Design, synthesis, and actions of a novel chimeric natriuretic peptide: CD-NP

Abstract

Objectives: Our aim was to design, synthesize and test in vivo and in vitro a new chimeric peptide that would combine the beneficial properties of 2 distinct natriuretic peptides with a biological profile that goes beyond native peptides.

Background: Studies have established the beneficial vascular and antiproliferative properties of C-type natriuretic peptide (CNP). While lacking renal actions, CNP is less hypotensive than the cardiac peptides atrial natriuretic peptide and B-type natriuretic peptide but unloads the heart due to venodilation. Dendroaspis natriuretic peptide is a potent natriuretic and diuretic peptide that is markedly hypotensive and functions via a separate guanylyl cyclase receptor compared with CNP.

Methods: Here we engineered a novel chimeric peptide CD-NP that represents the fusion of the 22-amino acid peptide CNP together with the 15-amino acid linear C-terminus of Dendroaspis natriuretic peptide. We also determined in vitro in cardiac fibroblasts cyclic guanosine monophosphate-activating and antiproliferative properties of CD-NP.

Results: Our studies demonstrate in vivo that CD-NP is natriuretic and diuretic, glomerular filtration rate enhancing, cardiac unloading, and renin inhibiting. CD-NP also demonstrates less hypotensive properties when compared with B-type natriuretic peptide. In addition, CD-NP in vitro activates cyclic guanosine monophosphate and inhibits cardiac fibroblast proliferation.

Conclusions: The current findings advance an innovative design strategy in natriuretic peptide drug discovery and development to create therapeutic peptides with favorable properties that may be preferable to those associated with native natriuretic peptides.

Figure 1. AA Sequence and Structure of a CNP and Novel Chimeric Natriuretic Peptide C-Terminus of DNP and CD-NP

C-type natriuretic peptide (CNP) is a 22-amino acid (AA) endothelial-cell-derived natriuretic peptide, C-terminus of Dendroaspis natriuretic peptide (DNP) is a 15-AA chimeric natriuretic peptide, and CD-NP is a 37-AA-designed chimeric natriuretic peptide.

Figure 2. Effect of C-Terminus of DNP on UNaV and UV in Normal Dogs

Data are expressed as means ± SE. C-terminus, intravenous infusion of 42 ng/kg/min of C-terminus, n = 6. *p < 0.05 versus baseline. DNP = Dendroaspis natriuretic peptide; UNaV = urinary excretion of sodium; UV = urine flow.

Figure 3. Effect of CD-NP on MAP, RAP, and PCWP in Normal Dogs

Data are expressed as means ± SE. n = 6. *p < 0.05 versus baseline. CD-NP 10 = dose of CD-NP 10 ng/kg/min; CD-NP 50 = dose of CD-NP 50 ng/kg/min; CD-NP 100 = dose of CD-NP 100 ng/kg/min; MAP = mean arterial pressure; PCWP = pulmonary capillary pressure; RAP = right atrial pressure.

Figure 4. Effect of CD-NP on UNaV, UV, and GFR in Normal Dogs

Data are expressed as means ± SE. n = 6. *p < 0.05 versus baseline. GFR = glomerular filtration rate; other abbreviations as in Figures 2 and 3.

Figure 5. Effect of CD-NP on Proximal and Distal Fractional Sodium Reabsorption in Normal Dogs

Data are expressed as means ± SE. n = 6. *p < 0.05 versus baseline. DFRNa = distal fractional sodium reabsorption; PFRNa = proximal fractional sodium reabsorption; other abbreviations as in Figure 3.

Figure 6. Effect of CD-NP and Equimolar Doses of Human BNP in 2 Groups of Normal Dogs

Data are expressed as means ± SE. n = 6 in each group. *p < 0.05 versus baseline; †p < 0.05 between groups. Solid bars = CD-NP; open bars = B-type natriuretic peptide (BNP). Abbreviations as in Figures 3 and 4.

Figure 7. Effect of CD-NP on cGMP Generation and Antiproliferative Actions of CD-NP in Human CFs

(A) Cyclic 3′5′ guanosine monophosphate (cGMP) generation in human cardiac fibroblasts (CFs). Data are expressed as means ± SE. *p < 0.05 versus no treatment; **p < 0.05 versus CP-NP 10⁻¹¹M; *p < 0.05 versus CD-NP 10⁻⁸ M. (B) Antiproliferative actions of CD-NP in human CFs. Bar graph reports colormetric bromodeoxyuridine (BrdU) uptake in optical density units as measured by colorimetry. Data are expressed as means = standard error. *p < 0.05 versus control. Cardiotrophin-1 = proliferation of fibroblasts by cardiotrophin-1; Cardiotrophin-1 + CD-NP = CD-NP added to cardiotrophin-1 stimulated fibroblasts; Control = untreated human cardiac fibroblasts.