Live cell imaging: Visualization of [Ca 2+ ] i fluctuation Dual-wavelength methods –Alternating...
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Transcript of Live cell imaging: Visualization of [Ca 2+ ] i fluctuation Dual-wavelength methods –Alternating...
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
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
Some examples: fluorescence basedCa2+ measurement
GGqqPCRPCR
IP3
q
PIP2
DAG
PKC
PLC
Ca2+
Dye
Dye
FLUO4 DyeFLUO4 Dye
loading
Hit to lead
FLIPRFLIPR® ® systemsystemHit to lead
FlFluorometricuorometricIImagingmagingPPlatelateRReadereader
Generated data
FlFluorometricuorometricIImagingmagingPPlatelateRReadereader
Generated data
sGDP
sGTP
iGTP
iGDP
Ligand 1 Ligand 2
Some examples: cAMP measurementHit to lead
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
Questions?
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
Screening on cellsI. Cell Production
II. Cell treatment &/or compound exposure
III. Signal development/capture
Hit to lead
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
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
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
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
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
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
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
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
Surprise, surprise!!
Clopidogrel is acting on P2Y12!!!
Hit to lead
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
And now what we do?
Hit to lead
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
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