Exam Questions Pharmacogenetics: Which one of the following statements is CORRECT with regard to warfarin? A. The cytochrome P450 CYP2D6 is responsible for the metabolism of warfarin B. Warfarin exerts its anticoagulant effect by stimulating vitamin K epoxide reductase complex (VKORC1) C. Patients with CYP2C9 or VKORC1 gene variations may require lower d oses of warfarin to achieve the desired anticoagulant effect D. Healthcare professionals are now required to test patients for their CYP2C9 or VKORC1 genetic information prior to prescribing warfarin E. Warfarin dose requirements is irrelevant to age, ethnicity, and other drugs. Genetic variations of pharmacokinetics and pharmacod ynamics‐related genes are associated with variations in drug responses. Give an example to describe how the existence of specific genetic variants affects pharmacokinetic and pharmacodynamic responses to a drug. (10 min) Variation in CYP2C9 genotypes can result in an effect in the metabolism of warfarin, where patients with variant alleles CYP2C9*2 or CYP2C9*3 can have a decreased warfarin metabolism, which would increase the effects of taking a regular dose of warfarin. When warfarin is not metabolised effectively, the anticoagulation effects it provides can be exacerbated to dangerous levels, to a point po int where CCGX is down-regulated so much that glutamic a cid is no longer used to produce carbox yglutamic acid and then on to produce clotting factors (II, IV, IX, X). If a patient taking warfarin possesses the decreased metabolising alleles then a lower dose is required for a therapeutic effect, otherwise the blood will be thinned too much and risks lifethreatening bleeding. At risk patients can now b e genotyped to accurately prescribe dosages that will be metabolised effectively, and warfarin dosages are started in the lower therapeutic ranges and monitored using patients’ INR scores. Transcription/Nuclear Factors: MCQ: Which ONE of the following statements regarding glucocorticoids is CORRECT? A. They can reduce expression of anti-inflammatory genes B. Their actions occur immediately C. Glucocorticoid receptors are zinc finger transcription factors D. In the absence of ligand, glucocorticoid receptors are predominantly located in the nucleus E. The steroid-receptor complex is able to bind to the glucocorticoid response element (GRE) on the RNA
SAQ:
Describe how progesterone interacts with its receptors and how this leads to modulation of gene expression Progesterone, a type I nuclear receptor, exists in the cytosol as a homodimer, and binds 2 hormone response elements (HREs) that have inverted repeat se ctions (this is how the nuclear receptor determines selectivity over HREs that have simple repeat sections). The binding of the nuclear receptor to the HRE allows it to transactivate or transrepress the gene expression for multiple genes. Progestrone receptors are indicated by cancer and osteoporosis. Protein Structure 1.Translate the amino acid sequence of the peptide below into one letter code. Would you expect this peptide to be charged at neutral pH in water? Indicate any charges the peptide may have at pH 7. Which amino acid in the peptide is the N terminus?
Glu-Leu-Asn-Ala-Ser-Phe-Lys.
E-L-N-A-S-F-K 2. Identify potential groups in a protein that can form hydrogen bonds with a serine side chain. Explain your answers. 3. Proteins that span biological membranes often contain a-helices. Given that the insides of membranes are highly hydrophobic, predict the type of amino acids to be found in such a helix, give examples. Why is an a-helix particularly suited to exist in the hydrophobic environment o f the membrane interior? 4. Glycine is a highly conserved amino acid residue in the evolution of proteins. Why? 5. Choose the correct answer for questions a) to h) from the list below. Not all of the answers will be used. L-amino acids, water, protons, zwitterions, secondary structure, tertiary structure, Ramachandran, cysteine, extracellular, histidine, proline, Sanger, D -amino acids a) Chiral type of building blocks found in proteins. b) Disulfide bonds are formed by pairs of which amino acid? c) The amino acid with a pK near neutral pH. proline d) When a peptide bond is formed, what molecule is also made? e) The amino acid with a distinct structure, causing it to affect protein backbone structure. f) Name of the plot that allows one to investigate the likely orientation of certain amino acid pairs. g) The type of structure to which a helices, β sheets, and turns belong. h) The overall structure of a polypeptide chain is referred to as...
