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Developing Parallel Functional Assays for Opioid Receptors

Miao Tan, Radhika Venkat and Helena ManceboMultispan, Inc.

26219 Eden Landing Road , Hayward , CA 94545-3718 Correspondence: info @ multispaninc.com

Opioid receptors belong to the large superfamily of seven-transmembrane G-protein coupled receptors. They are activated by endogenous opioid peptides and by a series of synthetic compounds used in the clinic. There are four major subtypes of opioid receptors – delta (DOR), mu (MOR), kappa (KOR), and nocipeptin (NOP). The opioid receptors are important targets in drug discovery, widely distributed in the brain, spinal cord and digestive tract. Drugs against these receptors are known as narcotic analgesics and used in the treatment of severe pain. The majority of the drugs developed against opioid receptors are limited by liabilities such as respiratory depression and drug dependence. Therefore, there has been considerable interest in developing new ligands for opioid receptors. All opioid receptors are coupled through pertussis toxin-sensitive Gαi protein. In this report, we present optimized parallel functional assays such as cAMP, calcium, ERK, internalization and GTPγS from cells stably expressing opioid receptors or membranes in response to agonist and antagonist stimulations. These optimized assays from Multispan can be used for screening compounds that go through di�erent signaling pathways and discovering new therapeutic interventions with improved potency and e�cacy while having reduced side-e�ects.

ABSTRACT INTRODUCTIONG-protein coupled receptors (GPCRs) play critical roles in human physiology and are prime targets for drug discovery in the pharmaceutical industry. GPCRs are involved in many diseases, and are also the targets of approximately 30% of all medicinal drugs. GPCRs mediate signaling of stimuli as diverse as light, ions, small molecules, peptides and proteins and are the targets for many pharmaceutical drugs. These receptors are mainly coupled through Gαq, Gαi or Gαs proteins. Advances in high-throughput detection technologies have resulted in an increased use of cell-based functional assays in early drug discovery, in particular for GPCRs. Multispan has optimized parallel functional assays such as cAMP, calcium, ERK, internalization and GTPγS assays for wide range of GPCRs. The opioid receptors KOR, MOR DOR and NOP comprise of a prominent family of GPCRs and share extensive structural homologies among each other. In this report we present data for functional assays generated for opioid receptors. Our unique beta-arrestin dependent and independent internalization assay along with other functional assays can be used for high-throughput pro�ling and screening for pathway-speci�c compounds that modulate pain through the opioid receptors.

DOR KOR MOR NOP

ERK Assay

Gαi Gαqi5

GTP S

-arrestin independent

Degradation

Recycling -arrestin

cAMPAssayAssay Ca++ Assay Internalization

Assay

Compounds: Compounds used as agonists or antagonists were DAMGO (Phoenix, Cat # 024-10), Dynorphin B (Bachem, Cat # H2675), Deltrophin II (Sigma, Cat # T-0675), OFQ (Sigma, Cat # O4011) and Naltrexone (Sigma, Cat # N3136).

Stable Cell lines: MOR-CHO-K1(Multispan Inc., Cat # C1350-1a), KOR-HEK293T (Multispan Inc., Cat # C1354a), DOR-CHO-K1 (Multispan Inc., Cat # C1351-1) and NOP-HEK293T (Multispan Inc., Cat # C1352)

Calcium Mobilization Assay: Cells cultured overnight in Poly-D-Lysine-coated plates were incubated for one hour with bu�er containing calcium indicator dye FLIPR® Calcium 4 (Molecular Devices, R8142). Calcium �ux was monitored upon addition of compounds using FlexStation III (Molecular Devices).

cAMP Assay: Cells treated with compounds were subjected to cAMP assay using cAMP Hi-Range Kit (Cisbio, 62AM6PEC).

ERK Assay: Cells treated with compounds were subjected to ERK assay using Kit (Cisbio, 64ERKPEG).

