Exam 1 Flashcards
1
Q
Gastric ulcer treatment
A
Use of antibiotics to cure the ulcer (not only treat the symptoms)
> helicobacter pylori
2
Q
Development of aspirin + use
A
from bark extract > extracted active ingredient salicylic acid > synthesized aspirin
Prevents inflammation
> active against pain, fever, rheuma
Blocks enzyme cyclo-oxygenase
> generates prostaglandins
3
Q
Classification of drugs
A
By pharmacological effect
– Analgesics, antibiotics, etc
– Different chemicals with same effect
– Many mechanisms of action
By chemical structure
– Salicylates, penicillins, opiates
– Same structure, but not always
have the same effect!!
By target system
– Anti-prostaglandines, anti-
histamines
– Different possibilities to interact
By target molecule
– E.g. Cyclo-oxygenase inhibitor
4
Q
Curare: basic concepts of chemical transmission and cellular communication
A
Curare results in paralysis via blocking chemical signals between nerve and muscle cells
Acetylcholine is an agonist: resulting in contraction of muscle
Curare is an antagonist:blocking contraction of muscle by preventing acetylcholine from binding to the nicotineacetylcholine receptor
5
Q
General cell knowledge
A
Mitochondria are the source of energy production
Ribosomes are the cell’s protein ‘factories’
Endoplasmic reticulum is the location for protein synthesis
High Na+ conc outside cell mem
High K+ conc inside cell
5
Q
Generally, what types of molecules can drugs bind to
A
Proteins
DNA
Lipids
6
Q
Rocuronium (NMB)
A
Muscle relaxation during operation
Neuromuscular blockade
Antagonist
Competitive binding to nAChR binding site
How to reverse the effect:
Increasing the concentation of the agonist (ACh) can increase its competitiveness against the antagonist for the binding site
The concentration of ACh can be increased via an acetylcholine esterase inhibitor
OR
New approach:
A host molecule that ‘captures’ NMB so it cannot bind to the receptor
> host molecule is cyclic and has a pore that fits the NMB
Speeds up the reversal of effects which would otherwise occur extremely slowly via the bodys natural metabolism of the drug
7
Q
Drug target binding
A
Drug targets are often large molecules (macromolecules, drugs are usually way smaller than target molecule)
Drugs bind to target binding sites
> Binding sites are typically hydrophobic hollows or clefts on the surface of macromolecules
Mostly, equilibrium between bound and unbound
> but irreversible binding is also option
Binding involves intermolecular bonds
Drug functional groups interact with target
8
Q
What types of intermolecular interactions are there
A
Electrostatic or ionic bond
Hydrogen bond
Van der Waals
Dipole - dipole interactions
Ion - dipole interactions
Induced dipole interactions
9
Q
Electrostatic or ionic bond interactions
A
Takes place between groups of opposite charge
Strongest of all reversible bonds
> The strength is inversely proportional to the distance between the two
groups
> The strength of interaction drops off less rapidly with distance than with
other forms of intermolecular interactions
Ionic bonds are the most important initial interactions as a drug enters the binding site
10
Q
Hydrogen bond interactions
A
Vary in strength
> Weaker than electrostatic interactions but stronger than van der Waals interactions
A hydrogen bond takes place between an electron-deficient H and an electron-rich heteroatom (N or O)
> The electron deficient H is usually attached to a heteroatom (O or N)
The electron deficient H is called a H-bond donor
The electron rich heteroatom is called a H-bond acceptor
11
Q
Van der Waals interactions
A
Very weak interactions
> Interactions drop off rapidly with distance
Occur between hydrophobic regions of drug & target
> Interactions between non-polar groups
> often alkyl groups or aromatic rings
Drug must be close to the binding region for interaction
12
Q
Dipole-dipole interactions
A
occurs if drug & binding site have dipole moments
> Dipoles align as the drug enters the binding site
Dipole alignment orientates the molecule in the binding site
Strength: decreases with distance more quickly than with electrostatic interactions, but less quickly than with van der Waals interactions
13
Q
Ion - dipole interactions
A
The charge on one molecule interacts with the dipole moment of another
> Stronger than a dipole-dipole interaction
Strength: falls off less rapidly with distance than for a dipole-dipole interaction
14
Q
Induced dipole interactions
A
the charge on one molecule induces a dipole on another
> a quaternary ammonium ion and an aromatic ring
15
Q
H2O and intermolecular interactions
A
(De)solvation
- Polar regions of a drug and its target are solvated prior to interaction (preventing interactions)
- Desolvation is necessary (stripping of H2O so that interactions between drug + target can form)
- Desolvation costs energy
- The energy gained by drug-target interactions must be greater than the costs for desolvation (otherwise drug is ineffective - when E for desolvation is greater)
Hydrophobic interactions
- Hydrophobic regions of a drug and its target are not solvated
- Water molecules interact with each other and form an ordered layer next to hydrophobic regions
- Represents a “negative entropy” (increase in order) but nature likes to be disordered
- When hydrophobic regions of a drug and its target interact these water molecules are freed
> Results in an increase in entropy
> Beneficial to binding energy
16
Q
What are the 20 amino acids + characteristics
A
Alanine, Arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine, tyrosine, histidine, isoleucine, leucine, lysine, methionine, Phenylalanine, threonine, tryptophan, valine
Head group is a zwitterion
Residue/side chain determines the nature of the amino acid binding
OH groups can be phosphorylated, making it -vely charged = affecting function
17
Q
Primary protein structure
A
Order in which amino acids are linked together
