Abstract
Endothelium‐derived C‐type natriuretic peptide possesses cytoprotective and anti‐atherogenic functions that regulate vascular homeostasis. The vasoprotective effects of C‐type natriuretic peptide are somewhat mediated by the natriuretic peptide receptor C, suggesting that this receptor represents a novel therapeutic target for the treatment of cardiovascular diseases. In order to facilitate our drug discovery efforts, we have optimized an array of biophysical methods including surface plasmon resonance, fluorescence polarization and thermal shift assays to aid in the design, assessment and characterization of small molecule agonist interactions with natriuretic peptide receptors. Assay conditions are investigated to explore the feasibility and dynamic range of each method, and peptide‐based agonists and antagonists are used as controls to validate these conditions. Once established, each technique was compared and contrasted with respect to their drug discovery utility. We foresee that such techniques will facilitate the discovery and development of potential therapeutic agents for NPR‐C and other large extracellular domain membrane receptors.
Highlights
Non-reducing SDS-P AGE analyses of the protein batches used in these experiments indicated that FL natriuretic peptide receptor C (NPR-C) was primarily present as the active disulphide-bonded dimer (~130 kDa), whereas the extracellular domain (ECD) mainly existed as the monomer (~55 kDa; Figure 2)
It should be noted that natriuretic peptide receptor type-C (NPR-C) is an atypical G-protein-coupled receptors (GPCRs) in that it lacks a seven transmembrane domain, and ~90% of this protein is extracellular.[25,26,27,28]. These methods may be applicable for GPCRs but for other dissimilar receptors that possess a large and well-d efined ECD.[29]. We have shown this to be the case in the application of biophysical screening techniques for discovery of neuropilin-1 ligands, an immunoglobulin domain protein receptor for the vascular endothelial growth factor (VEGF).[30,31,32]
We propose that a combination of either surface plasmon resonance or fluorescence polarization as a primary screen, followed by a thermal shift assay to confirm binding, would function well as a mechanism to identify NPR-C ligands that could go forward into more advanced functional in vivo assays to characterize agonists or antagonists
Summary
Endothelium-d erived C-type natriuretic peptide (CNP) possesses cytoprotective and anti-atherogenic functions that regulate vascular homeostasis.[1,2,3] The vasoprotective effects of CNP are mediated, at least in part, by natriuretic peptide receptor type-C (NPR-C), which is widely thought. C-type natriuretic peptide produces vascular smooth muscle (SM) relaxation via NPR-C agonism, stimulating Gi protein and G-protein-coupled inwardly rectifying potassium channels (GIRKs), resulting in hyperpolarization (Figure 1).[6] administration of small molecule NPR-C agonists exerts a dose-dependent hypotension in vivo.[1]. Despite sophisticated in vivo characterization of the modulatory effects of CNP and a handful of small molecule agonists on NPR-C, no practical and robust screening cascade exists to identify and characterize more potent, bioavailable. Conditions for extracellular NPR-C SDS- PAGE: Novex Wedgewell 10%–20% Tris-G lycine 12 well (Invitrogen XP00102); markers: RunBlue prestained tricolour marker, Expedion NXA6050, 5 μl loaded; either 3 or 5 μg protein loaded under reducing or non-reducing conditions
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
