Pharmacokinetic and Pharmacodynamic Profiles of Glyco-Modified Atrial Natriuretic Peptide Derivatives Synthesized Using Chemo-enzymatic Synthesis Approaches.

Atrial natriuretic peptide (ANP) exerts beneficial pharmacological effects in the treatment of various cardiovascular disorders, such as acute congestive heart failure (ADHF). However, the clinical use of ANP is limited to the continuous intravenous infusion owing to its short half-life (2.4 ± 0.7 min). In the present study, we conjugated the glyco-modified ANP with a monoclonal antibody (mAb) or an Fc via chemo-enzymatic glyco-engineering using EndoS D233Q/Q303L. The most potent derivative SG-ANP-Fc conjugate extended the half-life to 14.9 d and the duration of blood pressure lowering effect to over 28 d. This new biologic modality provides an opportunity to develop outpatient therapy after ADHF.

Restriction of a peptide turn conformation and conformational analysis of guanidino group using arginine-proline fused amino acids: application to mini atrial natriuretic peptide on binding to the receptor.

Compounds (2S,4S)- and (2S,4R)-4-(2'-guanidinoethyl)proline have been synthesized as a conformationally restricted arginine. Their backbones fit the i + 1 position in a turn, and the side chains are restricted compared to that of arginine. These analogues were incorporated into mini atrial natriuretic polypeptide, which has an important turnlike conformation at Gly(6)-Arg(7)()-Met(8)-Asp(9). Structural analysis revealed that the size of the conformational space of Arg(7) on binding to the receptor was approximately one-third of the entire conformational space.

Synthesis and in vitro analysis of atrial natriuretic peptide-albumin conjugates.

Atrial natriuretic peptide (ANP) is a clinically useful anti-hypertensive hormone. Maleimide derivatives of ANP have been synthesized and conjugated to cysteine-34 of human serum albumin. The conjugates were analyzed to assess their stability, receptor binding affinity and ability to stimulate guanylyl-cyclase activity in rat lung fibroblasts.

Synthesis of phospho-urodilatin by combination of global phosphorylation with the segment coupling approach.

The chemical synthesis of biologically active phosphorylated urodilatin (CDD/ANP-95-126) was achieved by using a strategy of coupling protected peptide segments in solution. Three protected peptide segments corresponding to urodilatin (1-14) with side chain-unprotected Ser10, (15-24) and (25-32) were prepared manually using Fmoc chemistry on an aminopropyl polystyrene resin with the super acid-labile HMPB linker. For the coupling of segments, the carboxy group of the C-terminal segment (25-32) was converted into the tert-butyl ester by treatment with TBTA. The protected peptide segments were coupled in the presence of EDC/HOOBt or TBTU/HOBt to yield fully protected urodilatin with a free hydroxy function at Ser10. Introduction of the phosphate was performed with Et2NP(OtBu)2 and tetrazole followed by oxidation of the phosphite. Alternatively, a prephosphorylated protected segment (1-14) was used in the segment condensation. Our investigations indicate that both pathways, phosphorylation of protected urodilatin in solution and use of a prephosphorylated building block, are suitable methods to obtain a large phosphopeptide of high purity without formation of H-phosphonates or other by-products.

Synthesis of human C-type natriuretic peptide 22 using chlorotrityl resin and tetrafluoroboric acid deprotection.

Human C-type natriuretic peptide 22 (hCNP22), the third member of the natriuretic peptide family, was efficiently synthesized by Fmoc-based peptide chain construction on a 2-chlorotrityl (Clt) resin, followed by deprotection using tetrafluoroboric acid (HBF4). The use of Clt resin was effective in suppressing racemization at the C-terminal cysteine residue caused by the base treatment for Fmoc-cleavage. The disulfide bond of hCNP22 was constructed using the silyl chloride-sulfoxide method to avoid oxidation at the Met residue. Using amino acid- and dipeptide-resin derivatives, the effects of bases, protecting groups and resin supports on the racemization at the C-terminal Cys residue were examined in detail.

