Summary

There are currently a myriad of GPCR assays to consider when embarking on a HTS campaign—biochemical or cell based, functional, or binding, and so on. While all HTS assays would benefit from the use of appropriate downstream assays to eliminate off- t arget activities, functional assays effectively cast a larger net during HTS, identifying compounds with specific and nonspecific effects. Compounds identified by binding assays, however, do not always

TABLE 13.3 Comparison of GPCR Antagonist Potencies among Different HTS Assays

Sample No.

IC50 (nM)

Whole Cell Binding

FLIPR

BLA

Transfluor

8

320

103

50

730

9

ND

157

64

2800

10

182

187

80

550

11

ND

209

90

6782

12

1220

904

124

2200

13

607

751

140

3600

14

1430

220

163

2200

15

630

334

165

4800

16

ND

1500

200

5200

17

ND

492

209

10,000

18

1290

460

244

5200

19

2400

3300

252

8800

20

1850

1300

290

6300

21

1180

2700

375

29,000

22

2510

9800

418

13,500

23

1850

632

556

5600

Assay

Receptor Binding

HitHunter cAMP

PathHunter

Plate Format

384 Well

1536 Well

3456 Well

Sample

IC50 (nM)

IC50 (nM)

IC50 (nM)

1

11

2.5

23

2

3

0.85

13

3

18

5.3

9

4

18

2.3

200

5

0.49

6.8

176

6

9.3

165

24

7

6.1

31

24

8

739

111

31

9

865

259

177

10

28

33.5

170

11

255

781

171

12

83

7.5

599

translate into having functional efficacy or altering cellular physiology in a predictable manner. The assay of choice for HTS or uHTS should be selected with care depending on the signaling cascade and availability of activating ligands, with preference given to more proximal readouts (second messenger vs. reporter gene). Recent advances in GPCR biology also point to additional signaling cascades originating from activated GPCRs in addition to the classical second messenger-driven signaling [50] . The physiological effects driven by each of these signaling pathways need to be recognized and taken into account in the selection of suitable assays for HTS and uHTS.

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