Novel Bifunctional Natriuretic Peptides as Potential Therapeutics

Synthetic atrial natriuretic peptide (carperitide) and B-type natriuretic peptide (BNP; nesiritide) are used to treat congestive heart failure. However, despite beneficial cardiac unloading properties, reductions in renal perfusion pressures limit their clinical effectiveness. Recently, CD-NP, a chimeric peptide composed of C-type natriuretic peptide (CNP) fused to the C-terminal tail of Dendroaspis natriuretic peptide (DNP), was shown to be more glomerular filtration rate-enhancing than BNP in dogs. However, the molecular basis for the increased responsiveness was not determined. Here, we show that the DNP tail has a striking effect on CNP, converting it from a non-agonist to a partial agonist of natriuretic peptide receptor (NPR)-A while maintaining the ability to activate NPR-B. This effect is specific for human receptors because CD-NP was only a slightly better activator of rat NPR-A due to the promiscuous nature of CNP in this species. Interesting, the DNP tail alone had no effect on any NPR even though it is effective in vivo. To further increase the potency of CD-NP for NPR-A, we converted two different triplet sequences within the CNP ring to their corresponding residues in BNP. Both variants demonstrated increased affinity and full agonist activity for NPR-A, whereas one was as potent as any NPR-A activator known. In contrast to a previous report, we found that DNP binds the natriuretic peptide clearance receptor (NPR-C). However, none of the chimeric peptides bound NPR-C with significantly higher affinity than endogenous ligands. We suggest that bifunctional chimeric peptides represent a new generation of natriuretic peptide therapeutics. Synthetic atrial natriuretic peptide (carperitide) and B-type natriuretic peptide (BNP; nesiritide) are used to treat congestive heart failure. However, despite beneficial cardiac unloading properties, reductions in renal perfusion pressures limit their clinical effectiveness. Recently, CD-NP, a chimeric peptide composed of C-type natriuretic peptide (CNP) fused to the C-terminal tail of Dendroaspis natriuretic peptide (DNP), was shown to be more glomerular filtration rate-enhancing than BNP in dogs. However, the molecular basis for the increased responsiveness was not determined. Here, we show that the DNP tail has a striking effect on CNP, converting it from a non-agonist to a partial agonist of natriuretic peptide receptor (NPR)-A while maintaining the ability to activate NPR-B. This effect is specific for human receptors because CD-NP was only a slightly better activator of rat NPR-A due to the promiscuous nature of CNP in this species. Interesting, the DNP tail alone had no effect on any NPR even though it is effective in vivo. To further increase the potency of CD-NP for NPR-A, we converted two different triplet sequences within the CNP ring to their corresponding residues in BNP. Both variants demonstrated increased affinity and full agonist activity for NPR-A, whereas one was as potent as any NPR-A activator known. In contrast to a previous report, we found that DNP binds the natriuretic peptide clearance receptor (NPR-C). However, none of the chimeric peptides bound NPR-C with significantly higher affinity than endogenous ligands. We suggest that bifunctional chimeric peptides represent a new generation of natriuretic peptide therapeutics. The human natriuretic peptide system consists of atrial natriuretic peptide (ANP), 2The abbreviations used are: ANP, atrial natriuretic peptide; BNP, B-type natriuretic peptide; CNP, C-type natriuretic peptide; NPR, natriuretic peptide receptor; NPR-C, natriuretic peptide clearance receptor; DNP, Dendroaspis natriuretic peptide; GFR, glomerular filtration rate; hNPR, human NPR. B-type natriuretic peptide (BNP), C-type natriuretic peptide (CNP), and an N-terminally extended renal form of ANP called urodilatin (1.Potter L.R. Abbey-Hosch S. Dickey D.M. Endocr. Rev. 2006; 27: 47-72Crossref PubMed Scopus (784) Google Scholar). ANP, BNP, and urodilatin bind natriuretic peptide receptor (NPR)-A, whereas CNP binds NPR-B. Both receptors are transmembrane guanylyl cyclases, which when activated catalyze the synthesis of cGMP. The natriuretic peptide clearance receptor (NPR-C) lacks guanylyl cyclase activity and functions primarily to clear natriuretic peptides from the circulation. However, a signaling function has been proposed for this receptor as well (2.Rose R.A. Giles W.R. J. Physiol. (Lond.). 