> a.a are linked through their head groups by peptide bonds = forming a polypeptide chain or backbone
18
Q
Secondary protein structure
A
regions of ordered structure adopted by the protein chain
alpha helix: results from coiling of protein chain, peptide bonds making up backbone are able to form hydrogen bonds between eachother
or
beta sheet: layering of protein chains one on top of another, held together by hydrogen bonds between peptide chains
> chains can run in opposite directions (antiparallel) or in the same direction (parallel)
19
Q
Tertiary structure
A
Overall 3D shape of protein which is formed by VdW, Hydrogen, and Ionic interactions and repulsion makes it fold up in a way to make it energetically favourable
Thiol groups (-SH) like to be linked together
> cysteine becomes very rigid due to its thiol groups interacting
> done via covalent bonds, namely disulfide links
Change in protein structure = change in function
20
Q
Misfolding of proteins
A
DNA mutation causes the production of a protein that cannot fold when initially synthesized by a ribosome;
* Mutations cause the production of a protein that is destabilized and thus unfolds easier once folded;
* Stress during the lifetime of the protein modifies it, causing it to be destabilized and partially unfolded;
* Often leads to protein aggregates
> Insoluble & usually very large;
> Very difficult for cells to breakdown;
> Not easy to transport;
> Continues to grow over time and may
> even recruit properly folded protein;
> Often (but not always) toxic to cells;
21
Q
Huntingtons disease
A
Protein misfolding disease
Polyglutamine repeat in the Huntington protein causes self association of the
protein in neurons
Cytoplasmic inclusions are formed that kill nerve cells
22
Q
Quartenary protein structure
A
Only in proteins that are made up of a number of protein subunits
Protein subunits interacting e.g 2 diff proteins interacting via hydrogen bonding
23
Q
Proteomics
A
Large networks of proteins which can be measured by mass-spectrometry
24
Types of proteins that are preferred drug targets
GPCRs
Enzymes
Hormones
Unknown
Ion Channels
Nuclear Receptors
25
Amino acids in enzyme active site
They are involved in:
- binding of substrates: involves intermolecular bonds, and induced fit (i.e shape of active site alters to maximize binding interactions), orientation, bond weakening
- catalysis of a reaction:
* Acid/base catalysis
> Proton’s are shuttling via histidine
>
* Nucleophilic groups in enzymes
> Ser, Cys
> react with substrate to form intermediates that offer an alternative reaction pathway
26
Enzymes
- Provide a reaction surface and a suitable env
- being reactants together and position them correctly so that they easily attain their transition-state configurations
- weaken bonds in reactants
- participate in reaction mechanism
- form stronger interactions with the transition-state than with the substrate or product
27
How does a substrate bind in the active site
Ionic bonding
> Active site often near enzyme surface (polar)
Dipole-dipole interactions
H-bonds
Van der Waals interaction (hydrophobic)
Binding of substrates often not too strong
– Concentrations of substrates often high
* Medicinal chemist looking for inhibitor:
– Make perfect fit (high binding affinity)
– No reaction or activation of drug!
– Will not easily leave active site
– Can be overcome with more substrate
> i.e Competitive binding
normally active site is more hydrophobic than surface of enzyme
28
Alzheimers treatment
AChE-inhibitor: Tacrine
Prevents the breakdown of ACh, prolonging ACh presence in synapse
29
Drugs that act by irreversible non-competitive binding: characteristics and examples
Drugs that can form covalent bonds w/ biomolecule:
> contain E+ functional group (e.g alkyl halide) that react with Ser, Cys residues (OH, SH nucleophilic groups)
Nerve gasses (AChE inhibitor, sarin interacts w/ serine residue in active site, pralidoxime can stop its effects by binding to sarine causing it to dissociate from the serine in the active site)
Aspirine (acts on COX via interacting w/ serine residue)
30
Regulation of enzyme function
Intracellular regulation
- allosteric mechanism via binding co-factor
> non-protein substances needed for enzymatic reaction
> e.g ATP as energy source
External regulation
- external signal via receptors, kinases etc (affect enzyme functions)
- via diffusible chemical signals e.g NO
31
Signalling via NO
activated nerve terminal releases ACh which binds to receptor > Arginine synthesizes NO via NO synthase > NO exits cell and diffuses into target cell (smooth muscle cell) > binds to target protein (activating enzyme Guanylyl cyclase, producing cGMP which alters myosin) > relaxation of the muscle and decrease in blood pressure
NO is a gas that can diffuse through membranes
> local action (5-10sec half life)
> binds to intracellular target protein
> used in viagra
32
Viagra
enzyme (PDE5) inhibitor
sexual stimulation > release of NT that synthesize NO > activation of GC > cGMP levels rise >
cGMP converted to GMP via PDE5 (this step is inhibited via viagram)
> causes cGMP levels to stay high and accumulate
> causes cGMP to go down its other pathway
> other pathway:
cGMP activates protein kinase G which phosphorylates myosin filaments = causes arterial smooth relaxation = increased arterial flow = erection
32
Isoenzymes
same function, slightly different proteins with different properties and expression profile (for different tissues)
e.g COX1 and COX2
COX-1 vs COX-2 story
– 65% amino acid homology
– Near identical catalytic sites
* Aspirin can give bleeding of stomach
– Blocks beneficial effects of some prostaglandins in GI tract and platelets
– COX-1 always present in GI-tract/platelets.
– COX-2 is induced in inflammation (e.g. rheumatoid arthritis)
> Massive production prostaglandins
– Aspirin is 10-100 times more potent at COX
Selective COX2 inhibitors e.g Rofecoxib less GI irritation
33
Why do drugs act?
