Live cell imaging: Visualization of [Ca 2+ ] i fluctuation Dual-wavelength methods –Alternating...

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Live cell imaging: Visualization of [Ca 2+ ] i fluctuation Dual-wavelength methods Alternating excitation wavelength with fixed emission observation (Fura) Looking at different emission bands with constant excitation (Indo, GFP-FRET) Single-wavelength methods Fast sequential acquisition of fluorescent calcium indicators (Fluo 3, Fluo 4) Hit to lead

Transcript of Live cell imaging: Visualization of [Ca 2+ ] i fluctuation Dual-wavelength methods –Alternating...

Page 1: Live cell imaging: Visualization of [Ca 2+ ] i fluctuation Dual-wavelength methods –Alternating excitation wavelength with fixed emission observation (Fura)

Live cell imaging: Visualization of [Ca2+]i fluctuation

• Dual-wavelength methods– Alternating excitation wavelength with fixed emission observation (Fura)

– Looking at different emission bands with constant excitation (Indo, GFP-FRET)

• Single-wavelength methods– Fast sequential acquisition of fluorescent calcium indicators (Fluo 3, Fluo

4)

Hit to lead

Page 2: Live cell imaging: Visualization of [Ca 2+ ] i fluctuation Dual-wavelength methods –Alternating excitation wavelength with fixed emission observation (Fura)

Dual-wavelength ratiometric dyes vs single-wavelength dyes for HTS

Dual-wavelength• Pros

– Correction for variation in cell number

– Correction for efficiency loading

• Cons– Require UV excitation that

could result in significant autofluorescence

– Photolysis of photosensitive caged compounds

– Difficult to measure small variation in calcium

– More complex technology

Single-wavelength• Pros

– Dissociation constant ideal for measuring intracellular calcium variation (200 – 400 nM)

– Instrument more simple– Excited by wavelength in the

visible region.• Cons

– Cannot compensate for variability in the sample

– Cannot correct for efficiency loading

Hit to lead

Page 3: Live cell imaging: Visualization of [Ca 2+ ] i fluctuation Dual-wavelength methods –Alternating excitation wavelength with fixed emission observation (Fura)

Some examples: fluorescence basedCa2+ measurement

GGqqPCRPCR

IP3

q

PIP2

DAG

PKC

PLC

Ca2+

Dye

Dye

FLUO4 DyeFLUO4 Dye

loading

Hit to lead

Page 4: Live cell imaging: Visualization of [Ca 2+ ] i fluctuation Dual-wavelength methods –Alternating excitation wavelength with fixed emission observation (Fura)

FLIPRFLIPR® ® systemsystemHit to lead

FlFluorometricuorometricIImagingmagingPPlatelateRReadereader

Generated data

FlFluorometricuorometricIImagingmagingPPlatelateRReadereader

Generated data

Page 5: Live cell imaging: Visualization of [Ca 2+ ] i fluctuation Dual-wavelength methods –Alternating excitation wavelength with fixed emission observation (Fura)

sGDP

sGTP

iGTP

iGDP

Ligand 1 Ligand 2

Some examples: cAMP measurementHit to lead

Page 6: Live cell imaging: Visualization of [Ca 2+ ] i fluctuation Dual-wavelength methods –Alternating excitation wavelength with fixed emission observation (Fura)

Enzyme fragment complementation (EFC) technology-galattosidase (-gal) protein from E. coli is split in two fragments: a bigger one (acceptor enzyme, EA), and a smaller one (donor enzyme, ED). These fragments are inactive but when put together interact rapidly and form the active enzyme that hydrolyzes the substrates, producing a detectable signal.The cAMP from the cell lysates competes with the marked cAMP for the antibody (conjugated ED-cAMP). The non-linked ED-cAMP is free to complement the EA fragment, creating the active enzyme that will hydrolyze the luminescent substrate

Some examples: cAMP measurementHit to lead

Page 7: Live cell imaging: Visualization of [Ca 2+ ] i fluctuation Dual-wavelength methods –Alternating excitation wavelength with fixed emission observation (Fura)

Questions?

Page 8: Live cell imaging: Visualization of [Ca 2+ ] i fluctuation Dual-wavelength methods –Alternating excitation wavelength with fixed emission observation (Fura)

Reporter gene are DNA sequence coding for exogenous proteins which are either easy to detect (fluorescent) or produce products that are easy to detect.

Reporter gene technology

Examples:

GAL (-galactosidase) - hydrolize a substrate that become colored

- colorimetric assay

GFP (green fluorescent protein) - produce fluorescence under UV light

- fluorescence assay

LUC (luciferase)

- by oxidazing luciferine produces photons

- luminometric assay

Hit to lead

Page 9: Live cell imaging: Visualization of [Ca 2+ ] i fluctuation Dual-wavelength methods –Alternating excitation wavelength with fixed emission observation (Fura)

Screening on cellsI. Cell Production

II. Cell treatment &/or compound exposure

III. Signal development/capture

Hit to lead

Page 10: Live cell imaging: Visualization of [Ca 2+ ] i fluctuation Dual-wavelength methods –Alternating excitation wavelength with fixed emission observation (Fura)

Screening steps

• Creation of a cell line containing the gene of interest

• Standardization of the assay

• First screening of the library and identification of the putative hits

• Confirmation of the hits by performing a dose response

Hit to lead

Page 11: Live cell imaging: Visualization of [Ca 2+ ] i fluctuation Dual-wavelength methods –Alternating excitation wavelength with fixed emission observation (Fura)

SP1999 construct for FLIPR Screen

0

2500

5000

7500

RF

U

Co

ntr

ol

Co

-tra

nsf

ecti

on

Ch

imer

a

PLCPLC

Ca++

Gi Gi GqGq

CotransfectionCotransfection

Gi Gi GqGq

PLCPLC

Ca++

Fused receptor/Gi/Gq allows for transfection of a single moleculeand increases signal.