2008; 586: 353-366Crossref Scopus (162) Google Scholar). Both ANP and BNP reduce blood pressure by increasing natriuresis, diuresis, vasodilation, and endothelial permeability. They also prevent cardiac hypertrophy and suppress the renin-angiotensin-aldosterone systems. In 2001, BNP was approved by the United States Food and Drug Administration for the treatment of acutely decompensated heart failure. However, recent reports have suggested impaired renal function, which may be related to excessive hypotension (3.Sackner-Bernstein J.D. Skopicki H.A. Aaronson K.D. Circulation. 2005; 111: 1487-1491Crossref PubMed Scopus (660) Google Scholar, 4.Sackner-Bernstein J.D. Kowalski M. Fox M. Aaronson K. J. Am. Med. Assoc. 2005; 293: 1900-1905Crossref PubMed Scopus (600) Google Scholar). CD-NP represents a new class of natriuretic peptide drug that may circumvent the hypotensive nature of BNP and preserve or augment renal function in the congestive heart failure setting. CD-NP contains the full-length 22-amino acid human CNP fused to the 15-amino acid C-terminal tail of Dendroaspis natriuretic peptide (DNP), an endogenous vasoactive peptide found in the venom of the green mamba. The benefits of CNP are that it is venodilatory; less hypotensive than ANP and BNP; and unlike ANP and BNP, remains active in the failing heart (5.Dickey D.M. Flora D.R. Bryan P.M. Xu X. Chen Y. Potter L.R. Endocrinology. 2007; 148: 3518-3522Crossref PubMed Scopus (92) Google Scholar). However, CNP lacks the diuretic and natriuretic actions of ANP and BNP, which are critical for the improvement of patients with congestive heart failure. Because DNP possesses both natriuretic and hypotensive activities (6.Lisy O. Lainchbury J.G. Leskinen H. Burnett Jr., J.C. Hypertension. 2001; 37: 1089-1094Crossref PubMed Scopus (77) Google Scholar), CD-NP was engineered to combine the unique venodilatory and cardiac properties of CNP with the beneficial renal properties of DNP. In vivo application of CD-NP is promising because infusion of CD-NP into dogs increases natriuresis and diuresis while being less hypotensive than BNP. Furthermore, CD-NP increases the glomerular filtration rate (GFR) more than BNP (7.Lisy O. Huntley B.K. McCormick D.J. Kurlansky P.A. Burnett Jr., J.C. J. Am. Coll. Cardiol. 2008; 52: 60-68Crossref PubMed Scopus (141) Google Scholar). Although CD-NP infusion significantly increases cGMP concentrations in plasma, urine, and cultured fibroblasts, the ability of CD-NP to bind and activate each NPR was not determined. Here, we describe the molecular basis for the unique properties of CD-NP. In addition, we created two new variants of CD-NP that are even more potent activators of NPR-A. Surprisingly, the mutation of two amino acids in the ring of CNP transformed CD-NP into a superagonist of NPR-A. To our knowledge, this is the first description of peptides that are capable of activating both NPR-A and NPR-B at less than micromolar concentrations. Peptides and Reagents—Human ANP and CNP were purchased from Sigma. Human BNP, DNP, B-CDNP, and CDNP-B were synthesized by Phoenix Pharmaceuticals, Inc. (Burlingame, CA). CD-NP was manufactured by Clinalfa (Weil am Rhein, Germany). The lyophilized peptides were reconstituted in deionized water, aliquoted, and stored at -80 °C until used. cGMP radioimmunoassay kits were purchased from PerkinElmer Life Sciences. Cells—Human embryonic kidney 293 cells stably expressing rat or human NPR-A or NPR-B were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 200 μg/ml G418 or hygromycin