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. 2014 May;12(4):238-46.
doi: 10.1089/adt.2014.576.

A phenotypic high throughput screening assay for the identification of pharmacoperones for the gonadotropin releasing hormone receptor

P Michael Conn  1 , Emery Smith, Timothy Spicer, Peter Chase, Louis Scampavia, Jo Ann Janovick
Affiliations

A phenotypic high throughput screening assay for the identification of pharmacoperones for the gonadotropin releasing hormone receptor

P Michael Conn et al. Assay Drug Dev Technol. 2014 May.

Abstract

We describe a phenotypic high throughput screening (HTS) calcium flux assay designed to identify pharmacoperones for the gonadotropin releasing hormone receptor (GnRHR). Pharmacoperones are target-specific, small molecules that diffuse into cells, rescue misfolded protein mutants, and restore them to function. Rescue is based on correcting the trafficking of mutants that would otherwise be retained in the endoplasmic reticulum and unable to function correctly. This approach identifies drugs with a significant degree of novelty, relying on cellular mechanisms that are not currently exploited. Development of such assays is important, since the extensive use of agonist/antagonist screens alone means that useful chemical structures may be present in existing libraries but have not been previously identified using existing methods. Our assay utilizes cell lines stably expressing a GnRHR mutant under the control of a tetracycline (OFF) transactivator. This allows us to quantitate the level of functional and properly trafficked G protein coupled receptors present in each test well. Furthermore, since we are able to turn receptor expression on and off, we can rapidly eliminate the majority of false positives from our screening results. Our data show that this approach is likely to be successful in identifying hits from large chemical libraries.

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Figures

<b>Fig. 1.</b>
Fig. 1.
Gonadotropin releasing hormone (GnRH) receptor (GnRHR) pharmacoperone assay principle. The mutant GnRHR is retained in the lumen of untreated cells. Addition of pharmacoperone rescues GnRHR, and the receptors are trafficked to the plasma membrane. GnRHR on the plasma membrane is now responsive to agonism by GnRH, which is quantitated using Fluo-2 detection reagents (box). The level of functional GnRHR (mutant) is proportional to the magnitude of Gq modulated signaling, IP3 induction, and subsequent Ca2+ released and detected in the cells via Fluo2 using the FLIPR Tetra reader as illustrated via the kinetic traces shown in the lower inset diagram.
<b>Fig. 2.</b>
Fig. 2.
Responses of controls in the 384-well formatted GnRHR[E90K] and wild type pharmacoperone assay. (A) Mutant cells. In the absence of doxycycline (“Dox”), GnRHR[E90K] is synthesized and retained in the endoplasmic reticulum (ER). Following pretreatment with the pharmacoperone IN3, GnRHR[E90K] is rescued and trafficked to the plasma membrane, and a robust Ca2+ response to GNRH (500 nM) challenge is observed (●). Also included are the results of challenging the cells with GnRH, after preincubation with IN3 and doxycycline at 1 μg/mL (▲), as well as similar experiments without the GnRH challenge (■ and ▼). (B) Wild type cells. In the absence of Dox, wild type GnRHR is expressed and automatically trafficked to the plasma membrane. Upon GnRH challenge, the pharmacoperone IN3 decreases the amount of Ca2+ response in these cells, in a concentration-dependent manner, because IN3 acts as an antagonist in this format (●). Also shown are the control experiments in the wild type cells, similar to those done with the mutant cells. The data presented are means±standard deviation (sd) of quadruplicate wells (n=4).
<b>Fig. 3.</b>
Fig. 3.
Q89 Rescues the mutant GnRHR[E90K]. Cells were grown, and Q89 was used at different doses to rescue GNRHR. The EC50 for Q89 was determined to be 1.9 nM (●) in the presence of GNRH (500 nM) and had no effect when GNRH was absent (■). The data presented are means±sd of quadruplicate wells (n=4).
<b>Fig. 4.</b>
Fig. 4.
Scatter plot analysis of the high throughput LOPAC pilot screen. (A) All data from all assay plates (n=12, triplicate results), including controls, are displayed. The separation in activity (Z′=0.57±0.06, S/B=1.7±0.1) between wells dosed with IN3 (“high control”) and DMSO (“low control”) indicates a HTS assay that will allow for reproducible selection of hits. (B) Representative graph of IN3 pharmacoperone activity performed while running the LOPAC pilot screen. The EC50 (5.4 nM) indicates reproducible and expected assay sensitivity was achieved. The data presented are means±sd of quadruplicate wells (n=4).
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References

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