Research Article
No access
Published Online: 1 December 2017

A Homogeneous Cell-Based Halide-Sensitive Yellow Fluorescence Protein Assay to Identify Modulators of the Cystic Fibrosis Transmembrane Conductance Regulator Ion Channel

Publication: ASSAY and Drug Development Technologies
Volume 15, Issue Number 8

Abstract

Cystic fibrosis (CF), an inherited genetic disease, is caused by mutation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, which encodes an ion channel involved in hydration maintenance by anion homeostasis. Ninety percent of CF patients possess one or more copies of the F508del CFTR mutation. This mutation disrupts trafficking of the protein to the plasma membrane and diminishes function of mature CFTR. Identifying small molecule modulators of mutant CFTR activity or biosynthesis may yield new tools for discovering novel CF treatments. One strategy utilizes a 384-well, cell-based fluorescence-quenching assay, which requires extensive wash steps, but reports sensitive changes in fluorescence-quenching kinetic rates. In this study, we describe the methods of adapting the protocol to a homogeneous, miniaturized 1,536-well format and further optimization of this functional F508del CFTR assay. The assay utilizes a cystic fibrosis bronchial epithelial (CFBE41o-) cell line, which was engineered to report CFTR-mediated intracellular flux of iodide by a halide-sensitive yellow fluorescence protein (YFP) reporter. We also describe the limitations of quench rate analysis and the subsequent incorporation of a novel, kinetic data analysis modality to quickly and efficiently find active CFTR modulators. This format yields a Z′ value interval of 0.61 ± 0.05. As further evidence of high-throughput screen suitability, we subsequently completed a screening campaign of >645,000 compounds, identifying 2,811 initial hits. After completing secondary and tertiary follow-up assays, we identified 187 potential CFTR modulators, which EC50's < 5 μM. Thus, the assay has integrated the advantages of a phenotypic screen with high-throughput scalability to discover new small-molecule CFTR modulators.

Get full access to this article

View all available purchase options and get full access to this article.

References

1.
Lavelle GM, White MM, Browne N, et al.: Animal models of cystic fibrosis pathology: phenotypic parallels and divergences. Biomed Res Int 2016;2016:5258727.
2.
Molinski SV, Ahmadi S, Hung M, et al.: Facilitating structure-function studies of CFTR modulator sites with efficiencies in mutagenesis and functional screening. J Biomol Screen 2015;20:1204–1217.
3.
Mickle JE, Cutting GR: Genotype-phenotype relationships in cystic fibrosis. Med Clin North Am 2000;84:597–607.
4.
Egan ME: Genetics of cystic fibrosis: clinical implications. Clin Chest Med 2016;37:9–16.
5.
Pettit RS, Fellner C: CFTR modulators for the treatment of cystic fibrosis. P T 2014;39:500–511.
6.
Jensen TJ, Loo MA, Pind S, et al.: Multiple proteolytic systems, including the proteasome, contribute to CFTR processing. Cell 1995;83:129–135.
7.
Rowe SM, Verkman AS: Cystic fibrosis transmembrane regulator correctors and potentiators. Cold Spring Harb Perspect Med 2013;3:1–16.
8.
Lin S, Sui J, Cotard S, et al.: Identification of synergistic combinations of F508del cystic fibrosis transmembrane conductance regulator (CFTR) modulators. Assay Drug Dev Technol 2010;8:669–684.
9.
Gadsby DC, Vergani P, Csanady L: The ABC protein turned chloride channel whose failure causes cystic fibrosis. Nature 2006;440:477–483.
10.
Brodlie M, et al.: Targeted therapies to improve CFTR function in cystic fibrosis. Genome Med 2015;7:101.
11.
Van Goor F, Hadida S, Grootenhuis PD, et al.: Correction of the F508del-CFTR protein processing defect in vitro by the investigational drug VX-809. Proc Natl Acad Sci U S A 2011;108:18843–18848.
12.
Sermet-Gaudelus I: Ivacaftor treatment in patients with cystic fibrosis and the G551D-CFTR mutation. Eur Respir Rev 2013;22:66–71.
13.
Sui J, Cotard S, Andersen J, et al.: Optimization of a Yellow fluorescent protein-based iodide influx high-throughput screening assay for cystic fibrosis transmembrane conductance regulator (CFTR) modulators. Assay Drug Dev Technol 2010;8:656–668.
14.
Galietta LV, Jayaraman S, Verkman AS: Cell-based assay for high-throughput quantitative screening of CFTR chloride transport agonists. Am J Physiol Cell Physiol 2001;281:C1734–C1742.
15.
Yang H, et al.: Nanomolar affinity small molecule correctors of defective Delta F508-CFTR chloride channel gating. J Biol Chem 2003;278:35079–35085.
16.
Sondo E, Tomati V, Caci E, et al.: Rescue of the mutant CFTR chloride channel by pharmacological correctors and low temperature analyzed by gene expression profiling. Am J Physiol Cell Physiol 2011;301:C872–C885.
17.
Ehrhardt C, Collnot EM, Baldes C, et al.: Towards an in vitro model of cystic fibrosis small airway epithelium: characterisation of the human bronchial epithelial cell line CFBE41o-. Cell Tissue Res 2006;323:405–415.
18.
Ramsey BW, Davies J, McElvaney NG, et al.: A CFTR potentiator in patients with cystic fibrosis and the G551D mutation. N Engl J Med 2011;365:1663–1672.
19.
Ritz C, Baty F, Streibig JC, et al.: Dose-response analysis using R. PLoS One 2015;10:e0146021.
20.
Lovering F, Bikker J, Humblet C: Escape from flatland: increasing saturation as an approach to improving clinical success. J Med Chem 2009;52:6752–6756.
21.
Lipinski CA, Lombardo F, Dominy BW, et al.: Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv Drug Deliv Rev 2001;46:3–26.
22.
Kolb HC, Sharpless KB: The growing impact of click chemistry on drug discovery. Drug Discov Today 2003;8:1128–1137.
23.
Ding S, Gray NS, Wu X, et al.: A combinatorial scaffold approach toward kinase-directed heterocycle libraries. J Am Chem Soc 2002;124:1594–1596.
24.
Congreve M, Carr R, Murray C, et al.: A “rule of three” for fragment-based lead discovery? Drug Discov Today 2003;8:876–877.
25.
Baell JB, Holloway GA: New substructure filters for removal of pan assay interference compounds (PAINS) from screening libraries and for their exclusion in bioassays. J Med Chem 2010;53:2719–2740.
26.
Zhang JH, Chung TD, Oldenburg KR: A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 1999;4:67–73.
27.
Hughes JP, Rees S, Kalindjian SB, et al.: Principles of early drug discovery. Br J Pharmacol 2011;162:1239–1249.
28.
Smith E, Janovick JA, Bannister TD, et al.: Identification of potential pharmacoperones capable of rescuing the functionality of misfolded vasopressin 2 receptor involved in nephrogenic diabetes insipidus. J Biomol Screen 2016;21:824–831.
29.
Boyle MP, Bell SC, Konstan MW, et al.: A CFTR corrector (lumacaftor) and a CFTR potentiator (ivacaftor) for treatment of patients with cystic fibrosis who have a phe508del CFTR mutation: a phase 2 randomised controlled trial. Lancet Respir Med 2014;2:527–538.
30.
Madoux F, Tanner A, Vessels M, et al.: A 1536-well 3D viability assay to assess the cytotoxic effect of drugs on spheroids. SLAS Discov 2017;22:516–524.
31.
Jambrina E, Cerne R, Smith E, et al.: An integrated approach for screening and identification of positive allosteric modulators of N-methyl-d-aspartate receptors. J Biomol Screen 2016;21:468–479.