B. Whole Cell cGMP Elevation Assays—Cells were plated in 48-well plates. The day of the assay, the cells were incubated in serum-free medium for 4 h. The cells were incubated for 10 min at 37 °C in Dulbecco's modified Eagle's medium containing 25 mm HEPES (pH 7.4) and 1 mm 1-methyl-3-isobutylxanthine. This medium was then replaced with the same medium containing various concentrations of natriuretic peptides. The cells were stimulated for 1 or 3 min, and then the assay was terminated by aspirating the medium and adding 200 μl of ice-cold 80% ethanol. cGMP concentrations were estimated by radioimmunoassay as described previously (8.Abbey S.E. Potter L.R. J. Biol. Chem. 2002; 277: 42423-42430Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Membrane Guanylyl Cyclase Assays—Crude membranes were prepared and assayed as described previously (9.Bryan P.M. Xu X. Dickey D.M. Chen Y. Potter L.R. Am. J. Physiol. 2007; 292: F1636-F1644Crossref PubMed Scopus (50) Google Scholar). Briefly, cells from a 10-cm tissue culture plate were washed twice with ice-cold phosphate-buffered saline and then scraped in cold buffer containing phosphatase inhibitors. The cells were lysed by sonicating 1 to 2 s and membranes were precipitated by centrifugation. The crude membranes were resuspended in phosphatase inhibitor buffer. 20 μl of membranes containing 1 mm MgGTP were assayed for 3 min at 37 °C in the presence or absence of various concentrations of natriuretic peptide. The assayed were stopped with the addition of 400 μl of cold 50 mm sodium acetate buffer containing 5 mm EDTA and placed on ice. cGMP concentrations were determined on a fraction of the resulting solution by radioimmunoassay. Whole Cell Binding Assays—Cells were plated on 24-well plates precoated with polylysine. When the cells were 75–90% confluent, the growth medium was replaced with 0.2% bovine serum albumin in Dulbecco's modified Eagle's medium. Cells were incubated for 1–2 h in this medium at 37 °C. Binding medium containing 1% bovine serum albumin and 125I-ANP was prepared on ice. Binding media containing increasing concentrations of unlabeled ligand were added to the cells and incubated at 4 °C for 1 h. The cells were washed with ice-cold phosphate-buffered saline to remove nonspecifically bound tracer. 0.5 ml of 1 n NaOH was added to each well and incubated at room temperature to solubilize the cells. Radioactivity was determined in a Beckman 5500 γ-counter. Statistics—Each experiment was performed in triplicate in three separate assays. The data were graphed with Prism software and are presented as the average of all assays combined or as a representative experiment with error bars representing the S.E. of the individual experiment. Our goal is to develop natriuretic peptide variants that are unique co-activators of NPR-B and NPR-A and therefore may have therapeutic advantage beyond native natriuretic peptides that are specific for NPR-B (CNP) or NPR-A (ANP and BNP) for the treatment of cardiorenal disease syndromes such as heart failure. As such, new peptides should reduce cardiac load, decrease cardiac remodeling, and most importantly, enhance renal function. Recently, CD-NP was shown to be a more effective stimulator of GFR, natriuresis, and diuresis than BNP (sequence in Fig. 1) (7.Lisy O. Huntley B.K. McCormick D.J. Kurlansky P.A. Burnett Jr., J.C. J. Am. Coll. Cardiol. 2008; 52: 60-68Crossref PubMed Scopus (141) Google Scholar). However, the pharmacological basis for how CD-NP achieves this result has not been described. There are several possible explanations for why CD-NP elicits a greater GFR-enhancing effect than BNP. CD-NP could have a greater half-life than BNP, it could activate both NPR-A and NPR-B, or it could increase local concentrations of endogenous natriuretic peptides by binding more tightly to NPR-C, thereby reducing receptor-mediated degradation. These pharmacological questions related to the NPRs are addressed here. Activation of Human NPR-A—To unequivocally determine the ability