Tag –lite: assessment of compound
dissociation from GPCRs
Israel Ramos Villullas
Group leader of GPCRs and Ion Channels unit
BRADS Department
1
INTRODUCTION
Compound residence time at protein targets is an important parameter in the Drug
Discovery process:
Efficacy
Selectivity
Side effects associated to different aspects as epitope formation or target
associated toxicity.
To assess residence time, different indirect methods have been developed using
radioactive binding assays that combine the use of a radioligand excess with a dilution
or separation step after the compound is bound to the target.
These methods have several drawbacks such as the use of radioactivity, difficult
results interpretation, assay price and assay logistics that justify a switch to
alternative technologies.
Label free methods as SPR are also used to assess dissociation but they need:
Access to very expensive instrumentation
They are difficult for GPCRs because they need GPCR immobilization.
OBJECTIVE
To develop a dissociation assay :
Non radioactive
Simple
Cost effective
With a reasonable throughput
that does not require specialized equipment
The technology evaluated has been the new fluorescence-based Tag-lite® technology, from
Cisbio® Bioassays
A dissociation assay for human M3 receptor has been developed. Results and assay
advantages and disadvantages over the current methods, will be presented.
Radiolabeled dissociation assay
Membranes + Antagonists (5-20Ki)
Between 2 and 3 hours
Dilute well content + addition of radioligand excess
Dissociation starts
Take samples at different times
The antagonist’s dissociation from receptors
is estimated indirectly by measuring the radioligand
association to the receptor binding sites, as they
become available, after compound dissociation
Radiolabeled dissociation assays ( drawbacks)
Radioactive and heterogeneous
Membranes
Each data point needs a filtration plate:
Cost: Each plate costs 20 €
Assay logistics
Assay price:
58.4 € / compound (10 data points in duplicate)
2.9 € / data point
Radiolabeled dissociation assays ( drawbacks)
Dissociation
Preincubation
Dissociation is initiated by a dilution step combined with an excess of radioligand.
Preincubation
< 5 Ki
Preincubation
5 – 20 Ki
Preincubation
>20 Ki
< 0.5 Ki
0.5 ki – 2 ki
> 2 Ki
We use internal controls to validate results
If we run the assay at one compound concentration, 30% of the compounds assayed fail.
We always assay compounds at 2 concentrations and in some cases at three.
Ideal dissociation assays
General aspects:
Non radioactive, Physiological environment (cells), microplate compatible, cheap
Homogeneous
Obtain all time points from the same well
Preincubation:
High compound concentration > 20 Ki to ensure receptor occupancy > 90 % in a
relatively short time.
No controls. The compound concentration is high so approximately 100%
occupancy is ensured
Dissociation:
Initiate dissociation by complete compound removal from the well.
No controls. The compound concentration is too low to interfere in the dissociation
phase.
Easy results interpretation.
Tag-Lite: a new way to do dissociation assays
PREINCUBATION
Adhere cells to the plate
Add compounds after adhesion
Use 100 Ki.
Cells are adhered so
Compound can be removed afterwards
using a washer!!!!
Tag-Lite: a new way to do dissociation assays
DISSOCIATION
Assess dissociation meassuring changes in FRET in the same well
Tag-Lite: a new way to do dissociation assays
Assay development steps
Can we obtain several time points from the same wells?
Red ligand characterization
Red Ligand saturation assays:
Kd
Choose red ligand concentration for dissociation assay:
Red Ligand occupancy >80%
Kinetics: Determine Kon and check the association rate of the chosen red-ligand
concentration.
• There is a need to quickly reveal the binding sites as soon as they become available
Antagonist potency: To determine compound concentration in the preincubation step
Can we wash the cells?
Red ligand characterization
Kd and Kobs
Saturation studies were ran in two different modes:
Incubating the plate at RT on the bench
Reading assay plate in kinetic mode in the Envision from Perkin Elmer:
25 readings during 5 hours
To answer different questions:
Which is the Red-Ligand Kd?
Is it possible to read the same well several times without affecting the Red Ligand
potency?
Which is the ideal Red Ligand concentration to run dissociation assays?
Red ligand characterization
Kd and Kobs
Red Ligand saturation assay.
3 hours RT incubation. Plate on the bench
Technician 1 -------- Kd: 202 ± 47 (n=3)
Red Ligand saturation and association assay.
Kinetic read
Technician 1 --------Kd: 199 ± 5 (n=2)
Technician 2 --------Kd: 102 nM (n=2)
CISBIO ------- Kd: 70 nM
Time (Minutes)
Can we take several readings from the same
well?
Red ligand potency does not change when the same wells are read several times:
Plate incubated 3 hours on the bench and read one time on the Envision. Kd: 202 nM
Plate incubated in the Envision and read it in kinetic mode. Kd: 199 nM.
Total binding is stable more than 15 hours:
21000
20000
Ratio
19000
18000
17000
16000
15000
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Time (hours)
Red ligand characterization
Red ligand concentration for dissociation assays
Assay window: 1.3
Assay window: 2.2; Kobs: 0.14 min -1
Assay window: 2.2 ; Kobs: 0.07 min -
Time (Minutes)
Antagonists characterization
Potency
% Inhibition
Tiotropium
Ipratropium
Glycopirrolate
Log [Compound] (M)
Compound
Ki
nM
10 x Ki
nM
100 Ki
nM
1000 Ki
nM
Ipratropium
0.90
9
90
900
Glycopyrrolate
0.3
3
30
300
Tiotropium
0.30
3
30
300
Antagonists characterization
Dissociation
Compound concentration during pre-incubation step can be used at more than 10 Ki ensuring
receptor occupancy.
Antagonists characterization
Dissociation
Dissociation profiles for the antagonists tested are in good agreement with those obtained
internally using the radioactive method .
Compound
Radioactive
assay
Tag lite
assay
Ipratropium
t ½: 0.2 h
t ½: 0.2 h
Glycopyrrolate
t ½: 2.6 h
t ½: 2 h
Tiotropium
t ½: 16 h
t ½: 17 h
Tag lite dissociation assay
Advantages
Cellullar non radioactive assay.
Allows easy assay design and results interpretation
Assay logistics is simple
From one 384 plate, using two columns for controls (Total Binding and NSB), 352 compounds
can be assayed in kinetic mode.
20 time points / cmpd---------------------------- 7040 data points / 352 cpds
Considering plate, buffer, cells (10 K/well) and Red Ligand (800 nM final concentration)
list price:
10 € / compound.
0.5 € / time point compared with 2.9 € from radioactive assay.
Tag lite dissociation
Disadvantages
Assay window is low at high Red-Ligand concentrations.
Red-Ligand associates slowly to M3 receptor.
Tag lite dissociation
Pending
Eventhough preliminar data looks encouraging , cell handling must be improved:
Cell recovery is not 100%
Adhesion protocol need to be improved for HEK cells (Some cells are lost after washing):
Consider plate coating
Optimization of adhesion time.
This work has been done by Joaquim Botta Nodar
Questions?
Thanks for your attention!!!