1. Drug molecules bind proteins
> Receptors inside the cell (e.g. enzymes) or on membrane of the cell
2. Block/activate receptors
> Agonist activates, Antagonist blocks
33
How come acetylcholine exerts different effects
it binds to different receptors
e.g GPCRs (m1-m5), Ion channels (on skeletal muscle, neurons)
> Different receptors on different cells
> Same receptor, but different output, because of different signaling patterns
34
Simple biogenic amines (NTs) + how can molecules interact with them
dopamine, acetylcholine, noradrenaline, histamine, adrenaline, serotonin
a.a can interact via ionic interations and the +ve amine group (e.g serotonin and aspartate) (separate e.g, a prostaglandins COOH group can become COO- and interact ionically that way)
> they are all protonated at pH 7.4
35
Mechanisms of signalling
endocrine: Hormones (e.g. insuline) are released by gland (pancreas) in blood stream, find target cells, act throughout the body (liver) (long-distance)
paracrine: Local hormone signals are released to trigger neighboring cells (NO gas)
neuronal: Cell-cell interaction via release of NTs (e.g. ACh)
contact-dependent: membrane-bound signal molecule on signalling cell interacts with receptor on target cell (Embryonic development of cells depend on Delta-Notch interaction)
36
What do all signalling mechanisms rely on ?
Chemical messengers & their receptors
1) Intracellular nuclear receptors (slowest)
2-4: Membrane bound receptors
2) Ion channels (fastest)
3) G protein coupled receptors
4) Kinase-linked receptors
37
Nicotinic & muscarinic receptors
Nicotine and muscarine are ACh-like compounds (have a N+ amino group, and CH3 groups)
Muscarine m1-m5 receptors
- 5 GPCRs
Nicotine receptors
- Ion channels (many kinds!)
38
Receptor subtypes for biogenic amines + characteristics subtypes differ in
mainly GPCRs, but not always
Ach
– nicotine receptors (ion channels)
– m1-m5
DA
– D1-D5
HA
– H1-H4
Noradrenaline
- b1-b3
- a1-a2
Serotonine
– 5HT3 (ion channel)
– 5HT1, 5HT2, 5HT4-5HT7
Subtypes differ in:
- affinity for signal
- localisation
- signal transduction
39
Receptor families: basic structures
Kinase-linked receptors:
1 TM section, N-terminal outside, C-terminal inside with tyrosine kinase linked to it
ATP is required as a cofactor
GPCR:
7TM sections (+8th helix perpendicular to membrane) (charged groups at end of helices act as membrane anchors), N-terminal outside, C-terminal inside
G-protein binding site is intracellular
Ligand-gated ion-channels: (multisubunit)
4TM sections, N-terminal and C-terminal outside
Hydrophilic pore made up of 5 x 4TM hydrophobic regions
Intracellular steroid/nuclear receptor (multiprotein receptors, located in cytosol)
N terminal, DNA binding domain (DNA binds to DBD), Hinge region, ligand binding domain (ligand binds to LBD), C terminal
40
Steroid receptor family what binds to them?
Sex hormone receptors:
Estrogen (ER)
Progesterone (PR)
Adrenal hormone receptors:
Cortisol (GR)
Aldosterone (MR)
Testosterone (AR)
41
Activation of steroid receptors e.g cortisol
Cortisol passes plasma membrane >
once in the cell it binds to an intracellular receptor protein causing a conformational change >
Binding of hormone to LBD leads to translocation to nucleus: activated receptor-steroid complex moves into nucleus >
In nucleus DNA-BD binds to specific DNA recognition sequences: complex binds to the regulatory region of the target gene and activates transcription >
Binding leads to modulation of DNA transcription: transcription occurs = RNA >
mRNA leaves nuclear envelope via nuclear pore and is translated into a protein on a ribosome
Inherent slow responses (takes up to hours), actions of steroids actually need time to develop
42
Nuclear receptors and coactivators + changes in structure: agonist vs antagonist
When receptor is activated a hormone, the receptor releases corepressors and recruits coactivators, which help initiate transcription by recruiting RNA polymerase and other transcription machinery
> activated gene is then transcribed into mRNA
Binding of coactivator to receptor:
> Receptor with agonist: positioned well, leaving H12 (part of the receptor) in a good position that allows the coactivator to bind
> Receptor with antagonist: antagonist prevents H12 from sitting where it wants to, preventing a conformation that would allow the coactivator to bind = coactivator cannot bind and thus cannot signal for transcription
43
How to reach an intracellular steroid receptor?
Hormones (& drug molecules) need to pass the membrane i.e they need some hydrophobicity!
