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Creating a Soluble Binder to Endothelin-1 Based on the Natural Ligand Binding Domains of the Endothelin-1 (G-Protein-Coupled) Receptor

  • Arjun JainEmail author
  • Vidhi Mehrotra
  • Hannah Yong
  • Kirti Hiremath
  • Ashok Jain
  • Martin Johnson
  • Ira Jha
Article
  • 104 Downloads

Abstract

Since its discovery in 1989, there has been extensive research on endothelin (ET)-1 physiology, as well as pathology. Accordingly, there is considerable research on the discovery of therapeutics based around ET-1, amongst which current treatment options include endothelin receptor antagonists. These target the ET-1 receptors, which are G-protein–coupled receptors (GPCRs). We have effectively developed a soluble form of a GPCR that binds to ligands, by constructing a fusion polypeptide of different endothelin receptor ligand binding domains. Phage experiments identified strong binders to ET-1. We then constructed Fc-fusions of the top binders and further binding assays revealed a KD of 21.2 nM for the Fc-ETtr1 construct and KD of 77.3 nM for the Fc-ETtr2 construct. These constructs are soluble and have the ability to bind and potentially sequester elevated ET-1 levels that are prevalent in different diseases. These results provide a novel approach to targeting GPCR–binding ligands, and thereby to contribute to a very important class of therapeutics.

Keywords

ECL ETA receptor ETB receptor Endothelin-1 traps FFP (Fc-fusion proteins) GPCR (G-protein-coupled receptors) 

Abbreviations

ECL

Extracellular loop

ET-1

Endothelin-1

ETA

Endothelin A

ETB

Endothelin B

ETtr

Endothelin-1 traps

FFP

Fc-fusion protein

GPCR

G-protein-coupled receptor

HA

Epitope human influenza hemagglutinin epitope

LBD

Ligand binding domain

PBST

Phosphate buffered saline tween

SB

Super broth medium

STR

Streptavidin

Notes

Acknowledgements

We would like to thank DM; Ms. Leela Jain for all her support. We also thank RC for funding this project.

Author Contributions

AJ and VM participated in the research design. AJ, VM and HY conducted the experiments. AJ, KH, AJ and IJ contributed new reagents or analytic tools. VM and AJ performed the data analysis. AJ, VM, KH, AJ, MJ and IJ wrote or contributed to the writing of the manuscript.

Funding

This project was funded by private funding.

Compliance with Ethical Standards

Conflict of interest

The author(s) declare that they have no competing interests.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2017

Authors and Affiliations

  1. 1.Accelerate Cambridge, Judge Business SchoolUniversity of CambridgeCambridgeUK
  2. 2.Department of Physiology, Development and NeuroscienceUniversity of CambridgeCambridgeUK
  3. 3.University of DelhiDelhiIndia
  4. 4.National University of SingaporeSingaporeSingapore
  5. 5.Indian Institute of ManagementAhmedabadIndia

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