[Studies on development of disulfide bond forming reaction and the application to regioselective disulfide formation].

An efficient method for the disulfide bond formation in peptides by the silylchloride-sulfoxide system is described. Methyltrichlorosilane in trifluoroacetic acid, in the presence of diphenylsulfoxide, is found to cleave various S-protecting groups of cysteine to form cystine directly within 10 to 30 min. No side reactions were observed with nucleophilic amino acids such as Met, His, or Tyr, except for Trp, under the reaction conditions of the silylchloride-sulfoxide treatment. A chlorination of the indole moiety of unprotected Trp, rather than the sulfur-sulfur bond formation, is a dominant reaction when the peptide containing unprotected Trp is treated with the chlorosilane-sulfoxide. However, the disulfide bond can be formed efficiently with no modification at the indole ring by the treatment of the peptide having formyl-protected Trp residue with the silylchloride-sulfoxide system. The formyl group is removed by a brief treatment at basic pH without affecting the disulfide bond formed by the silylchloride-sulfoxide treatment. Total synthesis of human insulin, a two chain peptide containing three disulfide bonds, was achieved unambiguously by sequential and selective formation of disulfide bonds in the protein for the first time. The key reaction in the synthesis is regioselective formation of three disulfide bonds separately using the silyl chloride method described above. Prior to the insulin synthesis, it was confirmed by the syntheses of double-disulfide peptides: b-hANP, unnatural parallel dimer of a-hANP, and human endothelin-1 that no disulfide exchange occurred during the silyl chloride treatment. Using three orthogonal thiol protecting groups, Trt, Acm, and But, three disulfide bonds of human insulin were efficiently constructed by the successive reactions using thiolysis, iodine oxidation, and the sily1 chloride method. Each reaction for the stepwise disulfide formation proceeded within 15 to 60 min with no polymeric product and no solubility problem. The synthetic human insulin had the correct structure and was indistinguishable from natural human insulin.

Structure-function interrelation and clinical effect of atrial natriuretic peptide (ANP).

Atriopeptin III (AP III) and its six analogues were synthesized by solid phase method and their diuretic and hypotensive activities were determined. Among these analogues, analogue [D-Ala-5, D-Arg-23] AP III was nearly 10 times as potent as AP III in diuretic activity while its hypotensive activity increased only 50% of that of AP III. Analogue des [Ser-15Gly-16Leu-17Gly-18Asn-20Ser-21] AP III was 15% as potent as AP III in diuretic activity, but it still maintained about 60% of the hypotensive activity of AP III. Meanwhile, we tried analogue [D-Ala-5, D-Agr-23] AP III for the treatment of hypertensive syndrome in pregnancy and obtained some good results.

Synthesis, purification and biological activity of (Ser10-phosphatidyl)-urodilatin (phosphourodilatin).

Based on the global phosphorylation approach, a selective synthesis of (Ser10-phosphatidyl)-urodilatin (phosphourodilatin), which contains 32 amino acid residues and a disulfide loop is described. The peptide was assembled stepwise on a polyethyleneglycol-polystyrene support using Fmoc-chemistry. The phosphorylation was performed on-resin by phosphitylation with a large excess of di-tert-butyl-N,N-diethylphosphoramidite within 1 hour, followed by oxidation with tert-butylhydroperoxide to the protected phosphopeptide. After cleavage and deprotection the disulfide bridge was introduced without side reactions by iodine titration of the mono-acetamidomethyl protected crude peptide. During the synthetic pathway, the acylation with side chain-unprotected Fmoc-serine and the phosphitylation satisfactorily yielded the expected intermediates. In some phosphorylation experiments a by-product having a reduced mass corresponding to the H-phosphonate was observed. Illustrated with the synthesis of phosphourodilatin, this type of by-product, which could not be separated by HPLC, and the difficult amino acid sequence make the synthesis of a large phosphopeptide a more delicate task than the synthesis of short phosphopeptides, which do not contain oxidation-sensitive amino acids, difficult sequences or additional structural elements such as disulfide loops. The biological activity of phosphourodilatin was compared with non-phosphorylated urodilatin in two assay systems. Both peptides revealed a vasorelaxant effect on aortic smooth muscle strips and induced a cGMP-generation in RFL-6 cells with increasing dose dependency.