of various ligands to activate human NPR (hNPR)-A or hNPR-B, we stably expressed each individual receptor in human embryonic kidney 293 cells devoid of endogenous NPRs. Using these cells, we could be confident that any increase in natriuretic peptide-dependent cGMP concentrations results from the activation of the single stably expressed receptor. We first tested the ability of CD-NP to activate hNPR-A compared with the original parent peptides, CNP and DNP. We found that addition of the C-terminal portion of DNP to CNP resulted in a peptide that was a dramatically more potent activator of hNPR-A than CNP (Fig. 2A). Although extremely high concentrations of CNP (10 μm) yielded just detectable increases in cellular cGMP concentrations, CD-NP produced equivalent responses at concentrations that were >2 orders of magnitude lower. The calculated for CD-NP was Surprisingly, concentrations of CD-NP produced significantly less cGMP than of ANP, that CD-NP is a partial agonist of Because infusion of the C-terminal portion of DNP significantly increased cGMP in we tested this same could cGMP in cells. We found that this peptide at 10 not cGMP concentrations in cells expressing with a previous that an ring is for NPR-A activation H. PubMed Scopus Google Scholar). Activation of were on 293 cells stably expressing In these cells, CD-NP was less potent than the ligand for NPR-B, The for CD-NP was compared with for CNP (Fig. compared with CNP, CD-NP is a better agonist for NPR-A a agonist for NPR-B. the activity of CD-NP on NPR-B was higher than the ANP and DNP, which residues C-terminal to the ring As with the C-terminal portion of DNP was to (Fig. of CD-NP on Guanylyl Cyclase determine the cGMP in cells were due to activation of the receptor by CD-NP, guanylyl cyclase assays were membranes prepared from 293 cells expressing hNPR-A or were incubated with 1 mm and 5 mm in the presence or absence of various concentrations of natriuretic and the of cGMP a was determined (Fig. to the CD-NP was a dramatically more potent activator of hNPR-A than CNP (Fig. In the addition of the DNP tail only slightly the ability of CNP to activate (Fig. Binding of CD-NP in determine the of hNPR-A to CD-NP was due to an increase in receptor binding 125I-ANP binding were performed on 293 cells. CD-NP was not a for binding to hNPR-A because more CD-NP than ANP was to for 125I-ANP (Fig. However, more striking was the of CNP or the C-terminal portion of DNP to for 125I-ANP binding to hNPR-A even at 10 concentrations. the addition of the C-terminal tail of DNP to the of CNP dramatically increases affinity for with the that CD-NP, not CNP, is a NPR-A were binding to the purchased the for of the Binding of CD-NP in that CD-NP binds and However, that not that the renal and of CD-NP result from the activation of possible is that infusion of CD-NP the half-life of endogenous ANP BNP by for by the clearance receptor. To this we the ability of CD-NP, ANP, and CNP to for 125I-ANP binding to the clearance receptor (Fig. In contrast to the effect that the DNP tail had on the ability of CNP to bind NPR-A, it had any effect on binding to NPR-C because concentrations of all three peptides were to binding of 125I-ANP to this receptor. the addition of the C-terminal portion of DNP to CNP increases affinity for NPR-A not previous binding assays suggested that DNP not bind NPR-C M. 2006; PubMed Scopus Google Scholar), whereas 125I-ANP binding assays that DNP bind NPR-C 2007; PubMed Scopus Google Scholar). To this we tested the ability of DNP to 125I-ANP binding to NPR-C compared with ANP, BNP, and As shown in Fig. DNP bound less than CNP and less than ANP and BNP to the clearance receptor. our data the in the the ability of DNP to bind NPR-A it bind NPR-C, it binds NPR-C less tightly than it binds NPR-A. Activation of by ability of CD-NP to activate the rat of NPR-A and NPR-B was also determined. Here, we two unlike the human in which high concentrations of CNP had effect on hNPR-A (Fig. high concentrations of CNP were