Enzyme Modulation Given the kinetic data in the table on the next slide, how would you determine if the enzyme obeys Michaelis-Menten kinetics? What kind of inhibition? Hint: Use Lineweaver-Burk plots: 1/v
0
= (K
/v
[S]) + (1/v
M max
)
max
[S] (μM)
Velocity (μmoles/min) No inhibitor
Velocity (μmoles/min) With inhibitor
3
10.4
4.1
5
14.5
6.4
10
22.5
11.3
30
33.8
22.6
90
40.5
33.8
Volatge-gated Ion Channels 1. Describe the similarities and differences in topology and quaternary structure of the voltage-dependent sodium, potassium and calcium channels. Topology: Sodium channel has an inactivation gate between subunit III and IV, an S4 voltage sensor in subunit I as well as a pore loop between S5 and S6 Potassium has an S4 voltage sensor and S5/6 pore loop in subunit I Calcium has an S4 voltage sensor and S5/6 pore loop in subunit I Quaternary structure: Sodium: quaternary monomer, four tandem repeats in a single protein Potassium: Tetramer, four separate subunits Calcium: quaternary monomer, four tandem repeats in a single protein 2. What role does the fourth transmembrane segment play in the gating mechanism of potassium channels?
The fourth transmembrane segment is the voltage sensor, the molecular component that responds to changes in membrane potential. The sensor can detect minute changes in current that will open or close the K-gated ion channel, and from -40mV to 0mV the channel will be opened to help regulate the amount of intracellular potassium. 3. Describe the relationship between the number of v oltage sensor domains and the number of kinetic (voltage-dependent) steps required before the potassium channel opens. The number of kinetic steps required to activate a potassium channel is directly proportional to the number of voltage sensing domains the channel has. The simplest form, a 1 step process, has C ->/<- with O with rate constants of k f for the forward reaction and k b for the backwards reaction. Based on recorded activation currents it h as been shown that at least 4 voltage dependents steps are required to open a potassium voltage-gated channel Ligand-gated Ion Channels 1. Describe the topology and quaternary structure of the nicotinic acetylcholine receptors. What structures are responsible for forming the ion channel pore and the orthosteric ligand binding site? The nAChR is a pentamer consisting of two alpha subunits and three b eta subunits. There is a variable intracellular loop between M3/4 and the agonist binding sites are located between an alpha subunit and its adjacent beta subunit. The orthosteric binding site is in the extracellular domain of the channel, and the allosteric site further down towards the plasma membrane. 2. For a simple mechanism with two agonist binding sites for a ligand -gated ion channel, describe the actions of a competitive inhibitor and the consequences for channel function. As we know, competitive inhibitors compete with a gonists for binding at the orthosteric site of the channel. The binding of a competitive inhibitor does not reduce the overall probability that a channel will be open, but it does cause a right-shift of the probability curve, so that an increased agonist concentration is required to cause the re gular ligand-binding effects. Catalytic Receptors Describe the mechanism by which a receptor tyrosine kinase, such as the insulin receptor; (i) transmits a signal to the nucleus after the binding o f insulin Most RTKs exist as monomers, the insulin receptor exists as a d imer that consists of two pairs of polypeptide chains that are linked by disulphide bonds. When a growth factor binds to a regular RTK (not insulin receptor), the binding causes a conformational change dimerisation, which leads to a tyrosine autophosphorylation and the binding of SH2-domain protein. Grb2 is then phosphorylated and the Ras GDP/GTP exchange is activated, causing a kinase cascade that leads to a regulation of gene transcription. (ii) how this signalling is terminated Since phosphorylated proteins to not hydrolyse spontaneously, phosphatases are required to remove phosphate groups. Enzymes such as protein tyrosine phosphatase-1B (PTP1B) are used in either a site-selective or non-specific form to either cause a second signalling state or to turn the RTK off. PTP1B is used to remove the phosphate groups from the dimerised RTK and turn the signalling off. GPCR Families