Synthesis and biological activity of atriopeptin III and its small molecular analog.

Atriopeptin III (AP III) and its small molecular analog were synthesized manually by stepwise solid-phase method. The peptides were oxidized with iodine in 30% acetic acid at very high dilution to form intramolecular disulfide bridge and purified to homogeneity by conventional means, including Sephadex G15, dialysis and reversed-phase HPLC. Bioassay study demonstrated that the synthetic AP III possesses potent bioactivities identical to those of the same product of Peninsula Lab both in vitro and in vivo; whereas the linear peptide, having the same primary structure as AP III, showed very limited bioactivity. The small analog, with Ser-Ser-residue deleted from the N-terminal of AP III, was equipotent as AP III while exhibiting a longer half-life in vivo resulting from the peptide modification.

Complexes of Cu(II) with Asn-Ser-Phe-Arg-Tyr-NH2; an example of metal ion-promoted conformational organization which results in exceptionally high complex stability.

The pentapeptide fragment of ANF, Asn-Ser-Phe-Arg-Tyr-NH2, coordinates to Cu(II) using the same four nitrogen donor centers as simple pentapeptides such as pentaalanine yet the complexes are of much higher stability as a result of a highly organized side-chain structure which is present in the complex but absent from the free ligand.

Vasonatrin peptide: a unique synthetic natriuretic and vasorelaxing peptide.

This study reports the cardiovascular and renal actions of a novel and newly synthesized 27-amino acid peptide termed vasonatrin peptide (VNP). VNP is a chimera of atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP). This synthetic peptide possesses the 22-amino acid structure of CNP, which is a cardiovascular selective peptide of endothelial origin and is structurally related to ANP. VNP also possesses the five-amino acid COOH terminus of ANP. The current study demonstrates both in vitro and in vivo that VNP possesses the venodilating actions of CNP, the natriuretic actions of ANP, and unique arterial vasodilating actions not associated with either ANP or CNP.

Syntheses and biological activities of selenium analogs of alpha-rat atrial natriuretic peptide.

alpha-Rat atrial natriuretic peptide (7--28) (rANP (7--28)) and a series of its analogs in which half cystine residue(s) were substituted with half selenocystine residue(s) were synthesized by using the Fmoc-based solid-phase method followed by cyclization by means of dimethylsulfoxide (DMSO)-trifluoroacetic acid (TFA) oxidation. These analogs possess comparable activities in both receptor binding and cGMP accumulation in rat vascular smooth muscle cells to those of rAMP (7--28).

Development of natriuretic peptide analogs selective for the atrial natriuretic factor-R1A receptor subtype.