to activate rat NPR-A (Fig. whereas CD-NP was a partial agonist of it was a full agonist of rat NPR-A (Fig. In CD-NP had on rat and human NPR-B not the addition of the C-terminal tail of DNP to CNP has a greater effect on hNPR-A because this receptor is to CNP even at pharmacological concentrations. of within the CD-NP tested within the ring to further increases in the for hNPR-A of ANP, BNP, and CNP of amino acid the a acid just the first of the a acid to the C-terminal and the C-terminal tail (Fig. The of the C-terminal of the natriuretic peptides is addressed with the in the pharmacological of CNP addition of the C-terminal portion of DNP. tested the effect of adding the C-terminal tail of ANP or BNP to we not this of the peptides we on of the amino acids in the ring of CD-NP to further enhance potency affinity for We the corresponding for BNP into the described and the effect of the on the to activate the The were on BNP, not ANP, because BNP and NPR-A Google Scholar). We used the same assays to determine the ability of the new chimeric peptides to activate hNPR-A and as we to CD-NP. However, in these we used BNP than ANP as the representative NPR-A agonist because the for the new peptides were from BNP. ANP and BNP have to activate NPR-A, human ANP is a slightly better activator than BNP for ANP in Fig. 2 for BNP in Fig. As shown in Fig. the mutation of the first of amino acids to residues within BNP resulted in a in the of the peptide to cGMP in cells. This called B-CDNP, was slightly more potent than human BNP, and unlike CD-NP, it was a full of the of amino acids within the ring of CNP to CDNP-B also resulted in a peptide with increased full agonist potency CD-NP, the effect was less both variants potency for NPR-B (Fig. because their for NPR-B were the in potency was greater than that for CD-NP. binding were to determine the in potency of the CD-NP CD-NP was due to increased affinity of the peptides for the of NPR-A. The binding shown in Fig. is with the receptor activation shown in Fig. the increased potency of the CD-NP for NPR-A results from increased affinity for the receptor. the ring are specific for NPR-A because had or no effect on binding to NPR-C (Fig. The addition of the C-terminal tail of DNP to CNP, which is not resulted in a peptide that is and GFR-enhancing while being less hypotensive than BNP (7.Lisy O. Huntley B.K. McCormick D.J. Kurlansky P.A. Burnett Jr., J.C. J. Am. Coll. Cardiol. 2008; 52: 60-68Crossref PubMed Scopus (141) Google Scholar). The of renal activities by the chimeric peptide is for therapeutic Synthetic BNP is approved for the treatment of acutely decompensated heart failure. However, infusion of in blood pressure in which renal perfusion pressure and may reduce (3.Sackner-Bernstein J.D. Skopicki H.A. Aaronson K.D. Circulation. 2005; 111: 1487-1491Crossref PubMed Scopus (660) Google Scholar). the ability of CD-NP to these activities hypotension may be extremely beneficial in the clinical setting. the molecular of of CD-NP was the goal of this with specific on Our unequivocally determined that CD-NP is a activator of both hNPR-A (Fig. and (Fig. This is in contrast to CNP, which has no ability to activate the human form of NPR-A at concentrations to 10 (Fig. 2A). The potency of CD-NP compared with ANP and BNP is by at pharmacological concentrations, it activate the receptor. The mutation of amino acids in the ring portion of the peptide to corresponding residues in BNP resulted in a chimeric B-CDNP, which activated hNPR-A with a potency to human BNP (Fig. and maintained ability to activate NPR-B, higher concentrations were to of activation (Fig. which residues within these chimeric peptides binding to NPR-A and NPR-B be the of The potency of CD-NP for NPR-A could the less hypotensive infusion of CD-NP in dogs (7.Lisy O. Huntley B.K. McCormick D.J. Kurlansky P.A. Burnett Jr., J.C. J. Am. Coll. Cardiol. 