1. The GPCR superfamily can be divided into 6 families. (i) List each of these families, Family A, B, C, D, E and F (ii) for 4 of the families discuss the differences between each of them in terms of Nterminus, orthosteric binding sites A: binding site is in the transmembrane domain, small N-terminus B: binding site is near the N-terminus and ex tracellular loops, larger N-terminus, contain EGF-like domains C: binding site is in the venus flytrap domain o f the N-terminus, very large N-terminus (~560 AA) F: binding site is in cysteine-rich domain of N-terminus, large N-terminus that has a cysteine-rich domain. (iii) give a named example of a member of each family. A: rhodopsin B: secretin C: metabotropic glutamate/pheromone F: Frizzled/smoothened 2. Discuss the evidence that suggests that Family C GPCRs act as dimers The notion that the GABAB receptor can only be activated by trans-activation suggests that family C GPCRs act as dimers. This is shown b y agonist binding to the GABAB1 VFT and leading to the activation of the GABAB2 7TM. mGluRs can be activated by both cis- and trans-activation, but this does not necessarily disprove the theory that family C GPCRs act as dimers. GPCR Motifs 1. G-protein coupled receptors can be subdivided into six families i) Compare and contrast the structural features of the n-termini of the members of Family
A,
B&C A: small N-terminus, B: larger N-terminus that contains EGF-like binding domains, C: very large N-terminus (~560 AAs) that is in the “venus flytrap” shape. ii) Discuss the difference in the sites and modes of ligand binding between each of these families A: ligands bind to the transmembrane helices or t o extracellular loops B: B family GPCRs have large N-termini, and the re are several structures within it that help to identify the binding sites, the GCGR ECD has both an alpha-helix and antiparallel beta-sheets, and the orthosteric binding domain is below in the GCGR linker. (GCGR is the glucagon receptor precursor) 2. Describe the structural changes that occur whic h lead to Family A GPCRs receptor activation. Protease activated receptors (PARs) is a mode of family A GPCRs, where the cleavage site in the N-terminus is cleaved by thrombin, which reveals that tethered ligand that is able to activate the receptor.
Second Messengers List major second messenger molecules used by GPCRs. Choose one example to discuss in detail how this second messenger molecule transmits and amplifies the first messenger signal to the downstream signalling pathway. cAMP: cAMP is the second messenger for both the beta adrenergic receptor and the M2 muscarinic receptor. Each of these pathways follow the same signalling cascade, but have opposite effects. For example, the beta adrenergic receptor is bound by noradrenaline (its endogenous ligand), the receptor then binds a g-protein consisting of Galpha-s subunit and beta and gamma subunits. The binding of this receptor leads to an upregulation of adenylyl cyclase, which the increased the amount of intracellular cAMP, which in turn upregulates protein kinase A (PKA) and ultimately leads to increased protein ph osphorylation within the cell. G-Proteins Give an example of large G‐ proteins; describe its structural characteristics, how it switches between active and inactive states and how its mutation may result in a diseased phenotype. Heterotrimeric g-proteins are members of the large g-protein family. These proteins consist of three subunits: alpha, beta and gamma. The alpha subunit is used in signalling, however the full function of the beta and gamma subunits is not known. The G-protein causes a switch between inactive and active states when it is bound to a receptor. When this receptor binds with its agonist it activates the G-protein and causes GDP to be exachanged for GTP in the alpha subunit. The activated alpha subunit then interacts with the target (such as an enzyme) and is considered to be in the “on” state. The activated subunit of the G-protein is switched off with the help of the constitutive GTPase that is in the subunit, which signals for it to disengage from the effectors and bind with the ßy subunit once again. Loss of function of the Gas subunit can lead to diseases such as Albright hereditary osteodystrophy (AHO), which can cause developmental and physical disabilities, such as short stature, depressed nasal bridge and also mental retardation.
Regulation of GPCR Signalling MCQ When an agonist activates a receptor for a certain period of time, it can result in receptor phosphorylation. Which ONE of the following terms BEST describes this action? A. Dependence B. Desensitization C. Internalization D. Sensitization
E. Tolerance LAQ: (20 min) A. Describe the steps involved in the classical model of G-protein coupled
receptors desensitization 3 steps: - Phosphorylation of the receptor – through GRK-mediated phosphorylation, the phosphorylated receptor then interacts with intracellular ß-arrestin, which leads to uncoupling - Internalisation of the receptor – clathrin mediated endocytosis causes sequrestration of the agonist-occupied receptor - Down-regulation of the total number of receptors – decreased synthesis and increased degradation. Once receptors are delivered to lysosomes they cannot be recycled and must have new receptors synthesised. B. Compare and contrast the differences between heterologous and homologous desensitization. In your answer include ex amples of proteins that modulate these pathways.
