theme 2: target identification and validation

Question 1

give 4 examples of screening technologies

Answer 1

FLIPR- ca2+, nanoBRET, Reporter genes, label free

Question 2

what is FLIPR screening

Answer 2

this is a functional signalling assay for GPCRs. it measures intracellular calcium levels within intact cells.

Question 3

what is a BRET (bioluminescence resonance energy transfer) assay

Answer 3

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.

Question 4

how are reporter genes used in screening

Answer 4

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.

Question 5

what are label free technoligies

Answer 5

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.

Question 6

list the order of processes in molecular target screening

Answer 6

• disease selection
• target identification
• assay development
• hit to lead
• lead optimisation
  -preclinical development
• clinical trials

Question 7

list the processes involved in phenotypic screening

Answer 7

• disease selection
• assay development
• hit to lead
• target identification
• lead optimisation
  -preclinical development
• clinical trials

Question 8

what is the difference between molecular target and phenotypic screening?

Answer 8

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

Question 9

in what screening type is the molecular target of the disease to be known

Answer 9

molecular target screening

Question 10

what is the method of lead conformation in molecular target screening?

Answer 10

a direct binding assay is used or x-ray, crystallography
lead conformation needs to take place in cell based and phenotypic assays

Question 11

give an example of a primary cell type used in phenotypic screens

Answer 11

thyrocytes
bronchial epithelial cells

Question 12

what does IPS stand for

Answer 12

induced pluripotent stem cells

Question 13

give four examples of IPS cells

Answer 13

neurons, cardiomyocytes, hepatocytes, endothelial cells

Question 14

what does the orexin system play a key role in

Answer 14

wakefullness. narcoeleptic patients have very few orexin producing neurons

Question 15

define allosteric modulator

Answer 15

molecule imposng allosteric change on gpcr

Question 16

define orthosteric ligand

Answer 16

Molecule binding to the same site as the endogenous ligand that senses the altered receptor site

Question 17

define biased signalling

Answer 17

Different ligands can stabilise specific conformations of the receptor that favour coupling to the g protein or g protein independent signalling proteins

Question 18

define homodimer

Answer 18

Dimerisation with an identical receptor - can lead to cooperativity or signalling bias

Question 19

define heterodimer

Answer 19

dimerisation with different receptor - biased signalling and cooperativity

Question 20

define an orphan receptor

Answer 20

An orphan receptor is a protein that has a similar structure to other identified receptors but whose endogenous ligand has not yet been identified.

Question 21

how are orphan receptors used in GPCR target identification

Answer 21

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

Question 22

how can viruses lead to cancer

Answer 22

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

Question 23

give an example of a receptor tyrosine kinase

Answer 23

HER2 human epidermal growth factor receptor 2
EGFR
HER4
HER3

Question 24

What is HER2

Answer 24

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