Excello Pharmaceutics has identified and validated a new target (Receptor R) for the

treatment of inflammatory conditions such as arthritis. Receptor R is a G-protein coupled

receptor that couples to the Gq type of G-proteins. The selective and stable agonist for the R
receptor is A. Activation of R by A leads to activation of Gq proteins, resulting in increased
formation of second messengers inositol triphosphate (IP3) and intracellular Ca2+. A has
limited therapeutic use as it has low selectivity. The company is in the process of developing
assays to identify a lead compound. They have started tool development and have CHO
cells stably transfected with the receptor R and the radioligand [3H]-A is commercially
available.
They have decided to use a heterogeneous receptor binding assay for their primary high
throughput screen (HTS).
A. Discuss the advantages and disadvantages of the use of a heterogeneous verses a
homogeneous receptor binding assay.
B. Outline the assay conditions they will need to optimize prior to starting the screen. How
will these factors influence the final data?
C. Prior to the HTS the pharmacologists at Excello Pharmaceutics performed a saturation
binding assay.
(i) How will they use the data obtained from this experiment?
(ii) From the Scatchard plot results, shown below, determine the KD value and the receptor
density Bmax in CHO cells. Give units for both parameters.
Scatchard Plot

Total binding
Specific binding
Non-specific binding

75000

Specific bound/Free

Count per minute (CPM)

Saturation

50000

25000

0.15

0.10

0.05

0.00
0

400

800

1200

1600

10

20

30

40

50

60

70

Specific bound (pM)

[ H]-A (pM)

Scatchard plot analysis results (output from a program such as Prism):

Y-intercept = 0.1134
X-intercept = 65.55
1/slope = -578
correlation coefficient r = 0.90
D. Describe the type of assay you would choose for the secondary screen. Include in your
answer the role of a secondary screen and advantages and disadvantages inherent in the type
of assay you chose.

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