Homologous desensitisation – agonist specific. GRKs preferentially phosphorylate agonistactivated GPCRs. ßy subunits activate GRKs Heterologous desentisiation – adenylate cyclase bound to the alpha s subunit causes ATP to be converted to cAMP, which regulates PKA and causes agonist-activation independent phosphorylation. Receptor Internalisation and Alt Signalling Pathways 20 min Question: A. Compare and contrast the desensitisation process for “Class A” and “Class B” GPCRs. (10 marks) Class A: no colocalisation with ß-arrestin in endosome. GPCRs are rapidl y recycled to the cell surface after forming clathrin coated vesicles. ß-arrestin uncouples and the vesicle compartment becomes acidified and the GPCR can either be rapidly recycled or degraded. Class B: colocalisation with ß-arrestin in endosome is present, clathrin coated vesicle is formed after ß-arrestin binds, but it does not uncouple. The Class B GPCR is then most likely degraded, or in some cases slowly recycled. B. Describe the role of β-arrestin in non-G-protein mediated signalling by GPCRs (5 marks) It has been recently discovered that ß-arrestin can be involved in GPCR signalling when the receptor is not coupled to a G-protein. Arrestin signalling has a slower onset, sustained duration and is signalsome-dependent. Arrestin-induced signalling is through the pathway o f TK, MAPK and E3 ligase formation. C. The graph below shows the data collected in an experiment where COS-1 cells, transfected with β2-adrenergic receptors, were pre-treated with pertussis toxin (PTX) or monodansylcadaverine (MDC), and then stimulated with the agonist isoprenaline (Iso). Following stimulation the cells were solubilised and the proteins run on S DS-PAGE and
western blotted for phospho-ERK. What conclusions can be drawn from this data in regards to β2-adrenergic receptor signalling? (5 marks) PTX and MDC are antagonists, as they both reduce the fold increase in pERK, but this kind of assay cannot show if they are competitive or non-competitive.
Allosteric Modulation 10 min question (i) Describe the mechanism of action of allosteric modulators. Allosteric modulators bind to a site that is alternate to the regular endogenous ligand binding site for a receptor (these are the orthosteric sites). Allosteric modulators generally don’t have their own independent action; it only regulates the action of a hormone or neurotransmitter. Generally, the allosteric binding causes a conformational change in the receptor that affects the binding affinity of the orthosteric ligand. (ii) Define the following terms that are used to quant ify allosteric effects; α, β and τ Alpha – effect of an AM on the affinity of a protein for another molecule Beta – effect of an AM on the efficacy of an agonist binding to a receptor protein Tau – measure of efficacy (iii)Describe the type of allosteric modulation that a ligand with an α value of 0.2 would display Positive allosteric modulators (PAMs) increase the binding affinity of a protein for a molecule. PAMs have an alpha value of greater than 1. AMs with a value less than 1 are negative allosteric modulators (NAMs), and they reduce the affinity of the protein for a molecule. Side note: ß effects can affect the overall efficacy, and can reduce maximal responses. An allosteric activator does which ONE of the following?
A. Binds to an enzyme away from the active site and changes the conformation of the active site, increasing its affinity for substrate binding B. Binds to the active site and blocks it from binding substrate C. Binds to an enzyme away from the active site and changes the conformation of the active site, decreasing its affinity for the substrate D. Binds directly to the active site and mimics the substrate E. Binds to the active site of an enzyme inhibiting the formation of the transition state complex Constitutively Active Receptors Design an experiment to test if Drug X is a M3 receptor inverse agonist or an antagonist using the isolated guinea pig ileum setup Organ bath setup, contains Kreb’s solution to block Ach release from neurons. Pre-treating the ileum with Drug X can help establish if it is an inverse agonist, because if after it is treated with an agonist such as carbachol then there will be a noticeable drop in the basal levels of contractility, which can help determine Drug X’s mode of action. Instead of measuring the contractility (which could be imprecise due to tissue difficulties etc.) you c ould also measure the production of second messengers such as IP3 or Ca2+, as they are both associated with M3 muscarinic receptor activation.