Information & Authors

Information

Published In

cover image ASSAY and Drug Development Technologies
ASSAY and Drug Development Technologies
Volume 15Issue Number 8December 2017
Pages: 395 - 406
PubMed: 29172645

History

Published in print: December 2017
Published online: 1 December 2017
Published ahead of print: 27 November 2017

Permissions

Request permissions for this article.

Topics

Authors

Affiliations

Emery Smith*
Department of Molecular Medicine, The Scripps Research Institute Molecular Screening Center, Scripps Florida, Jupiter, Florida.
Kenneth A. Giuliano*
Proteostasis Therapeutics, Inc., Cambridge, Massachusetts.
Justin Shumate
Department of Molecular Medicine, The Scripps Research Institute Molecular Screening Center, Scripps Florida, Jupiter, Florida.
Pierre Baillargeon
Department of Molecular Medicine, The Scripps Research Institute Molecular Screening Center, Scripps Florida, Jupiter, Florida.
Brigid McEwan
Proteostasis Therapeutics, Inc., Cambridge, Massachusetts.
Matthew D. Cullen
Proteostasis Therapeutics, Inc., Cambridge, Massachusetts.
John P. Miller
Proteostasis Therapeutics, Inc., Cambridge, Massachusetts.
Lawrence Drew
Proteostasis Therapeutics, Inc., Cambridge, Massachusetts.
Louis Scampavia
Department of Molecular Medicine, The Scripps Research Institute Molecular Screening Center, Scripps Florida, Jupiter, Florida.
Timothy P. Spicer
Department of Molecular Medicine, The Scripps Research Institute Molecular Screening Center, Scripps Florida, Jupiter, Florida.

Notes

*
These authors contributed equally to this work.
Address correspondence to:Timothy SpicerDepartment of Molecular MedicineThe Scripps Research Institute, Scripps Florida130 Scripps WayJupiter, FL 33458E-mail: [email protected]

Disclosure Statement

No competing financial interests exist.

Metrics & Citations

Metrics

Citations

Export citation

Select the format you want to export the citations of this publication.

View Options

Get Access

Access content

To read the fulltext, please use one of the options below to sign in or purchase access.

Society Access

If you are a member of a society that has access to this content please log in via your society website and then return to this publication.

Restore your content access

Enter your email address to restore your content access:

Note: This functionality works only for purchases done as a guest. If you already have an account, log in to access the content to which you are entitled.

View options

PDF/EPUB

View PDF/ePub

Full Text

View Full Text

Media

Figures

Other

Tables

Share

Share

Copy the content Link

Share on social media

Back to Top