Drug Discovery 11

Question 0

what is a problem with traditional binding ?

Answer 0

the filtration stage of binding adds complexity to HTS protocol meaning more sophisticated protocols are needed

Question 1

which type of binding assay is good for HTS?

Answer 1

competition binding, saturation binding is not so good

Question 2

what are scintillation proximity assays ?

Answer 2

they have a transparent base withe each well composed of scintillants and polystyrene
light emission occurs due to bound radioactive ligand in close proximity to scintillant - it is the product that is activated by radioactivity and emits the light

Question 3

what is a problem with scintillation proximity assays ?

Answer 3

the use of radioligands are expensive and carry health and safety issues for pharma companies so they are only used if essential for screening

Question 4

what is fluorescence polarisation ?

Answer 4

a ligand is labelled with a fluorescent molecule and polarized light is passed through it
the fluorescent molecules are excited with the plane polarised light and this will emit light back into a fixed plane in the same phase
the light remais polarised if the molecules remain stationary during the excitation of the fluorophore

Question 5

in reality do molecules remain in a fixed position? and what does this mean ?

Answer 5

no they rotate and tumble

and therefore the planes into which light is emitted can be very different from the plane used for initial excitation

Question 6

why does the size of the molecule affect fluorescence polarisation ?

Answer 6

larger molecules rotate and tumble more slowly so planes into which light is emitted are similar to the plane used for initial excitation
small labelled ligand will tumble less when it binds to a larger receptor and hence the complex emits more polarised light- this can be used to quantify binding

Question 7

what can fluorescence polarisation be used for ?

Answer 7

if you bind a small labelled ligand to a big receptor this stops tumbling and so it tumbles less than the unbound ligand
detect how much polarised fluorescence we get and therefore more fluorescence polarised light produced the more ligand is bound to the receptor
- it gives you a method of competition binding without having to use a radiolabelled ligand

Question 8

what is the basis of GTP-gammaS assay ?

Answer 8

by adding GTP-gammaS GTP analogues to the system they are able to bind to alpha subunits when the receptor is activated however they are non-hydrolysable so they can block the cycle
the GTP-gammaS is radioactively labelled

Question 9

describe GTP-gammaS assays?

Answer 9

• traditional assays used radioactive [35S]GTP-gammaS but this comes with health and safety issues for pharma companies
  modern highthroughput approaches use time - resolved fluorescence - when you excite the atom, fluorescence occurs over a longer time so enables more time to calculate the amount of fluorescence

lanthanide chelates are used for their long fluorescence decay after excitation

Question 10

how does time resolved fluorescence work ?

Answer 10

the background fluorescence decays rapidly
and long lived fluorophores decay much more slowly- the delay betwee excitation and emission detection is recorded - the excited and emitted wavelengths are very different so therefore these energies are not confused

Question 11

what are the stages of time resolved fluorescence ?

Answer 11

add buffer components and GPCR membrane compounds to the acrowell filter plate and incubate with GTP-eu
then filter and wash to get just bound GTP-eu
then excite it at 340nm and measure emissions at 615nm

Question 12

what does FRET stand for ?

Answer 12

forster resonance energy transfer

Question 13

what happens in FRET ?

Answer 13

a CFP donor molecule is excited at 436nm and then emissions are measured at 480nm
if an acceptor is close, the donor emission excites it and the acceptor YFP fluoresces producing light at 535nm wavelength

Question 14

describe FRET:

Answer 14

• uses 2 fluorophores, a donor and an acceptor
• excitation of the donor by an energy source triggers an energy transfer to the acceptor
• but only if the acceptor is within a given proximity to the donor
• the acceptor in turn emits light at a different wavelength to the excitation and donor wavelengths

Question 15

how can the interactions between the donor and acceptor molecules be assayed?

Answer 15

by fusing the donor and acceptor to binding partners

Question 16

what is TR-FRET ?

Answer 16

time resolved forster resonance energy transfer

• uses both FRET and TRF
• uses TRF properties of lanthanide chelates as with GTP assay
• but uses this lanthanide as a donor such that its emission results in energy transfer resulting in fluorescence at an acceptor

Question 17

what is the lance cAMP assay principle?

Answer 17

eu-chelate is attached to streptavidin - this is a means of sticking eu-chelate onto cAMP
the eu-chelate streptavidin is attached to biotinylated cAMP
the eu-chelate is excited at 340nm and this produces emissions at 615nm
by adding alexa cAMP specific antibody this energy is transferred to the antibody and fluorescence is emitted at 665nm- by generating more cAMP, the more it will compete with the antibody

Question 18

how is the FRET removed from the cAMP assay and what does this mean ?

Answer 18

in the absence of cellular cAMP there is a signal from the acceptor
but
as agonist stimulation produces cellular cAMP it competes with the antibody acceptor and the FRET signal is reduced - this occurs in a concentration dependent manner

Question 19

other than TR-FRET cAMP assays what other cAMP assays are there?

Answer 19

reporter assays

• used less frequently now
• they have an engineered cell line that links GPCR pathway to the expression of a detectable protein - a specific gene is activated when you activate a specific pathway

Question 20

what happens when luciferase is reacted with oxygen ?

Answer 20

oxyluciferin and light are produced

- luciferase is a natural protein from fireflies

Question 21

what happens in reporter assays ?

Answer 21

a signalling pathway is activated which causes the expression of a reporter protein which can be easily detected however this may require hours of receptor activation because the cell has to transcribe the protein so it takes a long time to be able to detect sufficient protein

• the reporter protein may light up or cause a colour change
• it is not ideal for HTS

Question 22

what is the IP1 HTRF assay ?

Answer 22

by activating Gq it activates PLC which increases IP3
IP3 is broken down to IP2 and then to IP1 and then to myo ionisitol
however if the conversion of IP1 to myo ionisitol is blocked by LiCl the amount of IP1 builds up

Question 23

describe the stages of IP1 HTRF assay and the basis of it ?

Answer 23

1- cells are added to a microplate
2- cell adhesion occurs when the compounds and LiCl in Krebs buffer is added
3- this is then incubated with anti-IP1 cryptate and IP1-d2 - the antib-IP1 is a donor attached to antibody that recognises IP1 and he d2 is the acceptor attached to IP1
like with the lance assay the agonist induced activation results in a reduction of FRET - in this case the IP1 produced by the cells competes with the labelled IP1 for the antibody

Question 24

what is the calcium assay ?

Answer 24

uses fluorescent indicators to accurately measure intracellular calcium concentrations e.g. FURA-2
when FURA-2 is bound to calcium the excitation maximum is 340nm but when it is not bound to calcium its excitation maximum is 380nm
the 340/380nm excitation ratio (RFU) allows accurate measurements of the intracellular calcium concentration