ultra-hts using 3456-well plate and htrf technology in...

29th Sep 2016

Ultra-HTS Using 3456-Well Plate and HTRF Technology in GPCR Drug Discovery

Toyo Sakurai Hit Discovery and Cell Processing Research Group, Biological Research Department

2

Agenda

3456-well Plate

Miniaturization of HTRF cAMP Assay

HTS Campaign

HTRF Symposium in TOKYO 29SEP2016

3

How many wells are there in the plate?


～25 μl/well

～ 5 μl/well

～ 1 μl/well

3.7 mm

1.7 mm

1.1 mm

384-well plate 16 x 24



4 HTRF Symposium in TOKYO 29SEP2016

Required;

Plate supplier: Aurora (from Waken Btech) Liquid dispenser: Echo and BioRAPTR (non-contact) Plate reader: PHERAstar and ViewLux (high resolution)

Only Homogenous Assay！

Notes of 3456-well Plate

Assay well

Lid

Evaporation control well

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Issues of 3456-well Assay


Evaporation Evaporation of liquid causes terrible edge effect.

Dispensing & mixing It is extremely difficult to dispense <1 μl to very small wells with accuracy. It is necessary to consider the generation of bubbles and the mixing reagents.

Stacker It is not possible to use the stacker of plate reader, because 3456-well plate has no flange (or skirt).


3456-well Plate


HTS Campaign

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HTRF cAMP Assay


HTRF cAMP kit enables a quantitative measure of cAMP homogeneously.

cAMP concentration (Log)

Rat

io (

66

5 n

m/6

20

nm

x 1

04)

Competitive, homogeneous assay

8

Use of Suspension Cells for Homogenous Assay


cAM

P c

on

c.

Ligand

Homogeneous assay was available to evaluate the activity of GPCR-X using HTRF cAMP HiRange kit.

Low

High

GPCR-X

9

Miniaturization from 1536 to 3456-Well


Although miniaturization to 3456-well affected the ratio of HTRF signal slightly, it was enough sensitivity for HTS.

L ig a n d [ L o g ( M ) ]

Ra

tio

- 3 - 2 - 1 0 1 2

0

1 0 0 0

2 0 0 0

3 0 0 0

4 0 0 0

5 0 0 0

1 5 3 6 - w e l l

3 4 5 6 - w e l l

- 3 - 2 - 1 0 1 2

0

2 0 0 0

4 0 0 0

6 0 0 0

8 0 0 0

L ig a n d [ L o g ( M ) ]R

ati

o

1 5 3 6 - w e l l

3 4 5 6 - w e l l

GPCR-X GPCR-Y

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Protocol of GPCR-X cAMP Assay


Steps 1536-well 3456-well

Dispense compounds (Echo) ～ 30 nl ～ 7.5 nl

Seed cells (BioRAPTR) 3 μl

(1,500 cells/well) 0.8 μl

(400 cells/well)

↓ 37°C for 90 minutes

Add d2 & cryptate (BioRAPTR) 1 μl each 0.4 μl each

↓ Room temperature for 120 minutes

Measure HTRF signal (PHERAstar) 8 min/plate 17 min/plate

5 μl/well, total 1.6 μl/well, total

We established robust protocol with < 2 μl/well total volume in 3456-well format.

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Advantage of Miniaturization


384-well 1536-well 3456-well

Total (reagent) volume / well 20 (5) μl 5 (1) μl 1.6 (0.4) μl

Cells / well 6,000 1,500 400

Plates / batch 60 plates 30 plates 20 plates

Compounds / batch 19,200 38,400 64,000

Required for HTS campaign (e.g. 200,000 compounds)

Regent 1,200 ml 240 ml 87 ml

Cells 1.4 x 109 cells 3.6 x 108 cells 8.7 x 107 cells

Plates 625 plates 157 plates 63 plates

Batches 11 times 6 times 4 times

We achieved excellent performance by miniaturization to 3456-well format.

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Index of Assay Stability


Z’ = 1 – 3*(SD_high + SD_low) / (MEAN_high – MEAN_low)

Z = 1 – 3*(SD_high + SD_sample) / (MEAN_high – MEAN_sample)

Low High Mean 256 787

SD 7.0 55.7

S/B 3.1 Z' 0.65

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Robust Performance in 3456-well Format GPCR-X Agonist Assay


3456-well assay indicated high stability and flatness.

Z’ = 0.68 Z = 0.73

DMSO for control Ligand DMSO

GPCR-X


0 1 0 2 0 3 0 4 0 5 0 6 0 7 0

0

1 0 0 0

2 0 0 0

3 0 0 0

4 0 0 0

5 0 0 0

6 0 0 0

7 0 0 0

8 0 0 0

C o lu m n

Ra

tio

H ig h D a t a P o s it iv e c o n t r o l L o w c o n t r o lDMSO Ligand (EC80) with DMSO

Ligand (Emax) Ligand (EC80) with DMSO for control

Z’ = 0.57 Z = 0.57

Antagonist screening is also available.

Seed cells Add ligand Add d2& cryptate Measure

0.5 μl 0.25 μl 0.3 μl, each

Robust Performance in 3456-well Format GPCR-Y Antagonist Assay

GPCR-Y


About 3456-well Plate


HTS Campaign

16

Evaluation of Test Screening


3456-well format showed good reproducibility.

Inhibition (%), replicate 1

Inh

ibit

ion

(%

), r

eplic

ate

2

GPCR-Y Antagonist Reproducibility = 76%


HTS campaign using 3456-well plate was performed with good stability and quality.

Z’ = 0.60 ± 0.05

Stability of HTS Campaign

GPCR-Y Antagonist

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Dose-Response Test for 3 Target (species, selectivity)

Hit Profiling of GPCR-Y Antagonist


3456-well format is also quite helpful at the hit profiling stage.

384-well

much data point…

3456-well

19

Conclusion


Miniaturized ultra-HTS using 3456-well plates showed sufficient stability, cost effectiveness and time saving.

HTS campaigns for GPCR-X agonists and GPCR-Y antagonists were implemented with robustness.

cAMP HTRF assay is applicable to 3456-well format. Now we apply other HTRF technologies to 3456-well format.

ultra-hts using 3456-well plate and htrf technology in...

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