of the cell • Protein chain winds back and forth through the cell membrane 7 times hence 7TM assigned Roman numbers I to VII from the N-terminal • 3 extracellular loops and 3 intracellular loops fairly constant in length except that which connects V and VI which varies depending on the specific receptor • N-terminal extracellular.

noradrenaline. dopamine.Ligands • Monoamines e. acetylcholine (muscarinic) • Nucleotides • Lipids • Hormones • Glutamate • Ca++ ©1 .g. histamine.

Ligand binding • Despite the large variety of GPCR’s their overall structure is similar • Thought to have a common binding site with different chemical messengers that could fit in different ways but… • Different structural ligand groups fit specific receptors ©1 .

top of TM helices + extracellular loops+ N-terminal chain C) Hormones .pocket in TM helices B) Peptide hormones .extracellular loops + N-terminal chain D) Glutamate .Ligand binding site .varies depending on receptor type Ligand A B C D A) Monoamines .N-terminal chain ©1 .

growth factors and cytokines • Loss of function may lead to developmental defects or hormone resistance • Over-expression can result in malignant disorders • Important targets in the design of new cancer drugs ©1 .Kinase-linked receptors (1TM) • Activates enzymes directly and do not require a G-protein • Activated by a large number of polypeptide hormones.

Tyrosine kinase linked receptors Extracellular N-terminal chain Ligand binding region NH2 Hydrophilic transmembrane region (a-helix) Cell membrane Intracellular C-terminal chain Catalytic binding region (closed in resting state) C O2 H ©1 .

Tyrosine kinase receptor • Example: Insulin receptor (tetrameric complex) Phosphorylation Cell membrane HO OH OH OH ATP ADP PO OP OP OP Insulin binding site Kinase active site ©1 .

the inevitable consequence of this relative nonselectivity is that these agents also affect other proliferating tissues. • However.Nucleic acid as drug targets • The rationale for using cytotoxic agents such as the nitrogen mustards and their more recent derivatives is that they target DNA transcription and/or replication in rapidly proliferating tumors. ©1 .

has presented the future development of DNA-targeting molecules with both an opportunity and a challenge: to devise gene-selective molecules that are uniquely able to downregulate the expression of a single abnormally expressed or mutant gene • In general we classify the drugs which act on DNA as intercalating agents.• The elucidation of the sequence of the human genome. ©1 . as well as the specific identification of many cancer-related genes. alkylating agents and chain cutters.

Intercalating agents • Compounds which are capable of slipping between layers of nucleic acid and base pairs and disrupting the shape of the double helix • Prevents replication and transcription • Drugs must be flat in oder to fit between the base pairs • These ligands are mostly polycyclic. ©1 . and therefore often make good nucleic acid stains. and planar. aromatic.

rhabdomyosarcoma). • DNA intercalators are used in chemotherapeutic treatment to inhibit DNA replication in rapidly growing cancer cells. proflavine.Intercalating agents • Intensively studied DNA intercalators include berberine. doxorubicin. and dactinomycin (used in Wilm's tumor. • Examples include doxorubicin (adriamycin) and daunorubicin (both of which are used in treatment of Hodgkin's lymphoma). ©1 . Ewing's Sarcoma. and thalidomide. ethidium bromide.

Right: DNA strand intercalated at three locations (red areas). ©1 . Left: unchanged DNA strand.Intercalating agents Intercalation induces structural distortions.

©1 .Intercalating agents Ethidium intercalated between two adeninethymine base pairs.

Alkylating agents • Highly electrophilic compounds reacting with nucleophiles to form strong covalent bonds • DNA has several nucleophilic groups • Alkylating agents involve reactions with guanine in DNA. ©1 . • This in turn inhibits their correct utilization by base pairing and causes a miscoding of DNA. • These drugs add methyl or other alkyl groups onto molecules where they do not belong.

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Alkylating Mechanism of Mechlorethamine With Guanine Base ©1 .

