Lecture 6.3 (FLIPR)

Question 1

What could trigger the expression of luciferase in luciferase reporter gene assays for GPCRs?

Answer 1

Upon GPCR activation, stimulated transcription factors bind onto specific promoters of the reporter gene to trigger luciferase expression

Question 2

What could trigger the expression of luciferase in luciferase reporter gene assays for Gs-coupled receptors (a type of GPCR)?

Answer 2

-Gs-coupled receptor activated
-stimulation of adenylyl cyclase
-increase in cyclic AMP (cAMP) levels
-CREB activation
-gene transcription

Question 3

What are Gs-coupled receptors?

Answer 3

-a subtype of G-protein-coupled receptors (GPCRs)
-have specific interactions with the Gs protein subunit and play a distinct role in cellular signaling pathways.

Question 4

What is CREB?

Answer 4

cAMP response element binding proteins

Question 5

What cells are used for luciferase assays for Gs-coupled receptors?

Answer 5

GPCRs are transfected into HEK-293 cells stably containing cAMP responsive luciferase reporter.

Question 6

Luciferase activities behave in dosedependent manner for Gs
-coupled receptors.

True or false

Answer 6

True

Question 7

What is the principle of Fluo-3 Based Ca2+ Flux Assay in the FLIPR?

Answer 7

1. cells incubated with membrane-permeant Fluo-3-acetoxymethyl ester
2. AM form of Fluo-3 is hydrolyzed by intracellular esterases –> trapped in cells as fluorescent free acid form
3. Elevation in intracellular calcium triggered by receptor or ion
   channel signaling increases the Fluo-3 fluorescence due to cellular Ca2+ binding to trapped Fluo-3 free acid

Question 8

Apart from luciferase reporter gene assays, what other assay could be used for screening (HTS) assays for GPCRs?

Answer 8

Calcium (Ca²⁺)-based high-throughput screening (HTS) assays for Gq-coupled receptors

Question 9

State the principle of Ca2+-Based HTS Assays For GPCRs (Gq-coupled receptor)?

Answer 9

1. ligand binds to the Gq-coupled receptor
2. activates G protein by exchanging GDP for GTP on the αq subunit
3. αq subunit PLCβ
4. PIP₂ Cleavage into two 2nd messengers –> IP₃ and DAG
5. Ca²⁺ Release
6. Ca²⁺ detection using Fluo4 or Aequorin

Question 10

Name calcium detection methods

Answer 10

Fluo3, Fluo4, Aequorin

Question 11

What are some HTS assays for GPCRs?

Answer 11

1. Fluo3/Fluo4/Aequorin for Gq-coupled receptor for calcium detection
2. luciferase assays for Gs-coupled receptors (cAMP responsive luciferase reporter)

Question 12

What is the principle of radioligand binding assays in HTS?

Answer 12

measure how well test compounds compete with a radiolabeled ligand for binding to a receptor.

–> to identify lead compounds with high receptor affinity and selectivity

Question 13

What are the 2 types of radioligand binding assays in HTS?

Answer 13

1. displacement analysis where the radioliabeled ligand is displaced by test compounds to identify lead comppunds
2. saturation analysis

Question 14

Why are cellular imaging assays more in focus now in drug discovery?

Answer 14

lower cost and higher biological relevance

Question 15

What are some targets in target based assays in imaging?

Answer 15

GPCRs
Kinases
Proteases
Biological pathways

Question 16

What is the screening protocol for one compound/well?

Answer 16

1. primary screening (n=1, 10uM)
   –> identify +ves with
   %inhibition>cutoff
2. Confirmation screening (n=4,
   10um) –> conform +ves
3. dose-response curves (n=4)
   –> hits
4. confirmed chemical structures
   –> validated hits

Question 17

What is the screening protocol for one mixtures?

Answer 17

1. primary screening of mixtures (n=1, 10uM for each cpd)
2. Deconvolution screening
   (n=4, 10 mM, one cpd/well)
3. Dose-response curves (n=4)
   –> hits
4. confirmed chemical structures
   –> validated hits