Identification of small molecule NPR-B antagonists by high throughput screening — potential use in heart failure

Abstract

We found previously that stimulation of natriuretic peptide receptor (NPR)-B by C-type natriuretic peptide (CNP) in failing rat ventricle potentiates β1-adrenoceptor (β1-AR)-mediated inotropic response to noradrenaline through cGMP-mediated inhibition of phosphodiesterase (PDE) 3, thereby enhancing cAMP-mediated signalling. Increased cAMP-mediated signalling is deleterious in chronic heart failure (HF; basis for the use of β-blockers in HF) and we propose to consider NPR-B antagonists as new HF treatment in addition to conventional therapy. Since there is no NPR-B-selective antagonist available for clinical studies, we aimed at identifying a novel small molecule (non-peptide) NPR-B antagonist. An assay was developed and high throughput screening performed on a chemical library of about 20,000 small molecule compounds (<500 Da) to identify NPR-B antagonists based on inhibition of CNP-stimulated cGMP production in NPR-B-expressing HEK293 cells. The screen revealed several potential NPR-B antagonists, of which six were selected for further studies. Three showed selective NPR-B vs NPR-A inhibition and three were partially selective. The compounds mediated reversible, noncompetitive inhibition and most likely act as allosteric modulators binding outside the agonist binding site of NPR-B. In rat ventricular muscle strips, the potentiating effect of CNP upon β1-AR-evoked inotropic effects could be attenuated by at least one of these compounds. We identified several small molecule NPR-B antagonists by high throughput screening and show in a functional heart preparation that blocking NPR-B stimulation with a small molecule compound can reduce the potentiating effect of CNP on the β1-AR-mediated inotropic response to noradrenaline.

Different Compartmentation of Responses to Brain Natriuretic Peptide and C-Type Natriuretic Peptide in Failing Rat Ventricle

“…The effects of CNP in the heart are complex, as we previously showed increased b 1 -adrenergic receptor-induced, cAMP-mediated inotropic responses in the presence of CNP (Qvigstad et al, 2010), a potentially harmful effect. Thus, when evaluating new drugs that target NP signaling, direct effects on the heart should be taken into consideration (Levy, 2013;Bach et al, 2014).…”

Identification and characterization of small molecular NPR-B receptor antagonists

“…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 silico and were tested for activity and selectivity towards NPR-A and NPR-B.…”

Novel enhancers of guanylyl cyclase‐A activity acting via allosteric modulation

“…QBIHEK293A ( RRID:CVCL_6910 ), HEK293T ( RRID:CVCL_0063 ) and HeLa ( RRID:CVCL_0030 ) cells were grown in Dulbecco's modified Eagle's medium (DMEM) (Gibco®, ThermoFischer Scientific) that was supplemented with 10% fetal bovine serum (FBS), 100 U·ml −1 penicillin and 0.1 mg·ml −1 streptomycin. QBIHEK293A cells were stably transfected with codon optimized human GC‐A or GC‐B, as previously described (Bach et al, 2014 ). For the stable GC‐A‐ and GC‐B‐expressing cells, 1 mg·ml −1 geneticin (G418) was added as the selection antibiotic.…”