ChimeraChimera

Hit to lead

Page 12: Live cell imaging: Visualization of [Ca 2+ ] i fluctuation Dual-wavelength methods –Alternating excitation wavelength with fixed emission observation (Fura)

Assay standardization

Hit to lead

2-MeS-ADP dose response

-11 -10 -9 -8 -7 -60

10000

20000

30000

40000

2-MeS-ADP Concentration [log M]

Re

lati

ve

Flu

ors

ce

nc

eU

nit

s (

ma

x-m

in)

EC50=1.59 nM

Dose response of standard agonist

Parameter to be standardized

-cell number per well-solvent concentration-dye loading time-incubation time-instrument parameter setting-Etc, etc

Page 13: Live cell imaging: Visualization of [Ca 2+ ] i fluctuation Dual-wavelength methods –Alternating excitation wavelength with fixed emission observation (Fura)

Example of screening resultsScreening resulted in 2926 hits of which 1310 were confirmed after 5-points IC50 runThese compouds were clustered in 200 groups

By combining the most active cluster members and the top 200 compounds (sorted by average IC50) were identified 338 compounds to be tested

Hit to lead

Page 14: Live cell imaging: Visualization of [Ca 2+ ] i fluctuation Dual-wavelength methods –Alternating excitation wavelength with fixed emission observation (Fura)

Example of hits

HN

O

O

OH

O

Ki 278 nM

HN

O

F

F

F

HO

Ki 848 nM

S HN

O

Cl

Ki 902 nM

NN

O

O

O

O

HO

O

Cl

Ki 592 nM

NN

N

NN

O

O

O

Cl F

Ki 796 nM

N

N

S

O

O

Br

O

O

O

O

Ki 775 nM

Hit to lead

Evaluation of compounds’ chemical tractabilityEvaluation of patent position

Identification of lead compound/series

Page 15: Live cell imaging: Visualization of [Ca 2+ ] i fluctuation Dual-wavelength methods –Alternating excitation wavelength with fixed emission observation (Fura)

Identification of the pathology of interest

• What would be better between an:– Antithrombotic?

– Antibleeding?

– Analgesic?

Knowles and Gromo, Nature Rev Drug Disc 2002

Anticoagulants are useful in primary and secondary prevention of deep vein thrombosis, pulmonary embolism, myocardial infarctions, and strokes

Page 16: Live cell imaging: Visualization of [Ca 2+ ] i fluctuation Dual-wavelength methods –Alternating excitation wavelength with fixed emission observation (Fura)

Reference standard for in vitro and in vivo

• In vitro:– to compare efficacy and potency at the receptor

• In vivo:– to compare efficacy in disease models– to evaluate side effects

Hit to lead

Page 17: Live cell imaging: Visualization of [Ca 2+ ] i fluctuation Dual-wavelength methods –Alternating excitation wavelength with fixed emission observation (Fura)

Commercially available drugs acting on platelets’ activation cascade

Aspirin

XResting platelet

ThrombinCollagen

ADP TxA2

PAFEpinephrine

GP IIb/IIIa

Activated platelet

X

Your drug

Orbofiban Integrilin Abciximab

X

Hit to lead

Clopidogrel: unknown site of action

Page 18: Live cell imaging: Visualization of [Ca 2+ ] i fluctuation Dual-wavelength methods –Alternating excitation wavelength with fixed emission observation (Fura)

Surprise, surprise!!

Clopidogrel is acting on P2Y12!!!

Hit to lead

Page 19: Live cell imaging: Visualization of [Ca 2+ ] i fluctuation Dual-wavelength methods –Alternating excitation wavelength with fixed emission observation (Fura)

Effect of clopidogrel on platelets aggregrations

Hit to lead

(Foster et al 2001)

Clopidogrel blocks ADP and Collagen-induced aggregration in wild type but not in P2Y12 KO mice

Page 20: Live cell imaging: Visualization of [Ca 2+ ] i fluctuation Dual-wavelength methods –Alternating excitation wavelength with fixed emission observation (Fura)

And now what we do?

Hit to lead

Page 21: Live cell imaging: Visualization of [Ca 2+ ] i fluctuation Dual-wavelength methods –Alternating excitation wavelength with fixed emission observation (Fura)

A close look to the drug profile

Clopidogrel profile:

-Clopidogrel is an effective antithrombotic drug that provides significant protection against heart attack and stroke.

- It is a prodrug that must be metabolized to an active species.- Responses in patients are variable. - It binds covalently to the receptor. - The drug must be given for several days before the maximum clinical effect is observed.- Elimination of the drug is slow.

Hit to lead

Page 22: Live cell imaging: Visualization of [Ca 2+ ] i fluctuation Dual-wavelength methods –Alternating excitation wavelength with fixed emission observation (Fura)

There is an unmet medical need!!

Improvement of available therapy

• To discovery an antithrombotic agents with improved efficacy, that binds reversibly to the receptor and has a better pharmacokinetic profile.

Hit to lead