Cenderitide: structural requirements for the creation of a novel dual particulate guanylyl cyclase receptor agonist with renal-enhancing in vivo and ex vivo actions

Abstract

Aims: Cenderitide is a novel dual natriuretic peptide (NP) receptor chimeric peptide activator, which targets the particulate guanylyl cyclase B (pGC-B) receptor and pGC-A unlike native NPs. Cenderitide was engineered to retain the anti-fibrotic properties of C-type natriuretic peptide (CNP)/pGC-B with renal-enhancing actions facilitated by fusion to the carboxyl terminus of Dendroaspis NP (DNP), a pGC-A agonist, to CNP. Here, we address significance of the DNP carboxyl terminus in dual pGC receptor activation and actions of cenderitide compared with CNP on renal function and cyclic guanosine monophosphate (cGMP) in vivo and ex vivo in normal canines.

Methods and results: In vitro, only cenderitide and not CNP or three CNP-based variants was a potent dual pGC-A/pGC-B activator of cGMP production (from 5 to 237 pmol/mL) in human embryonic kidney (HEK) 293 cells overexpressing human pGC-A while in pGC-B overexpressing cells cenderitide increased cGMP production (from 4 to 321 pmol/mL) while the three CNP-based variants were weak agonists. Based upon our finding that the DNP carboxyl terminus is a key structural requirement for dual pGC-A/pGC-B activation, we defined in vivo the renal-enhancing actions of cenderitide compared with CNP. Cenderitide increased urinary cGMP excretion (from 989 to 5977 pmol/mL), net generation of renal cGMP (821-4124 pmol/min), natriuresis (12-242 μEq/min), and glomerular filtration rate (GFR) (37-51 mL/min) while CNP did not. We then demonstrated the transformation of CNP ex vivo into a renal cGMP-activating peptide which increased cGMP in freshly isolated glomeruli eight-fold greater than CNP.

Conclusion: The current study establishes that dual pGC-A and pGC-B activation with CNP requires the specific carboxyl terminus of DNP. In normal canines in vivo and in glomeruli ex vivo, the carboxyl terminus of DNP transforms CNP into a natriuretic and GFR-enhancing peptide. Future studies of cenderitide are warranted in cardiorenal disease states to explore its efficacy in overall cardiorenal homeostasis.

Keywords: C-type natriuretic peptide; CD-NP; Chimeric natriuretic peptide; canine.

Figure 1

Structures and amino acid sequences of CNP, Cenderitide, CA-NP, CB-NP, and C-MANP.

Figure 2

Generation of cyclic guanosine monophosphate. In vitro cyclic guanosine monophosphate generation in human embryonic kidney 293 cells stably transfected with either the particulate guanylyl cyclase A or particulate guanylyl cyclase B receptor in response to a 10⁻⁶ M dose of CNP, Cenderitide, CA-NP, CB-NP, and C-MANP compared with no treatment. Values are mean ± SEM. *P < 0.05 vs. no treatment; ^**P < 0.05 vs. CNP, CA-NP, CB-NP, and C-MANP; ^***P < 0.05 vs. CA-NP, CB-NP, and C-MANP.

Figure 3

Plasma cyclic guanosine monophosphate, urinary cyclic guanosine monophosphate excretion, and net renal generation of cyclic guanosine monophosphate at pre-infusion, at 30 and 60 min of peptide infusion, and post-infusion. Values are mean ± SEM. *P < 0.05 vs. CNP and ^†P < 0.05 vs. pre.

Figure 4

Urinary flow and urinary sodium excretion during pre-infusion, at 30 and 60 min of peptide infusion and post-infusion. Values are mean ± SEM. *P < 0.05 vs. CNP and ^†P < 0.05 vs. pre.

Figure 5

Cyclic guanosine monophosphate response in isolated canine glomeruli to CNP or Cenderitide 10⁻⁵ M.