A pharmacological characterization of subtypes of the atrial natriuretic factor (ANF) receptor ANF-R1, found in bovine adrenal cortex and rat papillary membrane preparations, has been carried out using various chimeric analogs based on rat ANF(99-126) [rANF(99-126)] and porcine brain natriuretic peptide 32 (pBNP32). Receptor binding and cGMP production assays in bovine adrenal cortex indicate that replacement of the amino-terminal segment of pBNP32 with that of rANF(99-126) enhances the affinity of the peptide for the ANF-R1A receptor subtype and its stimulation of associated guanylate cyclase activity. In rat kidney papillae, the substitution of amino- and/or carboxyl-terminal portions of pBNP32 with those of rANF(99-126) also results in a large increase in the affinity and agonistic potency for the ANF-R1A subtype but in only modest changes in those for the ANF-R1B receptor subtype. Interestingly, in this preparation the chimeric analogs could discriminate by their differential affinities and cGMP production potencies between the two receptor subtypes. In particular, pBNP1, obtained by combining the ring structure of pBNP32 with the amino- and carboxyl-terminal portions of rANF(99-126), is the most selective analog. pBNP1 displays higher affinity and agonistic potency for ANF-R1A receptor than for ANF-R1B receptor, with selectivity ratios between these two subtypes of 632- and 504-fold, respectively. Moreover, an excellent correlation is observed between the affinity of the peptides for the ANF-R1A receptor and their stimulation of particulate guanylate cyclase activity in bovine adrenal cortex (r = 0.99, p < 0.01) and rat papillary (r = 0.97, p < 0.01) membrane preparations. In addition, all the chimeric analogs in this study show affinities similar to those of rANF(99-126) and pBNP32 for the ANF-R2 receptor in NIH-3T3 membrane preparations. Importantly, the chimeric analogs pBNP1 and pBNP3, which contain the core of pBNP32 and the amino-terminal segment of rANF(99-126), display higher affinities for the ANF-R1A receptor type than for the ANF-R2 receptor type. These results indicate that the analogs combining the ring structure of pBNP32 with the amino- and/or carboxyl-terminal segments of rANF(99-126) are more selective for the ANF-R1A receptor subtype than are the natural peptides rANF(99-126) and pBNP32.

Powerful solvent systems useful for synthesis of sparingly-soluble peptides in solution.

Our maximum protection strategy for the synthesis of human parathyroid hormone(1-84) indicates that fully protected peptide segments in the form of Boc-peptide phenacyl (Pac) ester are relatively soluble in ordinary organic solvents such as DMF, NMP or DMSO, which are suitable for coupling segments. However, about 1% of such segments synthesized were found to be insoluble even in the most polar solvent, DMSO. Thus, a more powerful solvent which can be used for their peptide synthesis was pursued. Among the solvent systems tested, a mixture of trifluoroethanol (TFE) or hexafluoroisopropanol (HFIP) and trichloromethane (TCM) or dichloromethane (DCM) was found to be most powerful for dissolving such sparingly-soluble protected peptides. These solvent systems were confirmed to be useful for the removal reaction of the carboxy-terminal Pac esters from the sparingly-soluble segments. They were then tested for the coupling reactions of fully protected Boc-peptides with other sparingly-soluble peptide esters. The TFE/TCM or TFE/DCM system was extremely useful for coupling segments without danger of racemization and of trifluoroester formation, if WSCI was used as the coupling reagent in the presence of 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBt).

Highly efficient photoaffinity labeling of the hormone binding domain of atrial natriuretic factor receptor.

A high-efficiency photoaffinity derivative of atrial natriuretic factor (ANF) was developed for studying the peptide binding domain of the receptor protein and for better characterization of this receptor in tissues with a low density of binding sites. The position of the photosensitive residue was chosen on the basis of a molecular conformational model and on structure-activity relationship studies which both indicate that the carboxy-terminal end of the peptide is part of a hydrophobic pole likely to interact deeply within the ANF binding pocket of the receptor. Selection of the photoreactive residue p-benzoylphenylalanine (BPA) as a substitute for arginine in position 125 of the peptide sequence led to a photoaffinity derivative with a high (63%) efficiency of covalent incorporation to the receptor protein. This derivative (BPA-ANF) has a 10-fold lower affinity when compared with ANF, but it is a full agonist in stimulating cGMP production and inhibiting aldosterone secretion in bovine adrenal zona glomerulosa. Photoaffinity labeling with BPA-ANF specifically identifies ANF-R1 and ANF-R2 receptor proteins with a 10-fold higher efficiency than with azido derivatives of ANF or with cross-linking agents. This new ANF derivative therefore appears to be useful for studying ANF receptors in tissues with low levels of expression, for locating receptor following cellular internalization, and for tagging proteolytic fragments of the receptor amenable to amino acid microsequencing.

Small atrial natriuretic peptide analogues: design, synthesis, and structural requirements for guanylate cyclase activation.

