1ページ

What does Hamamatsu FDSS do?

Add 96,384,1536 well s at a time

Read fluorescence & luminescence

0.01Hz (100s) to 40Hz (25ms)

2ページ

- GPCR calcium influx assays All calcium wash or non-washing kits (Fluo-4 or Fura-2 based),

Premo™ Cameleon, Aequorin

- Ion Channel assays FMP, VSP, potassium channel (FluxOR™), sodium channel (SBFI),

chloride channel (YFP)

- Enzymatic assays Prolyl isomerase, GTPase, Kvb

- Transporter assays

- CytoStar-T

- Circadian clock

- Mosquito Odorant Receptors

- Light activated receptor or channel assays

Selected assays ran on FDSS

kinetic reader

3ページ

Use of Fura2 calcium dye in screening

buffer

10µM NTS143

100nM NTS143

Fluo4 Calcium NW dye

Fura2 Calcium NW dye

buffer

100nM NTS143

10µM NTS143

Agonist?

Antagonist!

1st addition carbachol

4ページ

K+-Channel openers suppress epileptiform activities induced by 4-Aminopyridine in cultured rat hippocampal neurons

Na+ indicator (SBFI), ex340/380, em540

J Pharmacol Sci 108, 517 – 528 (2008)

5ページ

New sodium dye_ANG-2

www.teflabs.com

6ページ

Potent and selective inhibition of the human Na + /H+ exchanger isoform HE1

European Journal of Pharmacology 420(2001) 1–8

BCECF

Fura2

7ページ

Tl+ assay for potassium chanels

Tl+ can permeate K channels and be detected by Tl+-sensitive dyes

8ページ

-10 -9 -8 -7 -6 -5 -4 -3 -2

1

2

3

4

5

Glutamate+

10 uM PHCCC

Glutamate

Log agonist, [M]

Slo

pe

(fo

ld o

f b

asa

l)

“GIRK” Assay for mGluRs

- HTS-compatible functional assays for Gi/o-coupled mGluRs has been challenging

- GIRK assay has enabled rapid development of quantitative, HTS-compatible assays of mGluR 2, 3, 4, 7, and 8

- Detection of agonists, antagonists and potentiators has been enabled

-10 -9 -8 -7 -6 -5 -4 -3 -2

0

25

50

75

100

125

LY

CPPG

MPPG

UBP1112

Log antagonist, [M]

Per

cen

t E

C8

0 g

luta

ma

te r

esp

on

se

mGluR4 mGluR8

Mol Pharmacol 73:1213–1224, 2008

9ページ

BacMam-hERG Expression/ FluxORTM

BacMam Delivery +

0.1 1 10 100 1000 100000.00

0.05

0.10

0.15

0.20

0.25

IC50 = 246 nM

01

Quinacrine dihydrochloride (nM)

max-m

in

0.1 1 10 100 1000 100000.00

0.05

0.10

0.15

0.20

0.25

IC50 = 64 nM

02

Clozapine (nM)

max-m

in

0.1 1 10 100 1000 100000.00

0.05

0.10

0.15

0.20

0.25

IC50 = 15 nM

03

Amitriptyline hydrochloride (nM)

max-m

in

0.1 1 10 100 1000 100000.00

0.05

0.10

0.15

0.20

0.25

IC50 = 31 nM

04

Histamine dihydrochloride (nM)

max-m

in

0.1 1 10 100 1000 100000.00

0.05

0.10

0.15

0.20

0.25

IC50 = >10,000 nM

05

Dipyridamole (nM)

max-m

in

0.1 1 10 100 1000 100000.00

0.05

0.10

0.15

0.20

0.25

IC50 = 278 nM

06

GBR-12909 dihydrochloride (nM)

max-m

in

0.1 1 10 100 1000 100000.00

0.05

0.10

0.15

0.20

0.25

IC50 = 16 nM

07

(+)-Chlorpheniramine maleate (nM)

max-m

in

0.1 1 10 100 1000 100000.00

0.05

0.10

0.15

0.20

0.25

IC50 = 37 nM

08

Cyproheptadine hydrochloride (nM)

max-m

in

0.1 1 10 100 1000 100000.00

0.05

0.10

0.15

0.20

0.25

IC50 = 38 nM

09

(±)-Chlorpheniramine maleate (nM)

max-m

in

0.1 1 10 100 1000 100000.00

0.05

0.10

0.15

0.20

0.25

IC50 = 36 nM

10

Clemizole hydrochloride (nM)

max-m

in

0.1 1 10 100 1000 100000.00

0.05

0.10

0.15

0.20

0.25

IC50 = 34 nM

11

Trimipramine maleate (nM)

max-m

in

0.1 1 10 100 1000 100000.00

0.05

0.10

0.15

0.20

0.25

IC50 = 118 nM

12

Chlorprothixene hydrochloride (nM)

max-m

in

0.1 1 10 100 1000 100000.00

0.05

0.10

0.15

0.20

0.25

IC50 = 336 nM

13

Fluphenazine dihydrochloride (nM)

max-m

in

0.1 1 10 100 1000 100000.00

0.05

0.10

0.15

0.20

0.25

IC50 = 10 nM

14

Benztropine mesylate (nM)

max-m

in

0.1 1 10 100 1000 100000.00

0.05

0.10

0.15

0.20

0.25

IC50 = 19 nM

15

Cyclobenzaprine hydrochloride (nM)