But not too much otherwise they get stuck in the membrane
> drugs are synthesized with OH groups (hydrophilicity) and also aromatic rings (hyrophobicity)
43
Neuronal signalling
Activated presynaptic nerve terminal releases NT into synaptic cleft > NT binds to receptor on the postsynaptic cell (a transmitter-gated ion channel) > ions entering causes changes in membrane potential (electrical signal)
very rapid response (msec)
Chemical signal > electrical
44
Ligand-gated ion channels (e.g ACh mechanism + structure)
ACh binds to binding site between 2 subunits causing conformational changes = TM2 segments rotate to open central pore of channel = Na+ ions can flow in
> Close structure is kinked
Made up of:
5 4TM proteins (i.e a transmembrane protein domain has 4 TM proteins, there are 5 of these)
Protein variations (lots of diff receptors can be made): a1-a10, b1-b4, gamma, delta, e
neuronal nAchR: a2b3 composition
Muscle specific: a1, gamma, delta, e
The 5 4TM domains form a pore (that can open and close): TM2 (1,2,3,4) of each protein subunit 'lines' the central pore
(Competitive) agonist / antagonist binding domain is found on the extracellular side
Cationic ion channels for K+, Na+, Ca2+ (e.g. nicotinic) = excitatory
Anionic ion channels for Cl- (e.g. GABAA) = inhibitory
45
How can you tell if a molecule is zwitterionic
if you can make positive and negative charged e.g
GAB: NH2 can have a positive charge, OH can have a negative charge
46
Colors of elements in diagrams
Nitrogen in blue
Oxygen in red
Carbon is black or gray
Hydrogen is white
47
E.g of drugs affecting ion channels
Diazepam: sleep aid
> blocks GABAa
Varenicline: stop smoking
> acts on nACh receptor
Ondansetron: nausea
> acts on 5HT3 receptor
48
Receptor-Tyrosine kinases: ligands and MoA
Insulin or growth factors (e.g EGF, VEGF, etc)
signal molecule (ligand) binds on N-terminus > catalytic domains of the Receptor-tyrosine kinase dimerize > kinase activity (on C-terminus) is stimulated = cross-phosphorylation of tyrosine subunit residues = activation of receptor tyrosine kinase > phosphorylated Tyrosine residues act as
“docking stations” for other proteins > intracellular signalling proteins bind to phosphorylated tyrosines forming signalling complexes > signal is relayed into the cells exterior exerting an effect (e.g protein synthesis, GLUT transporters)
49
Receptor-tyrosine kinase: dimers
Different options for dimers:
- EGF gives dimerization (it is a bivalent ligand which can bind 2 receptors at the same time), a single protein receptor that upon binding will dimerize (w/ another EGF-R)
- Insulin activates the receptor dimer, Insulin-R is already a dimer without the ligand, the ligand just activates it
50
Termination of effect of receptor-tyrosine kinase
Conducted via Tyrosine phosphatases
51
Drugs targeting Tyr-kinase linked recptors
lots of molecules can inhibit this receptor
e.g
EGFR inhibitor, VEGFR inhibitor
> have aromatic rings and amino groups that can be protonated for ionic interactions
52
Versatility of GPCR signalling (ligands)
Ca2+, proteins, small molecules (e.g NTs), light, odor, taste, infectious agents
53
GPCRs in disease
Not enough signal
> DA in Parkinson’s
Too much signal
> HA in allergy/release after mosquito bite
‘functional antagonism’
> relaxation airways in asthma via beta2- agonists
expression GPCR too low/absent
> vasopressin V2 receptor in Diabetus insipidus
GPCRs used by pathogens
> HIV/HHV-8
GPCR inactive/overactive after point mutation
54
GPCR halmark: use of rhodopsin
purified from cow eyes
- Responds to light
- High expression in eye
- inactive state in the dark
- Structural template for many GPCR models
55
Biogenic amine binding-pocket of GPCRs
e.g histamine N groups will interact with lysine N and asparagine O and aspartate O (from the binding pocket)
For larger molecules, e.g LH and FSH hormones, the ligand sits on top of the TMs
56
Thrombine binding to GPCRs
Thrombin uses its enzymatic function (protease) to cleave part of the extracellular part of the receptor, exposing a part of the receptor that can now activate the receptor (as it is free to move)
> converts fibrinogen into fibrin
57
GPCR polymorphism
Plays a role in HIV resistance
Individuals that are free from HIV despite repeated exposure:
> their leukocytes are resistant to HIV infection
> have a mutation in CCR5, a chemokine GPCR (GPCR with chemokine bound to it)
> a shift in the genetic code = stop codon earlier?
> CCR5 doesnt act as a receptor, its binding ability is gone so HIV cannot bind
58
First chemokine receptor antagonist
HIV binds to chemokine GPCRs
CCR5 antagonist prevents the binding of HIV
59
Other effects of genetic variation of CCR5
Transplant rejection
- CCL5, ligand for CCR5, increases in transplant
- white blood cells infiltrate transplant, inflammation
Delta32-CCR5 patients keep a transplant longer
- potential CCR5 antagonist in transplant rejection?
- D32-CCR5 patients are more West Nile Virus susceptible - WNV infects the brain, leading to inflammation and production of CCL5. This normally attracts T-cells to the rescue
60
Signal relaying: GPCRs
G-protein trimer relays the signal
> G protein has alpha, beta, gamma trimer, it is a guanine nucleotide binding protein
GDP is bound, it is phosphorylated to GTP, this cleaves the trimer into an activated beta-gamma complex and an activated alpha complex (with GTP bound)
Activated alpha subunit binds to target protein and activates it
Activated beta-gamma subunit can act on ion channels (opening them up)
Inactivation occurs via dephosphorylation of GTP into GDP by intrinsic GTPase activity in the alpha subunit
> all 3 protein subunits join back together
> returns GPCR back to basal state
61
GPCR receptor subtypes and signalling
Gs and Gi coupling
Gs - stimulatory
Gi - inhibitory
e.g DA receptors
Gs: D1 and D5
Gi: D2, D3, D4
62
G-protein subclasses and their functions
alpha-s: + adenylyl cyclase
alpha-i: - adenylyl cyclase, + K+ channels, - Ca2+ channels
alpha-q: + phospholipase C
