Dendroaspis natriuretic peptide and the designer natriuretic peptide, CD-NP, are resistant to proteolytic inactivation

Abstract

Designer natriuretic peptides (NPs) represent an active area of drug development. In canine and human studies, the designer natriuretic peptide CD-NP demonstrated more desirable therapeutic potential than recombinant B-type NP (BNP), which is known as nesiritide and is approved for treatment of acute decompensated heart failure. However, why CD-NP is more effective than BNP is not known. We previously reported that CD-NP is a poorer activator of human guanylyl cyclase-A (GC-A) and a better activator of human guanylyl cyclase-B than BNP. Here, guanylyl cyclase bioassays were used to compare the susceptibility of CD-NP verses ANP, BNP, CNP and DNP to inactivation by human kidney membranes. The half time (t(1/2)) for CD-NP inactivation was increased by factors of 13, 3 and 4 compared to ANP, BNP and CNP, respectively, when measured in the same assay. Surprisingly, DNP failed to undergo complete inactivation and was the most degradation resistant of the peptides tested. The neutral endopeptidase (NEP) inhibitor, phosphoramidon, blocked inactivation of CNP and CD-NP, but not BNP or DNP. In contrast, the general serine and cysteine protease inhibitor, leupeptin, completely blocked the degradation of BNP and CD-NP, but did not block CNP inactivation unless phosphoramidon was included in the assay. Thus, NPs with shorter carboxyl tails (ANP and CNP) are degraded by phosphoramidon-sensitive proteases and NPs with extended carboxyl tails (BNP, DNP and CD-NP) are resistant to NEP degradation and degraded by leupeptin-sensitive proteases. We conclude that DNP and CD-NP are highly resistant to proteolysis and that proteolytic resistance contributes to the beneficial cardiovascular properties of CD-NP. We suggest that this property may be exploited to increase the half-life of NP-based drugs.

Fig. 1

The C-terminal tail of DNP confers proteolytic resistance. The indicated natriuretic peptides were incubated with human kidney membranes at 37 °C for the indicated periods of time. Remaining peptide concentrations were determined as described under Materials and methods. The solid lines represent peptide concentrations determined using a single-phase exponential decay model. The broken line represents a two-phase exponential decay model. (A) Degradation of CNP and CD-NP where n=8. (B) Degradation of ANP and CD-NP where n=4. (C) Degradation of BNP and CD-NP where n=4. (D) Degradation of DNP and CD-NP where n=8.

Fig. 2

Leupeptin, but not phosphoramidon, blocks renal membrane degradation of CD-NP and BNP. The indicated natriuretic peptides were incubated with crude human kidney membranes at 37 °C for 30 min for panels A and B and 120 min for panel C in the absence or presence of the indicated protease inhibitors. Remaining peptide concentrations were determined as described under Materials and methods. (A) Proteolytic inactivation of CNP and CD-NP where n=8. (B) Proteolytic inactivation of BNP and CD-NP where n=4. (C) Proteolytic inactivation of DNP and CD-NP where n=4. Abbreviations: act, actinonin; leu, leupeptin; phos, phosphoramidon. *p≤0.05, **p≤0.005, ***p≤0.0001.