max-m

in

0.1 1 10 100 1000 100000.00

0.05

0.10

0.15

0.20

0.25

IC50 = 3.4 nM

16

Ketotifen fumarate (nM)

max-m

in

10ページ

Compound profiling for hERG channel activities

60 compounds

per 1536 plate,

12 concentrations

in duplicate

11ページ

Highly lipophilic natural ligand activating its GPCR

1st Assay Plate

2nd Assay Plate

1st addition 2nd addition

With one dispenser, full stimulation

of cells on next assay plate occurred,

due to carryover of sticky ligand

Using two dispenser protocol, a

HTS campaigne of 154,000

compounds, 445 assay plates

resulted Z‘-factor of larger than 0.5

Screen for agonists, antagonist, and

positive allosteric modulators with

sticky peptidic ligand

12ページ

Premo™ Cameleon Calcium Sensor

• Calcium sensor packaged in

BacMam virus

• Transduces hard-to-transfect cells

• Frozen storage of transduced cells

• True ‘no wash’ – screen in full

medium

+Ca2+

–Ca2+

13ページ

EC50=13.47uM EC50=8.36uM

Suspension HPK-CHO cell stimulated by ATP

14ページ

0 20 40 60 0.5

0.8

1.1

1.4

1.7

5.6

17

50

positive control

buffer

KCl (mM)

Time (sec)

RF

U/R

FU

(#1)

OFF

0 20 40 600.5

0.8

1.1

1.4

1.7

5.6

17

50

Positive Control

Buffer

KCl (mM)

Time (sec)

RF

U/R

FU

(#1

)

ON

Plate shaking to reduce artifact for channel assays

15ページ

YFP assay to identify modulators of CFTR-Δ F508

Control

30%

40%

50%

60%

70%

80%

90%

100%

0 10 20 30 40

Time (s)

No

rmalize

d F

luo

rescen

ce

In

ten

sit

y DMSO

FSK/Gen

+ I-172

FSK

FSK/Gen

10 mM C4

30%

40%

50%

60%

70%

80%

90%

100%

0 10 20 30 40

Time (s)

No

rma

lize

d F

luo

res

ce

nc

e In

ten

sit

y

DMSO

FSK/Gen

+ I-172

FSK

FSK/Gen

Plate cells

24h 37C

YFP reading

24h 27C

Corrector potentiator

16ページ

Low Mg2+-induced synchronized repetitive calcium oscillation in cultured neurons

40 ref & 28,000 cmpds @ 10 μM 150 cmpds selected for acute in

vivo antiepileptic effect (MES @

30 mg/kg ip Mouse)

17ページ

Characterizing the phenotype of iPS cardiomyocytes

Amplitude (a.u.) Rate (min-1) baseline 0.1% DMSO

baseline 1uM TG Baseline: t = 0.93s (n=4)

TG: t = 1.97s (n=4; p<0.005) Decay

SR

RyR RyR

Ca2+ ATP

SERCA

Ca2+ ATP

SERCA thapsigargin Lacking in

neonatal

myocytes

18ページ

HTS to identify Prolyl Isomerase Inhibitors

Journal of Biomolecular Screening. 14(4) p419-422. 2009

Suc-Ala-Ala-Pro-Phe-MCA, cis

Cyclophilin A Cyclosporin A

chymotrypsin

Suc-Ala-Ala-Pro-Phe-MCA, trans

Suc-Ala-Ala-Pro-Phe + AMC

thermo

365nm/460nm

plate shaking function

10Hz data collection

19ページ

Light as a GPCR agonist in HTS

0.20

0.38

0.05

1.5

0.75

0 30 60 90 120 150-0.1

0.0

0.1

0.2

0.3

0.4

0.5

0.6 3

Time (sec)

Net

Ch

an

ge R

ati

o340:3

80

20ページ

BacMam aequorin measured by photon counting camera and ImagEM

-12 -11 -10 -9 -8 -7 -6 -5 -40

250000

500000

750000Histamine

Methylhistamine

EC50

Histamine

3.8659e-009

Methylhistamine

3.8809e-006

EMCCD

Log [Agonist] M

Inte

gra

ted

RL

U

-12 -11 -10 -9 -8 -7 -6 -5 -4-5000

20000

45000

70000

95000

Histamine

Methylhistamine

EC50

Histamine

7.6494e-009

Methylhistamine

4.4401e-006

PC

Log [Agonist] M

Inte

gra

ted

RL

U

-12.5 -10.0 -7.5 -5.0-10000

90000

190000

290000

390000

490000

590000

Pyrilamine

Triprolidine

Chloropheniramine

Log [Antagonist] M

EC50

Pyrilamine

4.1970e-009

Triprolidine

4.3479e-009

Chloropheniramine

1.3801e-008

EMCCD

Inte

gra

ted

RL

U

-12.5 -10.0 -7.5 -5.0-2000

10000

22000

34000

46000

Pyrilamine

Triprolidine

Chloropheniramine

Log [Antagonist] M

EC50

Pyrilamine

2.3087e-009

Triprolidine

5.1425e-009

Chloropheniramine

8.4570e-009

PC

Inte

gra

ted

RL

U

21ページ

Kinetically analysis of endogenous b2AR full and partial agonists in HEK293 cAMP GloSensor cell line

Forskolin

Isoproterenol

Metaproterenol

Salmeterol

Dopamine

22ページ

Multiplexed analysis of second messenger signaling in live cells

23ページ

BRET: kinetic read on FDSS