alpha-12: + small G-proteins
63
Galpha-s-mediated signaling
Adenylate cyclase system
Gs binds to adenylyl cyclase and activates it = synthesis of cAMP (secondary messenger) which activates PKA = phosphorylation and activation of further enzymes = effects/function e.g relaxation smooth muscle In lung with beta2 agonist
64
cAMP production & breakdown
Production: ATP into cAMP (cyclized) via adenylyl cyclase
Breakdown: cAMP into 5'-AMP via phosphodiesterase (breaks cyclic bond)
Bidirectional control of AC (adenylyl cyclase) via Gs and Gi coupling (gs stimulates, gi inhibits)
65
physiological actions of cAMP following GPCR activation
Adrenaline > heart > increase in heart rate
Adrenaline > muscle > glycogen breakdown
Adrenaline > fat > fat breakdown
Adrenaline > lung > smooth muscle relaxation
Histamine > stomach > increase in acid secretion
Histamine > heart > increase in heart rate
Histamine > lung > smooth muscle relaxation
Actions of cAMP: cell-type dependent
- different PKA protein substrates
66
Cholera
Gs-protein disease
Bacteria vibrio cholerae
- ingested via water
Multiply in intestine
Epithelial cells leak fluid
- diarrhoea
Activates Gs
67
Galpha-q-mediated signaling
phospholipase C activation
Ligand binds to receptor
> activates Gq protein
> Activated Gq-alpha interacts with phospholipase C (PLC)
> PLC cleaves PIP2 into DAG and IP3
IP3 binds to IP₃ receptors on the endoplasmic reticulum (ER) = Ca²⁺ release into the cytoplasm
DAG (and Ca2+) activates PKC = phosphorylates various target proteins
Once IP3 and DAG have completed their tasks they are recombined to form PIP2
68
Method to measure PLC activation
via measuring Ca2+ levels inside the cell
Calcium can be measured using intracellular fluorescent calcium dyes that change fluorescence upon binding of Ca2+
> peak in flourescence (release of intracellular stores via IP3) and then slowly returns back to basal levels via influx of Ca2+ back in ER via Ca2+ channels
69
Calmodulin-Ca2+ binding
Released Ca2+ ions from IP3 activation bind to Calmoduline (a ca2+ binding protein) change conformation
> activate calmodulin-dependent protein kinases that phosphorylate and activate other enzymes
70
CaM-kinase dependent NO synthesis
1: Acetylcholine binds muscarinine receptor
(GPCR). Signalling via PLC gives Ca2+
2: Ca2+ binds calmoduline
3: Calmoduline activates CaM-kinase
4: CaM kinase activates NO-synthase, etc etc
71
Photo-receptive cells
2 types: rods and cones
3 million cones: distinguishing color
100 million rods: sensitive to light
the outer segment is full of discs which are full of photoreceptors i.e rhodopsin
72
Rhodopsine
GPCR for light
> doesn't require a ligand, it is already present and irreversibly bound (in the transmembrane section)
> The chromophore is bound to Lysine
> 11-cis-retinal is irreversibly covalently bound to lysine residue (inactive confirmation), when hit by a photon it converts to 11-trans-retinal (active conformation) which activates the GPCR
membrane outer segment has cation
-selective ion channel
> Open in the dark, Na+ influx, depends on cGMP levels
photon inhibits Na+-influx
> Activation of cGMP-specific PDE via eye-specific G-protein (called “transducin”)
> Membrane hyperpolarization (more negative inside)
Hyperpolarization transferred to synapse
> 1 photon closes 100’s of channels and gives ± 1 mV hyperpolarization
essentially open to closed depends on modulation of cGMP
73
breakdown and production of cGMP
GTP to cGMP via guanylate cyclase
cGMP to 5'-GMP via cGMP phosphodiesterase
74
Rhodpsin-transducin system
In the dark
Rhodopsin contains 11-cis retinal (inactive form)
High cGMP levels keep cGMP-gated Na⁺ channels open, allowing Na⁺ ions to enter the cell.
This results in a depolarized membrane potential and continuous release of glutamate (Glu) at the synapse.
Depolarization keeps the cell active in the dark
In the light:
Photon absorption (light) converts 11-cis retinal into all-trans retinal, activating rhodopsin.
Activated rhodopsin stimulates transducin (T), which then activates phosphodiesterase (PDE).
PDE breaks down cGMP, leading to a drop in cGMP levels.
Without cGMP, the Na⁺ channels close, stopping Na⁺ entry.
The cell hyperpolarizes, reducing glutamate release.
75
How can we see different colors
due to different rhodopsin receptor for different wavelengths
> red cone, green cone, blue cone
76
GPCRs: taste
Gustducin is the specific G-protein for taste
Neurons in the nose have GPCRs on neuron endings, when odorants bind to receptors the olfactory receptor cells are activated they send electrical signals to the brain
e.g of ligands (odorants)
D-limonene: smells like orange
L-limonene: smells like pine needles
77
Various forms of signal termination at different levels of the GPCR signalling
Transmitter > breakdown/re-uptake
G protein > intrinsic GTPase activity
Second messengers > breakdown
Proteins > dephosphorylation
Receptor > Regulation of function & localization via phosphorylation
78
Photopharmacology
modulation of drug targets with light-sensitive molecules
> Dynamic, reversible manner
> Spatially restricted, complementary to optogenetics
e.g Azobenzene
> isomerizes upon illumination
> Difference in molecular shape & polarity
79
Stereochemistry and drug action
Drug-protein interactions
– ”it should fit perfectly”
– Often large differences in activity for stereoisomers
Important characteristics for drugs that different between stereoisomers
– Absorption
– Metabolism
– Receptor action
– Excretion
80
Agonists design
Synthetic agonists bind reversibly to the binding site and produce the same conformational change as the natural ligand
Similar intermolecular bonds formed as with natural messenger
Agonists are often similar in structure to the natural messenger
- Identify important interactions in natural messenger
- Agonists are designed to have functional groups making the same interactions
- Usually required to make same number of interactions
81
Beta2 agonists in asthma
adrenaline binds to ADRB2 (GPCR)
