Development of Reporter Gene Assay for Calcitonin Receptor

The calcitonin receptor (CTR) is a GPCR that binds the peptide calcitonin (CT) and protects the skeleton by inhibition of bone resorption by osteoclasts (Sexton, Findlay, and Martin 1999). In order to develop a stable cell line expressing human CTR, the Freestyle 293F cell line (Invitrogen) was chosen because it tested negative for expression of receptor activity-modifying proteins (RAMPs) 1 through 3 in a bDNA analysis experiment. RAMPs are a family of integral membrane proteins that associate with CTR to generate high affinity amylin receptors, AMY1 and AMY2, that display different ligand affinity and specificity compared to CTR (Hay, Poyner, and Sexton 2006). The 293F cells were transfected with a plasmid containing the CTR gene and a CMV promoter. As CTR is coupled to Gas and its activation increases the intracellular level of cAMP, the cells were also transfected with a plasmid containing the ^-lactamase reporter gene under control of a CRE in its promoter region. Thus, activation of CTR by human CT (hCT) is expected to increase the level of cAMP in the CTR-CRE-bla cells and this will stimulate expression of the b-lactamase enzyme.

Following transfection, the CTR-CRE-bla cells were grown in the presence of appropriate antibiotics for 2 weeks and sorted using a flow cytometer. In these preliminary experiments, the pool of cells was divided into two groups, only one of which was stimulated with 1 nM hCT. After 4 hr, both groups of cells were incubated in the presence of CCF4 (1 pM) for 2 hr. When excited by light at 409 nm on a flow cytometer, cells that expressed b-lactamase emitted a blue fluorescent signal. A pool of responders was collected along with four 96-well plates of single cell clones.

We found that 25% of cells in the hCT-stimulated group expressed b-lactamase, compared to <1% of cells in the unstimulated group. The single cell clones were grown in the 96-well plates until there were sufficient cells for continued maintenance in culture and functional testing in a concentration-response experiment using hCT. We were most interested in the clones that displayed both the highest blue to green fluorescence ratio as well as EC50 values for hCT that were in close agreement with literature values. These clones were subjected to further analysis using additional CTR agonists such as amylin to identify the best clone, which we considered to be the one displaying the correct rank order of ligand pharmacology and the highest S/B ratio.

Having selected the best CTR-CRE-bla clone, we determined its performance at different cell densities using the protocol described in Figure 4.3A. Cells were seeded at 2,500, 5,000, 10,000 or 20,000 per well in poly-D-lysine coated 384-well black clear-bottomed plates (BD Biosciences, BioCoat™, #354663) in assay medium [Dulbecco's modified Eagle minimal essential medium (D-MEM) with 10% dialyzed fetal bovine serum (FBS), 25 mM HEPES (pH 7.3), 0.1 mM non-essential amino acids, penicillin 100 units/mL, and streptomycin 100 pg/mL]. The plates were kept in an incubator at 37°C with 5% CO2 in air for 16 to 20 hr and subsequently various concentrations of hCT (10 pL) were added to each well and the cells incubated for a further 4 hr. Subsequently, 10 pL of CCF4 (1 pM) were added to each well and then the plate was kept in the dark at room temperature for 1.5 hr prior to reading on an Analyst-GT. Figure 4.3B shows the hCT-induced b-lactamase activity in the best CTR-CRE-bla clone; response ratios were calculated according to Equation 4.1:

We found no significant difference in the response ratio between cells plated at 2,500, 5,000 or 10,000 cells per well. However, when the cells were plated at 20,000 cells per well, we observed a lower response ratio due to a higher unstimulated background. We chose a cell density of 10,000 cells for all subsequent assay optimization steps because it revealed the least well-to-well variation compared to the other cell densities.

To determine the optimal CCF4 loading time, the cells were stimulated with various concentrations of hCT for 4 hr. Subsequently, the signal from each well on the plate was read 1, 1.5, and 2 hr

Calcitonin Receptor P-Lactamase Reporter Gene Assay Seed cells in 384-well plates (40 ^L)

^ Incubate at 37°C overnight

Add compounds and hCT diluted in assay buffer (10 ^L) ^ Incubate at 37°C for 4 hr

Add p-lactamase substrate CCF4 (10 ^L)

Incubate in darkness at ^ room temp Read plate on Analyst-GT

Effects of Cell Density per Well

■ 20 K cells/well • 10 K cells/well v 5.0 K cells/well o 2.5 K cells/well

■ 20 K cells/well • 10 K cells/well v 5.0 K cells/well o 2.5 K cells/well

CTR Agonists Dose-Response

CTR Agonists Dose-Response

CTR Antagonist Dose-Response

CTR Antagonist Dose-Response

-9 -8 -7 -6 -5 Log [sCT(8-32)] (M)

