Section 2 Flashcards
What are the 2 screening methods which can identify a target for drug discovery?
Target-based screening: Choose a novel genetically encoded target, identify drugs which interact with it, then look for a disease where drugs may have therapeutic utility.
Phenotypic screening: Find a model of the disease phenotype, find drugs which interfere with the phenotype, then identify the specific protein target or targets involved.
What is the difference between in vitro and in vivo phenotypic screens?
In vitro phenotypic screens involve monitoring the effect of a drug on an isolated organ or body part. For example, monitoring HR on a perfused heart or pH of the luminal contents of a perfused stomach.
In vivo phenotypic screens allow monitoring of parameters on an anaesthetised animal. In this case, the target, its gene sequence, and its protein structure are unknown.
How was in vitro phenotypic screening used to determine the mechanism of action of Cimetidine?
An isolated rat stomach was perfused with saline solution and the pH of the luminal contents was measured as it left the stomach in the presence of nothing, histamine alone, and histamine in combination with cimetidine.
How did M3-knockin mice have an effect on the effects of carbachol?
In normal mice, as the concentration of the muscarinic agonist carbachol increased, the bronchial lumen narrowed.
In the M3-knockin mice, the M3-mACHr gene was modified to express a muscarinic M3 receptor which had its intracellular phosphorylation sites changed from serine/threonine to alanine, thus preventing phosphorylation.
In these mice, the bronchial lumen was much less sensitive and constriction only occurred at very high concentrations.
What are molecular-based target screens?
A chemical library of millions of compounds is screened against a particular protein to identify a ligand with agonistic or antagonistic effects.
What is high throughput screening?
The use of automated equipment/robots to generate multiwell plates and rapidly test thousands to millions of samples for biological activity.
Usually done after screening of a diverse compound library to identify a smaller subset of potent compounds.
What is FLIPR?
FLIPR is a fluorometric imaging plate reader which monitors intracellular calcium changes.
A calcium sensitive dye (fluo4) emits a fluorescent signal upon calcium binding which can be monitored in real time. This measures intracellular calcium-reliant signalling and can monitor GQ coupled responses
How could FLIPR be used to investigate histamine H1 receptor antagonists?
When histamine activates a histamine H1 receptor (G-protein coupled recceptor) there is hydrolysis of membrane phospholipids and generation of inositol triphosphates. This binds to intracellular receptors on the ER and leads to release of calcium which activate aspects of the cell (e.g., contraction of a smooth muscle cell).
FLIPR will simultaneously deliver histamine to 256 well plates containing the ,cell. In the absence of an antagonist, histamine will bind to the H1 receptor and there will be a release of intracellular calcium, which will be bound by the fluorescent calcium-sensitive dye, fluo4. This fluorescence will be monitored. However, in the presence of antagonist, H1 receptor activation by histamine will be inhibited, thus preventing calcium release and fluo4 binding. This results in a lower fluorescent response.
What is NanoBRET?
A bioluminescence energy transfer assay which monitors ligand binding. This is done using a fluorophore attached to the ligand. This becomes fluorescent upon excitation at 630nm by the luminescent protein nanoluciferase bound to the N-terminus of the target receptor. Thus when the fluorescent ligand binds to receptor, it becomes excited and the fluoresence can be detected.
How do reporter genes indicate cell signalling?
Reporter genes are genes which encode a detectable protein (via its luminescence, fluorescence, or colour). These genes can be encoded into cells downstream of a promoter that responds to a particular signal. If this signal occurs, the promoter will respond by encoding the necessary genes, including the reporter gene. The successful cell signalling can be measured using the luminescence, fluorescence, or colour given off.
How does label-free technology work?
It measures changes in the local refractive index resulting from ligand-induced mass redistribution (DMR) within the bottom region of a cell monolayer. It is measured as a shift in wavelength.
Cells are grown on a surface containing a waveguide substrate which allows light to pass along the bottom of cell and gives of a reflected light of a particular wavelength.
In response to stimulation (e.g., an agonist), the positioning of organelles can change (aka mass distribution). If this occurs near the coverslip (within 150nm), it will be detected and there will be a local change in refractive index. The light from the broadband source will pass through the waveguide and be reflected at a different wavelength dependent on the change in refractive index. This appears as a change in colour of the reflected light.
What is the main difference between target-based and phenotypic screening?
The location of the target ID in the drug discovery cascade.
Target-based: target identified/decided on straight after disease selection.
Phenotypic: target is identified after a lead is decided on (work out what your lead drug actually works on).
What are the advantages and disadvantages of target-based screening?
Advantages:
Molecular target known.
Mechanism of action is known at outset.
Disadvantages:
Assay used may be less disease relevant.
Drug target may not be disease relevant; risk of lack of efficacy in clinical trials.
What are the advantages and disadvantages of phenotypic screening?
Advantages:
Don’t necessarily need to know the molecular target.
Multiple targets and signalling pathways can be targeted.
Screening is disease relevant.
Disadvantages:
Could be more expensive as screening is more complex.
Mechanism of action is unknown at outset.