beta2 agonist (salmeterol) binds = GDP phosphorylated to GTP
GTP > cAMP via adenyl cyclase = normal alveoli
Beta-AR Agonists
> weak activators vs full activators:
Agonists show similar, but distinct interactions with GPCR
Full agonist at beta receptor interact
with both serines (activation requires the 2 serine residues interacting)
82
What happens if a GPCR gets activated? + how can this be measured
GPCR activation leads to outside TM6 movement on the inside to allow G-protein docking & activation
> activation can be measured using flurophores and measuring flourescence
83
Antagonist design
Right shape to bind to receptor binding site but either fails to change shape of binding site or distorts it in wrong way
Most antagonist binds reversibly to the binding site and do not activate
Level of antagonism depends on strength of antagonist binding (affinity) and applied concentration
Messenger (agonist) is blocked from the binding site
Antagonists can form binding interactions with extra binding regions neighbouring the binding site for the natural messenger
84
ADRB2 antagonists
Against blood pressure
cAMP = high blood pressure
> antagonist inhibits GDP conversion to GTP and thus also cAMP production as the synthetic ADRB2 antagonist competes with the natural ADRB2 agonist
85
ADRB2: antagonist vs. agonist binding
Binding antagonist affected by
mutation of:
- D3.32
- N7.39
- S5.42
Binding agonist affected by
mutation of:
- D3.32
- N7.39
- S5.42
- S5.43 (seen in functional agonist, not seen in antagonism)
- S5.46 (seen in functional agonist, not seen in antagonism)
Same pocket, Agonist makes the pocket smaller
86
Design of oestrogen receptor antagonist
Oestradiol
hydrophobic skeleton
histamine interacts with OH group
Glutamine interacts w/ H on OH group
Arginine and H20 interact with O on OH group
His at one end, Glu and Arg at other end
Phenol and alcohol are important functional groups
Binding site is spacious and hydrophobic
* Phenol group of oestradiol is positioned in narrow slot
* Orientates rest of molecule
* Acts as agonist
Raloxifene is an antagonist (anti-osteoporesis agent)
* Phenol groups mimic phenol and alcohol of oestradiol
* Interaction with Asp-351 is important for antagonist activity
* Side chain prevents conformational change receptor
Asp with H of N+H group on side chain
Glu, Arg at on end, His at other end
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Irreversible antagonists
Antagonist binds irreversibly to the binding site
> Covalent bond is formed mostly with Ser/Thr or Cys
> Messenger is blocked from the binding site
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Allosteric ligands + e.g
Ligand binds reversibly to an allosteric binding site
Binding alters the shape of the receptor
Binding site is conformationally different
> can not bind endogenous signal
> Increases/decreases binding of effect endogenous signal
Cinacalet (Sensipar)
- Ligand binds Ca2+ sensing receptor
- Binding alters the shape of the receptor
- Used in problems with thyroid gland
- Regulates secretion of PTH, brings down Ca2+ levels
- Less bone fractures in “PTH-patients”
- N group and flourines
e.g m-glutamate receptors
> fly-trap allosteric binding site
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How to quantify drug target interaction
pKi, pKD, pEC50, pIC50
Affinity for target (“how strong does it bind”)
Functional activity
- potency (“at what concentration does it
have an effect”)
- efficacy (“how strong is the effect”)
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Michaels-Mentis CASE: the HIV protease reaction
HIV-I protease and viral polypeptide <> HIV-I protease / viral polypeptide complex > cleaved viral polypeptides
E + S <> ES > P
Inhibiting the activity of HIV-I protease is a strategy for combating the virus (Saquinavir)
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How to have an easy assay of a HIV protease?
Peptide with protease cleavage
site and fluorescent donor and
quencher
> HIV-I protease interacts, cleaving peptide = flourescence
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Plot initial rate v against [substrate]
Hyperbole
Tells us about Michaelis-Menten kinetics
Km = Vmax/2
Vmax
Vo = Vmax [S] / Km+[S]
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Target occupancy: Association rate constant, dissociation rate constant and equilibrium
Association constant: k+1 (A+R > AR)
Dissociation constant: k-1 (A+R < AR)
association rate = k+1 x [R] x [A]
dissociation rate = k-1 x [AR]
In equilibrium (at plateau of hyperbole)
association = dissociation
k+1 x [ A] x [ R] = k-1 x [AR]
Equilibrium dissociation constant = affinity
kd = k-1 / k+1
= [A] x [R] / [AR]
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How to quantify ligand-target occupancy ?
[AR] can be quantified using labeled ligands that emit flourescence or radioactivity
> separate into those free [A] and those bound [AR] via centrifugation and filtration, quantify bound
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Plot binding to target (AR) versus [ligand A]
Gives saturation binding – hyperbool, just like enzyme kinetics
* ligand affinity (Kd) at 50%
* total receptor number (Bmax)
Receptor number is finite = Rtotal
Rtotal = Bmax = [R] + [AR]
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Number of occupied receptors based on A affinity and concentration
When [A] >>> Kd
[AR] = Bmax
When [A] = Kd
[AD] = 1/2 Bmax
KD = [A] that occupies 50% of all receptors
e.g
Kd = 2*[A] , so f = 0.33, ie 33% of total
Kd = 0.1*[A] , so f = 0.91, ie 91% of total
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fractional occupancy: Reversible ligand-target binding under equilibrium conditions
[AR] = [A]xBmax / Kd+[A]
[AR] / Bmax = [A] / Kd+[A]
different fAR curves are due to diff compounds with diff Kd values
either linear scale (Kd, hyperbolic) or 10Log scale (pKd, sigmoidal)
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How to measure affinity of reversible unlabelled ligands?
