PROTEINS AS DRUG TARGETS 2:

Receptors and Nucleic Acids

Prof. Alicia Catabay Department of Pharmaceutical Chemistry

1. Receptor superfamilies

RESPONSE TIME

ION CHANNEL RECEPTORS

msecs
MEMBRANE BOUND

G-PROTEIN COUPLED RECEPTORS

KINASE LINKED RECEPTORS

seconds minutes

INTRACELLULAR RECEPTORS

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
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Ion channel receptors (Ligand gated ion channels)

General structure
Receptor
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

Families of receptors involved in the control of ion channels

A. 4-TM B.3-TM C.2-TM
(TM- transmembrane)

Structure and function of 4-TM

Includes: 1.Nicotinic acid receptor 2.5HT3 (serotonin) receptor 3. Glycine receptor 4.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

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

2xa, b, g, d subunits

Two ligand binding sites mainly on a-subunits

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

3xa, 2x b subunits

Three ligand binding sites on a-subunits

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

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

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

Link

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

3-TM ion channel receptor

Ion channels controlled by these receptor also contain 5 protein subunits; each has 3 transmembrane segments: TM1, TM2, and TM4 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

Calcium ion channels in the CNS

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

calcium channels.

Glutamate receptors

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

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

2-TM ion channel

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
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G-protein-coupled receptors (7-TM receptors)

Structure - Single protein with 7 transmembrane regions
Extracellular loops NH2 N-Terminal chain

Transmembrane helix
Membrane VII G protein binding region VI

IV

III

II

HO2C
C-Terminal chain

Variable intracellular loop

Intracellular loops

 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; C-terminal intracellular
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Ligands
Monoamines e.g. dopamine, histamine, noradrenaline, acetylcholine (muscarinic) Nucleotides

Lipids
Hormones

Glutamate
Ca++
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Ligand binding
Despite the large variety of GPCRs 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

Ligand binding site - varies depending on receptor type

Ligand
A B C D

A) Monoamines - pocket in TM helices

B) Peptide hormones - top of TM helices + extracellular loops+ N-terminal chain

C) Hormones - extracellular loops + N-terminal chain

D) Glutamate - N-terminal chain 1

Kinase-linked receptors (1TM)

Activates enzymes directly and do not require a G-protein Activated by a large number of polypeptide hormones, 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
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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

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

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.

However, the inevitable consequence of this relative nonselectivity is that these agents also affect other proliferating tissues.

 The elucidation of the sequence of the human genome, as well as the specific identification of many cancer-related genes, 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, alkylating agents and chain cutters.
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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, aromatic, and planar, and therefore often make good nucleic acid stains.
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Intercalating agents
Intensively studied DNA intercalators include berberine, ethidium bromide, proflavine, doxorubicin, and thalidomide. DNA intercalators are used in chemotherapeutic treatment to inhibit DNA replication in rapidly growing cancer cells.
Examples include doxorubicin (adriamycin) and daunorubicin (both of which are used in treatment of Hodgkin's lymphoma), and dactinomycin (used in Wilm's tumor, Ewing's Sarcoma, rhabdomyosarcoma).
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Intercalating agents
Intercalation induces structural distortions.
Left: unchanged DNA strand. Right: DNA strand intercalated at three locations (red areas).

Intercalating agents

Ethidium intercalated between two adeninethymine base pairs.

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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. These drugs add methyl or other alkyl groups onto molecules where they do not belong. This in turn inhibits their correct utilization by base pairing and causes a miscoding of DNA.
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Alkylating Mechanism of Mechlorethamine With Guanine Base

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
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 The calicheamicins are a class of enediyne antibiotics derived from the bacterium Micromonospora echinospora with calicheamicin 1 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. Calicheamicin 1 and the related enediyne esperamicin are the two most potent antitumor agents known.
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Calicheamicin

 In vitro, calicheamicins bind with DNA in the minor groove, where they undergo a reaction analogous to the Bergman cyclization, generating a diradical species. Like all enediynes, this diradical, 1,4-dehydrobenzene , then abstracts hydrogen atoms from the sugar backbone of DNA, which results in strand scission

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

Antisense therapy
a form of treatment for genetic disorders or infections. 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.
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 This synthesized nucleic acid is termed an "antisense oligonucleotides because its base sequence is complementary to the gene's messenger RNA (mRNA), which is called the "sense" sequence Example: a sense segment of mRNA 5'AAGGUC-3' - would be blocked by the anti-sense mRNA segment 3'-UUCCAG-5'

 Antisense drugs are being researched to treat cancers (including lung cancer, colorectal carcinoma,pancreatic carcinoma, malignant glioma and malignant melanoma), diabetes, and diseases such as asthma and arthritis with an inflammatory component. Most potential therapies have not yet produced significant clinical results, though one antisense drug, formivirsen (marketed as Vitravene), has been approved by the U.S. FDA as a treatment for cytomegalovirus
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There are several aspects of antisense therapy utilizing oligonucleotides that are potentially advantageous over traditional drug mechanisms. Oligonucleotides may be manufactured quickly, some within one week, and the sequence of a gene is all that is needed. Potential sensitivity to therapy may be easily measured Potential to produce longer lasting responses, versus just inhibition of protein typical with conventional therapies. Potential for enhanced binding affinity to target, as hydrogen bonding between oligonucleotide and target appears to exceed, by several orders of magnitude, Van der Waals and other forces used by standard agents to bind to protein targets.
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Gene therapy
Gene therapy is an experimental technique that uses genes to treat or prevent disease. In the future, this technique may allow doctors to treat a disorder by inserting a gene into a patients cells instead of using drugs or surgery. Researchers are testing several approaches to gene therapy, including: 1. Replacing a mutated gene that causes disease with a healthy copy of the gene. 2. Inactivating, or knocking out, a mutated gene that is functioning improperly. 3. Introducing a new gene into the body to help fight a disease. 1

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

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