Structure/activity studies on atrial natriuretic peptide ANP (1-28) have highlighted three portions of the native molecule as necessary for its biological responses. We have linked these three regions and excised the remaining segments to produce a family of small analogues (less than half the size of the parent) which demonstrate the full range of ANP's actions. Importantly, these compounds act at both major types of ANP receptor. Two critical modifications lead to more potent analogues; both involve expanding the cyclic portion of the molecule. Further optimization of one of these modified structures leads to A68828, a full ANP agonist which shows promise as a preventative agent against acute renal failure.

Truncated atrial natriuretic factor analogs retain full agonist activity.

The synthesis, receptor binding, and agonist activity of a series of truncated atrial natriuretic analogs (ANF) are described. These analogs incorporate two portions of the native 28 amino peptide, the eight amino acids C-terminal to Cys7, and two amino acids from the C-terminus (phenylalanine and arginine), into disulfide-bonded cyclic peptides. The inclusion of the C-terminal amino acids converted the ANF analogs from receptor ligands to full agonists, as measured by several methods, including the stimulation of cGMP biosynthesis in endothelial cells, inhibition of aldosterone biosynthesis in rat adrenal cells, and natriuretic-hypotensive activity in vivo. The most potent analogs have cyclohexylalanine (Cha) at position 8. The lead compound (Arg6,Cha8 ANF 6-15 Phe-Arg-Cys-NH2) is a tridecapeptide that integrates the C-terminal amino acids inside the disulfide ring. This peptide, designated as A-68828, has a binding affinity of IC50 = 120 nM, approximately 1/400 of ANF 1-28. However, this analog, in vivo, is only slightly less natriuretic (1/20-1/50) than ANF 1-28. Unlike the native peptide, A-68828 is only mildly hypotensive and at the highest concentration tested reduced blood pressure less than 15 mmHg (1 mmHg = 133.322 Pa). A-68828 inhibited ACTH-induced aldosterone release to a greater extent than ANF 1-28: 100 vs. 50%. The selective natriuretic activity of A-66828, relative to ANF, suggests clinical utility for the treatment of acute renal failure.

[Atrial natriuretic peptides. IV. Synthesis and study of shortened analogs]. / Atrial'nye natriiureticheskie peptidy. IV. Sintez i izuchenie ukorochennykh analogov.

New analogues of atrial peptides of rat were synthesized by classical methods of peptide chemistry in solution. They contain a D-amino acid residue in the C-terminal part and a residue of mercaptopropionic acid in the N-terminal part of the molecule. Biological activity of the new analogues was studied.

Synthesis and biological activity profiles of atrial natriuretic factor (ANF) analogs.

Several analogs of the atrial natriuretic factor (ANF) were synthesized by the solid-phase method using the acetamidomethyl (Acm) group for sulfhydryl protection. The compounds were tested in a receptor binding assay using bovine adrenal zona glomerulosa cell membranes and in the rat diuresis/natriuresis assay. Substitution of tyrosine in position 116 of ANF(101-126) and of the analog [3-Mpr105]ANF(105-126)(3-Mpr = 3-mercaptopropionic acid) did not alter the biological activity profiles and, therefore, these two analogs in radioiodinated form will be useful for enzymatic degradation and clearance studies. Replacement of 3-mercaptopropionic acid with 2-mercaptopropionic acid in [3-Mpr105]ANF(105-126) resulted in an analog with very low potency in both assay systems, presumably as a consequence of the steric bulk and/or local conformational restriction produced by the methyl group attached to the alpha-carbon in position 105. The analog [3-Mpr105,Nva109]ANF(105-126)(Nva = norvaline) showed very low affinity in the receptor binding assay but displayed considerable diuretic/natriuretic activity. The obtained biological activity profiles suggest that in comparison with other ANF peptides the des-amino ANF(105-126) analogs may have a somewhat longer half-life in vivo, or alternatively, may indicate a more complex situation of ANF receptor or binding site heterogeneity.