DMSO Effects

DMSO Effects

■ 1.0% DMSO o 0.5% DMSO □ 0.25% DMSO ♦ 0.0% DMSO

IA 1 i ■ *

Scatterplot ofaDMSO Plate

O DMSO a High control □ Low control

0 100 200 300 Well Number

FIGURE 4.3 Development of b-lactamase reporter gene assay for CTR. (A) Protocol for CTR b-lactamase reporter gene assay. (B) Concentration-response curves for hCT in the presence of different numbers of CTR-CRE-bla cells per well. (C) Concentration-response curves for four CTR agonists in the b-lactamase assay; rank order of potency was sCT > hCT > amylin > a-CGRP. (D) Concentration-response curve of CTR antagonist sCT(8-32) on hCT-induced b-lactamase activity. (E) Determination of DMSO tolerance of CTR assay. (F) Scatter plot of signals obtained in each well of 384-well plate. All wells in columns 1 through 23 contained 100 pM hCT and 0.4% DMSO; the 16 wells in column 24 contained DMSO only.

after the addition of the substrate. The EC50 values of hCT were similar at all three time points but a loading time of 2 hr yielded the largest response ratio, and so we chose a 2-hr loading time for all subsequent assay optimization steps.

Having established the optimal conditions for the b-lactamase assay, we determined the rank order of the potencies of selected peptide agonists of CTR. All peptides were dissolved in water before dilution in assay buffer. The cells were stimulated with various concentrations of hCT, salmon

CT (sCT), amylin, and a-calcitonin gene-related peptide (a-CGRP) for 4 hr at 37°C (Figure 4.3C). All agonists displayed similar maximum blue to green fluorescence ratios of ~1.7 and assay windows of ~8. All agonists produced complete concentration-response curves and we calculated the EC50 values of sCT, hCT, amylin, and a-CGRP to be 7, 14, 400, and 3700 pM, respectively, all of which are similar to literature values (Hilton et al. 2000).

The effect of the CTR antagonist peptide, sCT(8-32), was tested in the presence of 100 pM hCT to determine both the receptor specificity of the b-lactamase-dependent signal and the assay performance in the antagonist mode. The antagonist peptide was added along with hCT in the first addition step of the protocol, and they were incubated together for 4 hr at 37°C. We found that sCT(8-32) inhibited hCT-induced b-lactamase activity in a concentration-dependent manner with an IC50 of 25 nM (Figure 4.3D). Overall, our data demonstrate that the CTR-CRE-bla cells respond to known CTR modulators with expected potencies.

Cell-based assays are generally less DMSO-tolerant than biochemical assays. Due to the long compound incubation time of the b-lactamase assay, it is particularly important to pay attention to the effect of this agent on assay performance. Therefore, the EC50 value of hCT was determined in the presence of 0%, 0.25%, 0.5%, and 1.0% DMSO. The response ratio for b-lactamase activity in the presence of different DMSO concentrations is shown in Figure 4.3E. We found that DMSO reduced the response ratio in a concentration-dependent manner. For instance, the maximal response ratio was reduced by approximately 20% and 50% in the presence of 0.5% and 1% DMSO, respectively. The EC50 value of hCT was similar in 0% and 0.5% DMSO (8 and 7 pM, respectively). Based on these results, we determined the maximum permissible concentration of DMSO to be 0.5% during pharmacology and HTS experiments.

Figure 4.3F shows a scatter plot of the individual signals in 384 wells of a representative plate in a DMSO trial run (final concentration 0.4%); as in the GPR23 assay, this run was used to simulate assay performance during a screening run. All wells in columns 1 through 23 received 10 |L of hCT (500 pM) and DMSO (2%) in assay buffer during the first addition step of the assay protocol. The wells in columns 1 through 22 were considered to simulate the wells of a screening plate containing inactive test compounds. The wells in column 23 represented the high control wells of our plate. The wells in column 24 received 10 |L of assay buffer containing DMSO (2%) and represented the low control wells of our plate.

The plate was incubated for 4 hr at 37°C and then CCF4 (10 |L of 1 |M) was added to each well and left for 2 hr prior to reading. We found that hCT stimulated a robust b-lactamase signal with very little variation among wells. The 16 high and 16 low control wells revealed blue to green fluorescence ratios of 2.0 ± 0.1 and 0.4 ± 0.02 (mean ± S.D.), respectively. The blue to green fluorescence ratio for the 352 wells in columns 1 through 22 was 2.0 ± 0.1 (mean ± S.D.), which is similar to the high control ratio. We calculated the Z' value to be 0.9, which is indicative of an excellent assay (Zhang, Chung, and Oldenburg 1999). This b-lactamase assay could also be configured to identify agonists of CTR. In the agonist screening format, the wells in columns 1 through 22 would receive test compounds in the absence of hCT. Compounds that activate CTR and stimulate b-lactamase activity would be identified by higher blue to green fluorescence ratios compared to the low control wells.

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