Displacement (competition) binding
> Displace fixed [radioligand] from receptor by increasing concentrations unlabeled ligand
B: unlabelled
A: labelled ligand
Graph of fAR against log [B] conc
> inverted S shape (high plateau to low plateau): as B conc increases the fraction of AR decreases
IC50 is [B] giving 0,5 fractional AR occupancy
IC50: inhibitory conc at 50%, measurement of potency of B to displace A*
IC50 =/= affinity of B
At higher [A*], it is more difficult for
unlabeled ligand B to displace;
Higher [B] are needed to fully occupy the
target; IC50 value shifts to the right
IC50 value in binding experiments depends on experimental conditions and is not just a property of compound B
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Displacement (competition) binding – Cheng Prusoff
IC50 = Ki,B x ( ([A*]/Kd,A) + 1)
Convert IC50 into Ki value
Ki value: actual affinity constant KD, displacer B
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Agonist Dose-response curve: potency
% response against log [A]: sigmoidal
Potency of agonist: [A] to produce given response
EC50 = [A] giving half maximal response (-log EC50 also used)
> Partial agonists: reduced maximal response at 100% occupancy. A partial agonist needs to occupy all receptors to produce its (low) maximal response (e.g partial beta agonists)
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Occupancy theory of Clark
A + R <> AR > response (sigmoidal)
> anything that binds contributes to the effect
Response–[agonist] relationship is governed by agonist–receptor occupancy, i.e. how much AR is formed (Effect[A] = Emax x ([A]/Kd+[A]))
When all R is occupied: E = Emax
At 50% occupancy: E = 50% Emax
10% occupied = 10% response
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Ariens - Intrinsic activity (alpha) theory
Concept of intrinsic activity to help explain behavior of partial agonists (with lower Emax)
Effect [A] = alpha x ([A]/Kd+[A]) x Emax
alpha: coefficient of intrinsic activity
> a measure of efficacy (how strong is the effect)
alpha = Emax partial agonist / Emax full agonist
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Full and partial agonism: fractional response against potency
Partial agonists: reduced maximal response at 100% occupancy
Response is governed by target binding (EC50 = KD), i.e. ligand needs to occupy all receptors to give its maximal effect
> To get a max response of partial agonist you need 100% occupancy
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Alpha values for antagonists and agonists
when alpha = 0
> a molecule that binds but does not activate i.e an antagonist
Antagonists: no efficacy, alpha = 0
Full Agonist: alpha = 1
Partial agonist: alpha > 0, < 1
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Why are there different dose response curves for partial agonists in different cells even though it is the same molecule and same receptor?
Partial agonism is cell dependent: f(S)
> varying receptor expression levels
> varying G-protein expression levels
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Stephenson: evolution of occupancy theory
Not a strict link between binding and response, but between binding and “stimulus”
> a tissue determines how the stimulus (e.g receptor activation) affects the response
> stimulus is translated differently in different tissues to a response
A + R <> AR > stimulus > response
Stimulus S = e X ([A]/Kd+[A])
Effect E = f(S)
e = efficacy
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How can f(S) be different
e.g Burimamide can give a smallamount of cAMP in CHO cells with a lot of H2Rs
But no effect on heart, which has only low
levels of same GPCR = not enough levels of
cAMP will be made
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Receptor occupancy and signaling: Signal amplification
Signal amplification:
1 activated GPCR can activate multiple G-proteins, leading to a lot of cAMP molecules and etc the rest of the pathway (e.g with LH producing cAMP > testosterone)
Also, explains why f (S) from Stephenson will be different in different cells
Different levels of GPCRs, G-proteins etc…
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"spare receptors"
e.g HCG
HCG (an agonist) does not need to occupy all LH receptors for max stimulus generation for cAMP and testosterone production
Bmax determines:
> absolute amount of occupied receptors
> absolute stimulus at each ligand concentration
> location of full agonist DRCs (EC50) vs occupancy (KD)
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What happens when you lower receptor number with partial agonists + how can you decrease receptor number experimentally
lower Emax response at 100% occupancy
> If you lower the receptor number, you further reduce the Emax, but not the EC50 value
Reduce receptor number via irreversible antagonists
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Non-competitive insurmountable antagonist: Partial agonist and full agonist DRC in presence of an irreversible antagonist
Partial agonist:
Less receptor available for PA (partial agonist)
> leads to reduced response but EC50 is unaltered
Full agonist:
Less receptor available for A (agonist)
> EC50 shifts to right initially
> when it matches the occupancy curve, Emax will then drop as well
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Competitive (surmountable) antagonism: Agonist dose response in presence of an antagonist
Competition for common binding site
> Shift of EC50 agonist (shift to right)
> Emax is still obtained, if [A] is high enough
increasing [antagonist] shifts agonist DRC rightwards
Parallel shifts if similar increase in [antagonist B] is tested
How much it shifts indicates how well B (antagonist) binds
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Competitive antagonist: How to quantify strength of blockade?
Determine dose ratio (DR)
DR = EC50,A' / EC50,A
> EC50,A (no B present) and EC50,A’ (with B present)
Schild analysis
log (DR-1) = log [B] - log Kd,B
log (DR-1) = log [B] + pA2
Affinity of antagonist [B]:
pA2 = - log Kd,B
Linear relationship between log [B] and log (DR-1): i.e however much you increase [B], the DR increases by the same amount
> slope plot = 1
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Non-competitive antagonism
Effects agonist DRC like irreversible blocker
– Unsurmountable
Drug act at separate inhibitory site on protein
– Allosteric antagonism
More often drugs act via allosteric mechanisms
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How come some graphs show negative effect?
