Surface plasmon resonance using the catalytic domain of soluble guanylate cyclase allows the detection of enzyme activators

Filipa Mota,Charles K Allerston,Kathryn Hampden-Smith,John Garthwaite,David L Selwood

doi:10.1016/j.bmcl.2014.01.015

Abstract

Soluble Guanylate Cyclase (sGC) is the receptor for the signalling agent nitric oxide (NO) and catalyses the production of the second messenger cyclic guanosine monophosphate (cGMP) from guanosine triphosphate (GTP). The enzyme is an attractive drug target for small molecules that act in the cardiovascular and pulmonary systems, and has also shown to be a potential target in neurological disorders. We have discovered that 5-(indazol-3-yl)-1,2,4-oxadiazoles activate the enzyme in the absence of added NO and shown they bind to the catalytic domain of the enzyme after development of a surface plasmon resonance assay that allows the biophysical detection of intrinsic binding of ligands to the full length sGC and to a construct of the catalytic domain.

Highlights

We have discovered that 5-(indazol-3-yl)-1,2,4-oxadiazoles activate the enzyme in the absence of added nitric oxide (NO) and shown they bind to the catalytic domain of the enzyme after development of a surface plasmon resonance assay that allows the biophysical detection of intrinsic binding of ligands to the full length Soluble Guanylate Cyclase (sGC) and to a construct of the catalytic domain
Soluble guanylate cyclase is a heterodimeric enzyme endogenously activated by the binding of nitric oxide (NO) to a prosthetic haem group, leading to a conformational change that increases the rate of guanosine triphosphate (GTP) conversion into cyclic guanosine monophosphate (cGMP)
Each subunit is composed of multiple domains: The haem-nitric oxide binding domain of sGC is located at the N-terminus and Histidine-105 in the b-subunit is the proximal haem ligand; PAS-like domains are present in both subunits and are thought to direct preferential heterodimer formation; turnover of GTP into cGMP occurs at the C-terminus catalytic domain of the enzyme

Summary

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl. The catalytic domain of sGC (sGCcat) is easier to manipulate in an SPR assay—its smaller size (50 kDa) allows the use of a less dense surface (lower immobilisation level), which reduces steric effects and non-specific electrostatic binding whilst still providing a high enough signal/noise ratio. Both protein constructs, full length and catalytic domain only, were immobilised via amine coupling to a CM5 sensor chip of a Biacore T200 instrument in the presence of ATP and GTP to protect the binding sites.. Compounds 4 and 5 are close analogues, differing only in the position of a methyl group, and both give a high binding response

NN O

Findings

Additive response