2008; 52: 60-68Crossref PubMed Scopus (141) Google and could be an for therapeutic ANP (carperitide) and BNP This may CD-NP more beneficial than and in the peptides may be to several reports suggest that NPR-B is in cardiac hypertrophy in heart failure J. M. J. K. J. M. S. 2006; PubMed Scopus Google Scholar, H. K. K. J. Am. Coll. Cardiol. 2005; PubMed Scopus Google Scholar). The ability of CD-NP to activate both hNPR-A and may beneficial in to cardiac in heart failure Activation of NPR-A should natriuresis, diuresis, and to reduce blood pressure and on the and activation of NPR-B should the heart by Burnett Jr., J.C. Am. J. Physiol. PubMed Google as well as by on the heart to decrease hypertrophy and Although our activation that CD-NP not for the of CD-NP infusion in The increase in cGMP could be by activation of NPR-A or by an increased half-life of ANP or BNP due to with CD-NP for degradation. peptides are from the by and as well as by and degradation. We determined that CD-NP binds the clearance receptor with affinity to ANP and CNP (Fig. CD-NP for by NPR-C not any better than reports suggest that DNP is to by Lainchbury J.G. Burnett Jr., J.C. J. Am. Coll. Cardiol. 2002; PubMed Scopus Google addition of the C-terminal tail of DNP to CNP may the peptide less to and increase However, we not any of the ability of CD-NP to be by To be most effective CD-NP should have a this for chimeric natriuretic peptides have been In Burnett Jr., J.C. J. PubMed Scopus Google described the of peptide. This chimeric peptide contains the acid C-terminal tail of ANP to full-length natriuresis to ANP and is more potent than CNP in that the combined with a potent may hypotension to that with infusion of ANP or BNP. have advantage infusion of ANP or BNP in heart failure by being a less potent activator of NPR-A, CD-NP may or reduce of the of NPR-A be potent to the beneficial renal actions with DNP. The in CD-NP activation of NPR-A (Fig. is to that by B. J. PubMed Scopus Google in their to and the of the system when for therapeutic NPR-A is in rat and the full agonist partial agonist responses to CD-NP shown in Fig. are a function of the the human and rat receptors because CNP is in and The of all three human natriuretic peptides BNP, and are (Fig. 1) H. PubMed Scopus Google Scholar). CNP from the peptides in that it lacks a C-terminal Although the ring to be most critical for reports have determined that of amino acids C-terminal to the ring the potency of ANP K. J. Biol. Chem. Full Text PDF PubMed Google Scholar, D.M. PubMed Scopus Google Scholar). from the by H. Y. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google the of rat NPR-A bound to an ANP peptide of the amino acids for binding ANP and suggest that the C-terminal tail portion of ANP is for of it is that the C-terminal amino acids from DNP form critical with residues found in the of human not rat NPR-A. this also to the receptor used as the for the in vivo on CD-NP is not known. of the of and rat NPR-A that this is and to the human the critical hNPR-A and CD-NP are not we determine the are in the receptor at this of the amino acids from the of rat NPR-A by H. Y. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google no amino acid and NPR-A. We are the NPRs to the of these receptors to the human our data the the in vivo of CD-NP, we could not the infusion of the C-terminal portion of DNP. This C-terminal was to a cGMP in or cells at concentrations to 10 We also not any binding of the peptide to NPR-A or we that the peptide and of the endogenous peptides by or this remains to be determined. In CD-NP is a natriuretic peptide that represents a new drug for the treatment of heart failure and cardiorenal disease The unique of this chimeric peptide is that it both hNPR-A and in the amino acids in the ring the peptide a more potent NPR-A The of the three CD-NP peptides described to the benefits of a full NPR-A agonist a partial agonist in with the ability of these peptides to activate NPR-B may the therapeutic of the NPRs and result in the of a new of individual could be for on treatment of the of patients with congestive heart failure with and