Chain cutters • Cuts DNA strands and prevent enzyme DNA ligase from repairing damage • Acts by creating radicals on the DNA stucture: reacts with oxygen to form peroxy species and DNA fragments Examples: Drugs used in anticancer therapy Calicheamicine Binds to the minor groove o DNA and cuts it by producing highly reactive radical species ©1 .

• Calicheamicin γ1 and the related enediyne esperamicin are the two most potent antitumor agents known.• The calicheamicins are a class of enediyne antibiotics derived from the bacterium Micromonospora echinospora with calicheamicin γ1 being the most notable. • It was isolated originally from a rock collected by a Scripps Research Institute chemist while hiking in Texas. • It is extremely toxic to all cells and its analogues have been used as targeted therapy against cancer. ©1 .

Calicheamicin ©1 .

which results in strand scission ©1 . generating a diradical species. then abstracts hydrogen atoms from the sugar backbone of DNA. • Like all enediynes. where they undergo a reaction analogous to the Bergman cyclization. 1.4-dehydrobenzene .• In vitro. calicheamicins bind with DNA in the minor groove. this diradical.

The Docking and Triggering of Dynemicin A and Calicheamicin in DNA. ©1 .

©1 . • When the genetic sequence of a particular gene is known to be causative of a particular disease. it is possible to synthesize a strand of nucleic acid (DNA. RNA or a chemical analogue) that will bind to the mRNA produced by that gene and inactivate it. effectively turning that gene "off". • This is because mRNA has to be single stranded for it to be translated.Antisense therapy • a form of treatment for genetic disorders or infections.

which is called the "sense" sequence • Example: • a sense segment of mRNA 5'AAGGUC-3' .• This synthesized nucleic acid is termed an "antisense“ oligonucleotides because its base sequence is complementary to the gene's messenger RNA (mRNA).would be blocked by the anti-sense mRNA segment 3'-UUCCAG-5' ©1 .

malignant glioma and malignant melanoma). has been approved by the U.pancreatic carcinoma. diabetes. • Most potential therapies have not yet produced significant clinical results. and diseases such as asthma and arthritis with an inflammatory component. FDA as a treatment for cytomegalovirus ©1 . colorectal carcinoma.S. though one antisense drug.• Antisense drugs are being researched to treat cancers (including lung cancer. formivirsen (marketed as Vitravene).

and the sequence of a gene is all that is needed.There are several aspects of antisense therapy utilizing oligonucleotides that are potentially advantageous over traditional drug mechanisms. • Potential sensitivity to therapy may be easily measured • Potential to produce longer lasting responses. versus just inhibition of protein typical with conventional therapies. ©1 . • Potential for enhanced binding affinity to target. some within one week. • Oligonucleotides may be manufactured quickly. by several orders of magnitude. Van der Waals and other forces used by standard agents to bind to protein targets. as hydrogen bonding between oligonucleotide and target appears to exceed.

Inactivating. In the future. Replacing a mutated gene that causes disease with a healthy copy of the gene. 3. ©1 . or “knocking out. Researchers are testing several approaches to gene therapy. including: 1.Gene therapy • Gene therapy is an experimental technique that uses genes to treat or prevent disease. 2.” a mutated gene that is functioning improperly. this technique may allow doctors to treat a disorder by inserting a gene into a patient’s cells instead of using drugs or surgery. Introducing a new gene into the body to help fight a disease.

• Although gene therapy is a promising treatment option for a number of diseases (including inherited disorders. • Gene therapy is currently only being tested for the treatment of diseases that have no other cures. the technique remains risky and is still under study to make sure that it will be safe and effective. some types of cancer. and certain viral infections). ©1 .

the new gene will make a functional protein. ©1 .Gene therapy using an Adenovirus vector. If the treatment is successful. A new gene is inserted into an adenovirus vector. which is used to introduce the modified DNA into a human cell.

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