theme 2: target identification and validation Flashcards
give 4 examples of screening technologies
FLIPR- ca2+, nanoBRET, Reporter genes, label free
what is FLIPR ca2+ screening
this is a functional signalling assay for GPCRs. it measures intracellular calcium levels within intact cells. Fluorescent signal occurs when intracellular calcium is released. Way of measuring effective signalling of Gq coupled responses.
what is a BRET (bioluminescence resonance energy transfer) assay
a flourescent tag is added to the N-terminus of the receptor called NanoLuciferase. When a substrate binds to the receptor, the energy transfer means a blue light is given off of by the NanoLuciferase. This shows the expression of receptors.
Activation by a particular ligand can be measured using a flourecent labelled ligand. When this binds to the receptor the energy given off by the nano luciferase can excite the fluorophore on the flourecent labelled ligand and cause another colour signal to be given off alongside the blue nanoluciferase. This shows whether the receptor activation occurs by the flourecent labelled ligand or other non-labelled ligand.
how are reporter genes used in screening
Essentially, it measures the production of proteins by the expression of fourescence.
When the fluorescently labelled ligand is activated and excited, a colour is given off
Fluorescent labels can be encoded into cells downstream of a promotor region in a gene that detects a particular signal, such as an increase in Camp within the cell caused by gene expression.
The expression of the fluorescent label can then be measured as a measure of gene expression.
In drug discovery, a reporter gene (e.g., luciferase) linked to a specific pathway is used to screen compounds. A compound that activates or inhibits the pathway will alter luciferase expression, producing measurable luminescence to identify hits.
what are label free technoligies
this measures chnages in local refractive index resulting from the ligand induced mass redistribution (change in configurement of cell organelles) in the bottom region of the cell monolayer. It is manifested as a shift in resonant wavelength.
ie the redistribution of organelles changes the reflected wavelength given off when the broadband source is directed at the bottom of the cell.
ligand binding can change the distribution of organelles.
what is the process of target screening
- disease selection
- target identification
- assay development
- hit to lead
- lead optimisation
-preclinical development - clinical trials
Using genetic encoded target- then identifying drugs that interact with it.
Target ID comes close after disease selection- then lead compound is found.
what is the process of phenotypic screening
Picking the disease, then developing assay (manifestations of disease). High affinity ligand of interest is found and then target is identified.
- disease selection
- assay development
- hit to lead
- target identification
- lead optimisation
-preclinical development
- clinical trials
what is the difference between molecular target and phenotypic screening?
in molecular target screening. a novel genetically encoded target is chosen and drigs that interacted with it are identified. diseases where this may have application are then found.
In phenotypic screening, a disease phenotype is first found. drugs used here are then found and then specific protein targets are identified
in what screening type is the molecular target of the disease to be known
molecular target screening
what is the method of lead conformation in molecular target screening?
a direct binding assay is used or x-ray, crystallography
lead conformation needs to take place in cell based and phenotypic assays
give an example of a primary cell type used in phenotypic screens
thyrocytes
bronchial epithelial cells
what does IPS stand for
induced pluripotent stem cells
give four examples of IPS cells
neurons, cardiomyocytes, hepatocytes, endothelial cells
what does the orexin system play a key role in
wakefullness. narcoeleptic patients have very few orexin producing neurons
define allosteric modulator
molecule imposng allosteric change on gpcr
define orthosteric ligand
Molecule binding to the same site as the endogenous ligand that senses the altered receptor site
define biased signalling
Different ligands can stabilise specific conformations of the receptor that favour coupling to the g protein or g protein independent signalling proteins
define homodimer
Dimerisation with an identical receptor - can lead to cooperativity or signalling bias
define heterodimer
dimerisation with different receptor - biased signalling and cooperativity
define an orphan receptor
An orphan receptor is a protein that has a similar structure to other identified receptors but whose endogenous ligand has not yet been identified.
how are orphan receptors used in GPCR target identification
knowing the stricture and some drugs that target it can help to identrify endogenpous liogands and therefore drug develpment starting from the endogenous ligand can take placem
how can viruses lead to cancer
viruses infect cells by encoding their DNA into the host so proteins can be made. One of these proteins may be a GPCR that responds to endogenous chemokines that when bound to and activated, can induce transcription and cell growth and replication. if this occurs uncontrollably, a tumour can develop ie cancer
give an example of a receptor tyrosine kinase
HER2 human epidermal growth factor receptor 2
EGFR
HER4
HER3
What is HER2
a tyrosine kinase receptor that is amplified in some breast cancer cases and other cancers. it is targetted by antobodies and some drugs includig trastuzumab, pertuzumab, lapatinib
Describe the large scale industrialisation of screening?
Use of large chemical libraries- testing library of chemicals against particular protein target.
What is high throughput screening?
Method to identify smaller subset of potent active compounds- measuring for one response.
What is high content screening?
Microscopy-based screening- looking for a variety responses.
What is the use of BRET?
Monitors when fluorescent ligand is in close proximity to nanoluciferase (in binding site)
chlorocyclizine
What is an example of a phenotypic screen? (HCV)
Large library of already used drugs were screened for antiviral properties. Chlorcyclizine was discovered to inhibit viral fusion of HCV- viral RNA cannot be released into cytosol
What are some requirements for a drug target? (4)
Has to play a role in human disease, to what extent?, does its role differ substantially in pre-clinical studies, accessibility from ECF?