Inverse agonist that shifts equilibrium towards inactive conformation
(antagonist do not affect equilibrium (which naturally favours inactive conformation))
Agonist shift equilibrium towards active conformation
Alpha (intrinsic activity) of inverse agonist:
-1 to 0
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Constitutive active GPCR
equilibrium (between inactive and active conformation of receptors) favors the active conformation
constitutive activity may result from gene mutations
e.g LHR
hLH-R: LH-independent testosterone production = boosts pubertal development
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Strength of intermolecular forces
VdW < dipole - dipole < hydrogen bond < ion-dipole < covalent
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Most important bonding interactions in protein tertiary structure?
VdW and hydrogen bonding
> thus the centre of the protein must be hydrophobic and non-polar
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Role of the planar peptide bond in tertiary structure
Bond rotation is hindered due to trans conformation = number of possible conformations is restricted = more likely a specific conformation is adopted
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What is genetic polymorphism
a difference of one base pair in every thousand, results in a diff a.a being introduced into the protein
> usually no observable effect but sometimes can adversely affect proper functioning of an enzyme
> can alter sensitivity of an enzyme toward a drug
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How can drugs distinguish between the same receptors for the same ligand in different parts of the body
there a slight variations in amino acid composition, if the variations are in the binding site, drugs can be designed to distinguish between these
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Signal transduction in GPCRs
first stage (splitting of G-protein) is common to all of the 7-TM receptors
> subsequent stages depend on what type of G-protein is involved and which specific alpha-subunit is formed e.g alpha-s, alpha-i, alpha-q
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G-alpha-i signalling
Gi-protein interacts with different receptors than those interacting with Gs-protein
>alpha-i subunit binds to adenylyl cyclase and inhibits it
existance of Gi and Gs proteins = generation of secondary messenger cAMP is under dual control
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Can competitive inhibitors bind to the active site but not compete with the substrate?
Yes, via binding to the binding site of the cofactor
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What is a partial agonist
acts as an agonist + produces effect but effect is not as great as one would get with a full agonist
> conformational change induced may not be ideal = subsequent effects of receptor activation are decreased
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What is an inverse agonist
Same effect as an antagonist: i.e it binds to a receptor + fails to activate it
But some receptors are found to have an inherent activity even in the absence of a chemical messenger (constitutional activity)
> an inverse agonist is also capable of preventing this activity
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Desensitization and sensitization
Desensitization: drugs bind relatively strongly to receptor + switch it on but then block receptor after certain period of time
Sensitization
> cell synthesizes more receptors to compensate for the receptors that are blocked
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Intercalating drugs acting on DNA
Compounds containing planar or heteroaromatic features which slip between the base-pair layers of the DNA double helix
> aromatic rings are held there by VdW forces with base pairs above and below
> these drugs contain ionized groups which interact with the charged phosphate groups of the DNA backbone
Processes take place which may prevent replication and transcription leading to cell death
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Topoisomerase poisons (non-intercalating)
stabilize the normally transient cleavable complex that is formed between DNA and topoisomerase enzymes = inhibiting the rejoining of the DNA strand or strands
> anti-cancer agents
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Alkylating and metallating agents
alkylating agents: highly electrophilic and react w/ Nu-, forming strong covalent bonds
> Nu- groups on DNA: guanine
> If 2 E+ groups are present = interstrand and/or intrastrand cross-linking replication or transcription is disrupted
e.g nitrogen mustards (react w/ guanine groups to produce cross-linking), nitrosoureas, busulfan, cisplatin
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Chain cutters
cut the strands of DNA and prevent DNA ligase from repairing the damage
> act by creating radicals on DNA
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Chain terminators
act as 'false substrates' and are incorporated into the growing DNA chain during replication
> chain can no longer be extended and chain growth is terminated
Characteristics
1) they have to be recognized by the DNA template bu interacting with a nucleic acid base on the template strand
2) have a triphosphate group
3) impossibble for any further building blocks to be added
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Anti-sense therapy
uses oligonucleotides that are complementary to small sections of mRNA and prevent translation
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Transition-state analogues
They are enzyme inhibitors designed to mimic the transition state of an enzyme-catalyzed reaction mechanism
> bind more strongly than either the substrate or the product
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Suicide substrates
molecules that act as substrates for a target enzyme but are converted into highly reactive species which react with amino acid residues in the active site to form covalent bonds + act as irreversible inhibitors
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Transport proteins as drug targets
transport proteins transport polar molecules across hydrophobic cell membrane
> drugs can be designed to take advantage of this transport system in order to gain access to cell
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Viral structural proteins as drug targets
these drugs bind to structural proteins making up the capsid = prevent uncoating process
> antiviral agents
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Tubulin as a drug target
tubulin is involved in the polymerization and depolymerization of microtubules
> drugs can inhibit polymerization process by binding to tubulin (eg colchicine)
> or they can bind to microtubules, stabilizing them = inhibiting depolymerization (eg taxol)
leads to inability of cell division
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Inhibiting protein-protein interactions
Use of antibodies or protein-protein binding inhibitors
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Lipids as drug targets
1) disrupting lipid structure of cell membranes via building 'tunnels' e.g antifungal agent (a tunneling molecule)
> tunnel is lined with hydroxyl groups = hydrophilic = polar contents of cell drain away
2) drugs that act as ion carriers (e.g valinomycin)
> cyclic
> outward hydrophobic side chains interact via VdW with fatty lipid interior of cell membrane
> polar hydrophilic groups in center of ring can accomodate an ion
> disrupts ionic equilibriumm of the cell
3) or tethering drugs: tethered to the membranes of cells so that they interact more easily with molecular targets also tethered to the membrane
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Carbohydrates as drug targets
Use of antigens and antibodies
> to help w transplant acceptance