Advanced Pharmacodynamics What is a pA2 value? What is a Schild plot? How do you produce one? Pa2 is the negative log value of the EC50, and it is a more simple way to compare the potency of drugs than by using EC50s and concentration-response curves. A schild plot is a graph of the Log (DR-1) where DR is the dose ratio vs. the log of [B], the x-intercept is the pA2. Signalling Bias
10 min QUESTION (i) Compare and contrast how efficacy is described by the following two theories; classical receptor-occupancy and signalling bias. Classical Receptor-Occupancy Theory states that the occupancy of a ligand-receptor complex is governed by affinity, and that the activation of the ligand-receptor complex is governed by efficacy. Signalling bias theory is used to ex plain how the efficacy of certain drugs can vary based on different tissues and environments, introducing the idea of preferred signalling pathways for certain receptors. (ii) Provide an experimental example which can only be explained by signalling-bias. Signalling-bias theory is used to explain how different agonists stabilise different receptor conformations, such as Ghanouni et al (2001) that contrasts the probability of different ligandreceptor complexes existing at different times, and while the majority of the drugs prefer to exist in a particular conformer, there are measurable times when each drug induces a different conformer. (20 min) (i)
Discuss the evidence that G-protein and β-arrestin signalling occurs from GPCRs
located in endosomes. Give specific examples to support your answer By measuring Nb80-GFP we can see what level of B2-ARs are activated in the plasma membrane and the endosomes. Nb80-GFP is not accumulated in clathrin-coated pits or vesicles, but it still accumulated after their formation, which gives rise to the hypothesis that B2-AR endocytosis can contribute to a second phase of cellular cAMP response (ii)
Discuss the difference in cellular outcomes that can occur with signalling from caveolae and clathrin coated pits. Give specific examples to support your answer In the case of oxytocin, clathrin coated pit-mediated internalisation is induced after Ga-i signalling is used (this produces an anti-proliferative effect), however when located in the caveolae it signals through Ga-q, which leads to proliferation (iii) Describe how an ‘antagonist’ can produce β-arrestin mediated signalling. What is this phenomenon called? Receptor Theory (20 min)
The diagram below is a representation of the extended ternary complex model.
Alpha factor = binding of ligand affects receptor activation by this factor Beta factor = affects affinity for G-protein by this factor Gamma factor = affects binding of ligand with Gprotein Delta = the effect of synergy between any two factors
A) Describe what the characters circled in i, ii, iii and iv represent. i) ligand bound to activated receptor ii) equilibrium constant for ligand-activated receptor-g-protein complex and activated receptor-g-protein complex, modulated by a (affects receptor activation with ligand binding) and b factors iii) Activated receptor bound to g-protein iv) Equilbrium constant for the binding of g-protein to activated receptor, modulated by beta (affects affinity of g-protein) B) For the species in circles i and iii provide an example of experimental data that
supports
their existence. C) Define signalling-bias . D) Which of the factors found in the extended ternary complex model can lead to signalling-bias? Activated receptor-g-protein complex, that has been found to signal on a constituvely active basis.
New Concepts in Mol Pharm Describe, using examples, how to identify a signalling assay for use in orphan GPCR ligand screening. G-protein specific assays like yeast chimeras can be used to try and discover the constitutive coupling of an orphan GPCR, by removing the endogenous GPCR and introducing chimeras containing the last 5 amino acids of each human G-alpha. The strains of human GPCRs that couples with the orphan GPCR is then used in ligand screening. Reporter assays are useful in trying to identify orphan GPCRs, when different luciferase-fused response elements are transfected alongside the orphan GPC R and luciferase activity is monitored.
Surrogate ligands are now being used for screening of orphan GPCRs, because if an FDAapproved drug is discovered to have some affinity, then a lot is already known about it. This technique does not always succeed but when it does it allows information about the GPCR to be fast-tracked to more quickly allow for drugs to be made for it if needed be. Dendograms and other ‘family tree’-like relationships between GPCRs are now being used to discover information about GPCRs based on their families and what kinds of ligands they bind. Helps to explain unexplained off-target effects and also to predict previously unknown off-target effects.