Target identification may be required for confirmation of lead compound.
Give an example of a drug which was repurposed for use in new diseases?
Chlorcyclizine, an antihistamine, was found to exhibit activity against Hep C virus during a screen of a chemical library.
What screen was used to identify chlorcyclizines activity against the hepatitis C virus?
The screen involved a cell which contained a CAG promoter and luciferase reporter gene. Between this was a sequence containing a stop codon, which if expressed, would prevent luciferase being expressed. This sequence was bordered by a CRE recombinase LoxP site either side, which, if activated by CRE (encoded in the virus), would remove the sequence, allowing expression of the luciferase.
So, if a drug such as Chlorcyclizine was effective, the CRE would not be able to enter the cells, so the CRE recombinase wouldn’t be activated, so the sequence wouldn’t be removed, and the luciferase wouldn’t be expressed.
How does Chlorcyclizine work against the Hepatitis C virus?
Usually:
1. Hepatitis virus cell surface protein envelope glycoprotein E2 interacts with receptor on human cells.
2. Virus undergoes endocytosis and becomes sequestered in the cell.
3. Hepatitis virus uses glycoprotein E1 to fuse with endosome to release genetic material and continue infectious process.
Chlorcyclizine interferes with glycoprotein E1 and its fusion with the endosome, preventing release of viral RNA into the cytosol.
What are orphan receptors?
Receptors for which we don’t what their ligand is.
What type of receptors are OX1 and OX2?
G-protein coupled receptors.
What is the pathophysiology of narcolepsy?
Normally, orexin neuronal activity oscillates and is high during normal walking and falls during normal sleep.
Narcolepsy is a neurodegenerative disease where the orexin-producing cells in the hypothalamus become degenerated, causing patients to suddenly fall asleep and have disrupted sleeping patterns.
What did the discovery of the orexin system and OX receptors mean for potential drug discovery?
Orexin antagonists for the treatment of insomnia.
OX receptor agonists/orexin mimetics for treatment of narcolepsy.
Name 2 current orphan receptors and their use as therapeutic targets.
GPR35 - high levels of expression in spleen and peripheral leukocytes, as well as expression in inflammatory cells could indicate a good target for allergy or asthma.
GPR55 - expression in bone, CNS, and cancer cells so could be used to treat obesity, osteoporosis, cancer.
What are the 2 binding sites present on GPCRs?
Orthosteric site - binding site of endogenous ligand.
Allosteric site - distant site on receptor where other ligands can bind and impose allosteric/conformational changes which lead to differences in signalling or ligand binding.
What is the principle of biased signalling?
G-protein coupled receptors have 2 binding sites - an orthosteric and allosteric. Binding of a ligand to either site can cause conformational changes in the receptor which affect subsequent binding at the other site.
Since G-proteins bind at the allosteric binding site, binding of a particular ligand to the orthosteric site could induce conformational changes in the receptor which favours coupling to G proteins or G protein independent signalling proteins e.g., beta-arrestin.
What is beta-arrestin?
A G-protein independent signalling protein which interfere with G-protein binding and block G-protein mediated signalling, thus desensitizing GPCR signalling.
What is the calcium sensing receptor (CaSR)?
A class C GPCR which maintains calcium homeostasis by acting as negative feedback. Upon binding of calcium to the orthosteric binding site, a conformational change in the receptor occurs causing coupling of G proteins. This signals intracellularly to inhibit parathyroid hormone synthesis and release, therefore inhibiting calcium reabsorption in the kidney and controlling bone reabsorption and mineralisation.
How do calcimimetics work?
Cinaclet binds to calcium sensing receptor at orthosteric binding site to inhibit parathyroid hormone release and therefore reduce calcium reabsorption in the kidneys. Used in patients with hyperparathyroidism or CKD.
Give an example of a virus-encoded GPCR.
Human cytomegalovirus causes overexpression of viral GPCR US28 on the cell surface. This GPCR responds to endogenous human chemokines and initiates signalling which leads to increased activity of transcription factors involved in cell proliferation, therefore can lead to cancer.
What is the difference between an agonist, an antagonist and an inverse agonist.
Agonists - increase receptor signalling
Antagonists - do not change basal signalling but prevent increased receptor sigalling by an agonist.
Inverse agonist - has the opposite effect of an agonist i.e., activity inhibited below basal signalling.
How are EGFR and HER receptors involved in cancer progression?
- Binding of agonist causes conformational change which exposes dimerisation domain.
- Receptor dimerises which causes a conformation change leading to phosphorylation of tyrosine residues on the intracellular side of the receptor by tyrosine kinase.
- Intiation of cascade of intracellular signalling, particularly via MAPK and PI3K-Akt pathways.
- Activation of cell proliferation and migration.
How does HER2 play a role in breast cancers?
Unlike other EGFR receptors, HER2 doesn’t have any known agonists, it is always in an “activated” conformation with its dimerisation domain exposed.
HER2 is overexpressed in 15% of all breast cancers and results in a more aggressive phenotype and poor prognosis.