Membrane preparation: Membranes for GTPγS assay were prepared from stable cell lines MOR-CHO-K1 (Multispan Inc., Cat # MC1350-1a), KOR-HEK293T (Multispan Inc., Cat # MC1354a), DOR-CHO-K1 (Multispan Inc., Cat # MC1351) and NOP-HEK293T (Multispan Inc., Cat # MC1352) expressing full-length opioid receptors

GTPγS assay: Membranes obtained from stable cell lines expressing opioid receptors were subjected to GTPγS assay using SPA beads (Perkin Elmer, Cat # RPNQ0001).

DOR C1351-1

MOR C1350-1a

KOR C1352

NOP C1354a

Parallel Functional Assays for Opioid ReceptorsDeveloped by Multispan Inc.

-13 -12 -11 -10 -9 -8 -7 -6 -50

7

14

21

Dynorphin BDeltrophin II

EC50=1.5x10-7 M

EC50=7.2x10-9 M

Log [Ligand] M

RFU

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

10

20

30

40

50

60MORCHO-K1

Log [Dynorphin B] M

RFU

EC50=8.8x10-8 M

-12 -11 -10 -9 -8 -7 -6 -50

10

20

30 KORHEK293T

Log [Dynorphin B] M

RFU

EC50=2.6x10-9 M

-12 -11 -10 -9 -8 -7 -60

25

50

75EC50=1.5x10-9 M NOP

HEK293T

Log [OFQ] M

RFU

DOR MOR

KOR NOP

Figure 1. Dose-dependent intracellular Ca++ �ux was monitored upon treatment with control agonists. (A) DOR in CHO-K1 cells. (B) MOR in CHO-K1 cells. (C) KOR in HEK293T cells. (D) NOP receptor in HEK293T cells. Agonists showed expected EC50 values for all opioid receptors.

Validated Ca++ Assay for Opioid Receptors

A B

C D

KOR NOPA B

DOR MOR

NOP

A B

C

DOR* MORA B

KOR NOPC D

DOR MOR

KOR NOP

A B

C D

-13 -12 -11 -10 -9 -8 -7 -6 -5 -43000

4000

5000

6000

7000

8000EC50=3.5x10-9 M

Log [Dynorphin B]

Ratio

668

/620

x 1

0,00

0

-13 -12 -11 -10 -9 -8 -7 -6 -5 -43000

4000

5000

6000

7000

8000

9000EC50=1.2x10-8 M

Log [Dynorphin B]

Ratio

668

/620

x 1

0,00

0

-13 -12 -11 -10 -9 -8 -7 -6 -53000

4000

5000

6000

7000

8000EC50=1.2x10-9 M

Log [Dynorphin B] M

Rat

io 6

68/6

20 x

10,

000

-13 -12 -11 -10 -9 -8 -7 -6 -55000

6000

7000

8000

9000EC50=2.3x10-10 M

Log [OFQ] M

Rat

io 6

68/6

20 x

10,

000

Validated cAMP Assay for Opioid Receptors

Figure 2. Inhibition of forskolin-stimulated cAMP levels by control agonists in either CHO-K1 or HEK293T cells stably expressing the human opioid receptors. (A) DOR in CHO-K1 cells, (B) MOR in CHO-K1 cells, (C) KOR in HEK293T cells and (D) NOP receptor in HEK293T cells. Agonist compounds showed expected EC50 values.

-11 -10 -9 -8 -7 -6 -50

100

200

300

400

500

600

CHO-K1MOR

EC50=8.6 x 10-9 M

Log [DAMGO] M

cpm

-12 -11 -10 -9 -8 -7 -6 -50

100

200

300

400

CHO-K1DOR

EC50=7.5x10-9 M

Log [Deltrophin II] M

cpm

-11 -10 -9 -8 -7 -620

40

60

80

100

120

140EC50=6.6X10-9 M

Log [OFQ] M

cpm

NOPHEK293T

GTPγS Assay for MOR, DOR and NOP Receptors

Figure 3. Dose dependent GTPγS assay for MOR, DOR and NOP receptors expressed in either CHO-K1 or HEK293T cells with control agonists using 35S radioligand. (A). MOR, (B) DOR and (C) NOP receptor. Control agonists show expected EC50 values for all three receptors.

