C53: A novel particulate guanylyl cyclase B receptor activator that has sustained activity in vivo with anti-fibrotic actions in human cardiac and renal fibroblasts

Abstract

The native particulate guanylyl cyclase B receptor (pGC-B) activator, C-type natriuretic peptide (CNP), induces anti-remodeling actions in the heart and kidney through the generation of the second messenger 3', 5' cyclic guanosine monophosphate (cGMP). Indeed fibrotic remodeling, particularly in cardiorenal disease states, contributes to disease progression and thus, has been a key target for drug discovery and development. Although the pGC-B/cGMP system has been perceived as a promising anti-fibrotic pathway, its therapeutic potential is limited due to the rapid degradation and catabolism of CNP by neprilysin (NEP) and natriuretic peptide clearance receptor (NPRC). The goal of this study was to bioengineer and test in vitro and in vivo a novel pGC-B activator, C53. Here we established that C53 selectively generates cGMP via the pGC-B receptor and is highly resistant to NEP and has less interaction with NPRC in vitro. Furthermore in vivo, C53 had enhanced cGMP-generating actions that paralleled elevated plasma CNP-like levels, thus indicating a longer circulating half-life compared to CNP. Importantly in human cardiac fibroblasts (HCFs) and renal fibroblasts (HRFs), C53 exerted robust cGMP-generating actions, inhibited TGFβ-1 stimulated HCFs and HRFs proliferation chronically and suppressed the differentiation of HCFs and HRFs to myofibroblasts. The current findings advance innovation in drug discovery and highlight C53 as a novel pGC-B activator with sustained in vivo activity and anti-fibrotic actions in vitro. Future studies are warranted to explore the efficacy and therapeutic opportunity of C53 targeting fibrosis in cardiorenal disease states and beyond.

Keywords: C-type natriuretic peptide; C53; Fibrosis; NEP; Particulate guanylyl cyclase B receptor; cGMP.

Figure 1.

Generation of cGMP in HEK293 cells overexpressing human pGC-B (A) or pGC-A receptors (B) in response to 10⁻¹⁰, 10⁻⁹, 10^−8.5, 10⁻⁸, 10^−7.5, 10⁻⁷ or 10⁻⁶ M of CNP (red bars) or C53 (green bars), compared to vehicle. * p<0.05 vs vehicle.

Figure 2,

In vitro NEP degradation curve of C53 (green line) and CNP (red line) quantified by cGMP generating activity in HEK293 overexpressing human pGC-B. * p<0.05 vs 0 min, # p<0.05 vs CNP.

Figure 3,

Plasma CNP-like levels (CLL) with CNP (red bars) and C53 (green bars) infusion in vivo. Three equimolar doses (low = 4.55 pmol/kg/min, medium = 45.5 pmol/kg/min and high = 455 pmol/kg/min) of CNP or C53 was used and saline served as vehicle. * p<0.05 vs vehicle, # p<0.05 vs equimolar dose CNP.

Figure 4,

Plasma (A) and urinary (B) cGMP levels with CNP (red bars) and C53 (green bars) infusion in vivo. Three equimolar doses (low = 4.55 pmol/kg/min, medium = 45.5 pmol/kg/min and high = 455 pmol/kg/min) of CNP or C53 was used and saline served as vehicle. Urinary cGMP was calculated as cGMP excretion [urinary cGMP concentration (pmol/mL) X urinary volume rate (mL/min). * p<0.05 vs vehicle, # p<0.05 vs equimolar dose CNP.

Figure 5,

cGMP generation by C53 (green bars), at concentrations of 10⁻⁸, 10⁻⁷ or 10⁻⁶ M, in human cardiac fibroblasts (A) and human renal fibroblasts (B). Treatment buffer served as vehicle. * p<0.05 vs vehicle.

Figure 6,

Time-lapse imaging analysis of chronic inhibition of human cardiac fibroblasts (HCFs) proliferation over 3 days. (A) Quantification of the HCFs proliferation. HCFs proliferation was stimulated with TGFβ−1 (5 ng/mL) alone or with C53 at doses of 10⁻⁸, 10⁻⁷ or 10⁻⁶ M. (B) Representative phase objective confluence images, at 10X objective, of HCFs proliferation at time points 0, 24, 48, 72 hr. The upper panels are HCFs stimulated with TGFβ−1 alone and the lower panels are HCFs stimulated with TGFβ−1 in the presence of C53 treatment (10⁻⁶ M). # p<0.05 vs TGFβ−1.

Figure 7,

Time-lapse imaging analysis of chronic inhibition of human renal fibroblast (HRFs) proliferation over 3 days. (A) Quantification of the HRFs proliferation. HRFs proliferation was stimulated with TGFβ−1 (5 ng/mL) alone or with C53 at concentrations of 10⁻⁸, 10⁻⁷ or 10⁻⁶ M. (B) Representative phase objective confluence images, at 10X objective, of HRFs proliferation at time points 0, 24, 48, 72 hr. The upper panels are HRFs stimulated with TGFβ−1 alone and the lower panels are HRFs stimulated with TGFβ−1 in the presence of C53 treatment (10⁻⁶ M). # p<0.05 vs TGFβ−1.

Figure 8,

Human cardiac fibroblasts (HCFs) or human renal fibroblasts (HRFs) differentiation into myofibroblasts as determined by α-SMA expression in the presence of TGFβ−1 (5ng/mL), with or without C53 at concentration of 10⁻⁶ M, for 24 hours by immunofluorescence. Representative images of immunostaining images and quantitation for expression (red) and DAPI (blue) in HCFs (A and B) and HRFs (C and D) at 0 and 24 hours. Scale bar = 100um.

Figure 9,

Human cardiac fibroblasts (HCFs) or human renal fibroblasts (HRFs) differentiation into myofibroblasts as determined by α-SMA expression in the presence of TGFβ−1 (5ng/mL), with or without C53 at concentration of 10⁻⁶ M, for 24 hours by western blot analysis. Representative immunoblot of α-SMA protein expression and quantitation in HCFs (A and B) and HRFs (C and D) at 24 hours.