Identification and characterization of small molecular NPR-B receptor antagonists

Background

Natriuretic peptides increase in heart failure. Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) activate the natriuretic peptide receptor (NPR)-A and C-type natriuretic peptide (CNP) the NPR-B, causing production of cyclic 3’,5’-guanosine monophosphate (cGMP). Our group has previously shown that CNP potentiates β₁-adrenoceptor-mediated inotropic response by increasing cAMP levels. This is explained by a crosstalk between cGMP and cAMP, where cGMP produced by NPR-B inhibits phosphodiesterase (PDE) 3 from degrading cAMP. Increased β₁-adrenoceptor signalling is harmful in heart failure which is the basis for use of β-blockers in heart failure therapy. Further, PDE3 inhibition has been associated with increased mortality in heart failure. Thus, an NPR-B antagonist could eliminate the unwanted effects due to PDE3 inhibition and increased β₁-adrenoceptor signalling by CNP. There are no selective NPR-B antagonists available, and our aim is to identify and characterize novel non-peptide small molecular selective NPR-B antagonists for potential use in heart failure therapy.

Methods

Small molecular compounds were screened for activity and selectivity towards NPR-A and NPR-B, using a cGMP assay based on AlphaScreen technology. Based on this technology, we can identify compounds that interfere with BNP- and CNP-stimulated cGMP production through the NPR-A and NPR-B receptor, respectively. Antagonism properties can also be characterized.

Results and Conclusion

Potential NPR-B antagonists were identified by high throughput screening of about 20,000 compounds. Hits were tested against the NPR-A to determine selectivity, and characterized as non-competitive and reversible inhibitors of NPR-B response in HEK293 cells, displaying properties of negative allosteric modulators [1]. Information on structure and activity relationship (SAR), was collected and over 50 new compounds, clustered in five different groups of chemically related compounds, were designed in silicoand were tested for activity and selectivity towards NPR-A and NPR-B. From this testing, three out of five groups were found interesting. The ligands with the best antagonistic property from each of the 3 groups were used to select 60 more compounds by in silicodesign for further testing.