Log [Dynorphin B]M

% S

urfa

ce R

ecep

tor

-10 -9 -8 -7 -6 -5 -470

80

90

100

110

120

130

EC50 = 2.8 x 10-7M

-11 -10 -9 -8 -7 -6 -5 -46065707580859095

100105

Log [OFQ] M

% S

urfa

ce R

ecep

tor

EC50 = 3.6 x 10-8M

Validated Internalization Assay for KOR and NOP Receptors

Figure 4. Dose-dependent internalization assay for KOR and NOP receptors using control agonists. The percent surface receptors were monitored by FACS analysis. (A) KOR (B) NOP receptor. Reduction of the surface receptor was 36% and 35% for KOR and NOP respectively after agonists treatment. The EC50 values obtained for both the receptors are comparable with the published data.

-11 -10 -9 -8 -7 -6 -5 -440

60

80

100

EC50 = 7.29 x 10-8M

Log [DAMGO] M

% S

urfa

ce R

ecep

tor

-12 -10 -8 -6 -4

60

80

100

120

IC50 =4.4 x 10-9M

Log [Naltrexone] M

% S

urfa

ce R

ecep

tor

Figure 5. Internalization assay was monitored by FACS analysis for MOR using control agonist and antagonist. (A) Reduction of the surface receptor was 55.05% after agonist treatment. (B) MOR antagonist dose-dependently reversed the agonist-induced receptor internalization with potencies comparable to published data.

A Agonist Assay B Antagonist Assay

Validated Internalization Assay for MOR Receptorwith agonist and antagonist

Validated Phospho-ERK Agonist Assay for Opioid Receptors

Log [Deltrophin II] M

Rat

io 6

68/6

20 x

10,

000

-11 -10 -9 -8 -7 -6 -5 -44000

4500

5000

5500

6000

6500 EC50 = 4.6 x 10-9 M

-12 -10 -8 -6

2000

4000

6000

8000

10000

12000

14000

Log [DAMGO] M

Rat

io 6

68/6

20 x

10,

000

EC50 = 8.0 x 10-10 M

-11 -10 -9 -8 -7 -6 -5 -4500

1000

1500

2000

2500

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3500

Log [Dynorphin B] M

Rat

io 6

68/6

20 x

10,

000 EC50 = 9.9 x 10-9 M

Log [OFQ] M

Rat

io 6

68/6

20 x

10,

000

-10 -8 -6 -4800

1000

1200

1400

1600

1800 EC50 = 1.5 x 10-8 M

Figure 6. Phospho-ERK assay for opioid receptor using control agonists. (A) DOR (B) MOR, (C) KOR and (D) NOP receptors. The EC50 values obtained are comparable with the published data. * Further opitmization is in progress for DOR ERK assay.

• Opioid family receptors have been validated in cell based functional assays including cAMP, calcium, ERK, internalization and membrane GTPγS assays using known agonists and antagonists.

• Consistent EC50 and IC50 values similar to published reports have been obtained, demonstrating excellent pharmacological performance of all the opioid receptors.

• Our unique internalization assay directly quantifies the disappearance of the receptors from the cell surface capturing both beta-arrestin dependent and independent receptor desensitization, providing a highly desirable new way of screening compounds with high therapeutic e�cacy and little side e�ects that are known to the existing therapies targeting this receptor family.

• Screening and profiling using the composite of functional assays measuring different signaling pathways of opioid receptors provide important information on compounds during lead optimization in drug discovery using this target family.

Conclusions

Materials and Methods

Division- Arrested Cells MembranesGTPγS ERKInternalizationcAMPCa++ Catalogue # GPCR