Synthetic evaluation of disulphide-bonded sarafotoxin on a poly(oxy ether) grafted dendrimeric poly(alkyl amine) support for polymer assisted organic synthesis.

The present paper describes the synthesis, characterization and assessment of a novel class of insoluble polymeric polystyrene supports which combines polar poly(ethylene glycol)dimethacrylate as a cross-linker and poly(ethylene glycol) grafted poly(N,N-bisethylamine) as a dendritic template. Poly(N,N-bisethylamine) dendrimers were generated by a series of reactions such as Schiff base integration, acidolysis, diazotization and thionyl chloride treatment. The same successive sequences of reactions have been followed for second generation dendrimers also and subjected to PEGylation (PEG 600) to achieve the desirable physico-chemical properties. The applicability of the novel PEGylated dendrimer support was demonstrated by synthesizing linear as well as disulfide bonded peptides in high yields and purities.

Pharmacological and structural characterization of long-sarafotoxins, a new family of endothelin-like peptides: Role of the C-terminus extension.

Long-sarafotoxins (l-SRTXs) have recently been identified in both the venom of Atractaspis microlepidota and that of Atractaspis irregularis. They are characterized by different C-terminus extensions that follow the invariant Trp21, which plays a crucial role in endothelin-receptor binding. We initially determined the toxicity and three-dimensional structures of two chemically synthesized l-SRTXs that have different C-terminus extensions, namely SRTX-m (24 aa, including extension "D-E-P") and SRTX-i3 (25 aa, including extension "V-N-R-N"). Both peptides were shown to be highly toxic in mice and displayed the cysteine-stabilized α-helical motif that characterizes endothelins and short-SRTXs, to which a longer C-terminus with variable flexibility is added. To discern the functional and pharmacological consequences of the supplementary amino acids, different chimerical as well as truncated forms of SRTX were designed and synthesized. Thus, we either removed the extra-C-terminal residues of SRTX-m or i3, or grafted the latter onto the C-terminal extremity of a short-SRTX (s-SRTX) (ie. SRTX-b). Our competitive binding assays where SRTXs competed for iodinated endothelin-1 binding to cloned ET(A) and ET(B) receptor subtypes over-expressed in CHO cells, revealed the essential role of the C-terminus extensions for ET-receptor recognition. Indeed, l-SRTXs displayed an affinity three to four orders of magnitude lower as compared to SRTX-b for the two receptor subtypes. Moreover, grafting the C-terminus extension to SRTX-b induced a drastic decrease in affinity, while its removal (truncated l-SRTXs) yielded an affinity for ET-receptors similar to that of s-SRTXs. Furthermore, we established by intracellular Ca(2+) measurements that l-SRTXs, as well as s-SRTXs, display agonistic activities. We thus confirmed in these functional assays the major difference in potency for these two SRTX families as well as the crucial role of the C-terminus extension in their various pharmacological profiles. Finally, one of the chimeric toxin synthesized in this study appears to be one of the most potent and selective ligand of the ET(B) receptor known to date.

Application of topologically constrained mini-proteins as ligands, substrates, and inhibitors.

Protein-protein interactions are governed by a variety of structural features. The sequence specificities of such interactions are usually easier to establish than the "topological specificities," whereby interactions may be classified based on recognition of distinct three-dimensional structural motifs. Approaches to explore topological specificities have been based primarily on assembly of mini-proteins with well defined secondary, tertiary, and/or quarternary structures. The present chapter focuses on three approaches for constructing topologically well defined mini-proteins: template-assembled synthetic proteins (TASPs), disulfide-stabilized structures, and peptide-amphiphiles (PAs). Specific examples are given for applying each approach to explore topologically-dependent protein-protein interactions. TASPs are utilized to identify a metastatic melanoma receptor that binds to the alpha1(IV)1263-1277 region of basement membrane (type IV) collagen. A disulfide-stabilized structure incorporating a sarafotoxin (SRT) 6b model was examined as a matrix metalloproteinase (MMP)-3 inhibitor. PAs were developed as (a) fluorogenic triple-helical or polyPro II substrates for MMPs and aggrecanase members of the a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family and (b) glycosylated and nonglycosylated ligands for metastatic melanoma cells. Topologically constrained mini-proteins have proved to be quite versatile, helping to define critical primary, secondary, and tertiary structural elements that modulate enzyme and receptor functions.

An improved procedure for the preparation of the endothelin-B-receptor ligand sarafotoxin 6c.

Sarafotoxin 6c (S6c) is a specific agonist for the endothelin-B- (ETB) receptor, being largely used as a standard compound in pharmacological studies related to ETB-receptors. We have developed an improved peptide synthesis prncedure. Using the Fmoc chemistry methodology and an appropriate orthogonal protection scheme, we obtained a crude peptide with a major component identified as [Cys(Acm)(1,15)]S6c. A silver salt (AgBF4) treatment of this crude material completely removed the Acm moieties without the appearance of any major side-products. The free peptide was cyclized using a Fe3+/Fe2+ oxidizing method. Besides the formation of the bridges, no other modification was observed. Thus, the peptide purification was facilitated and an excellent yield of pure sarafotoxin was obtained. The material was characterized by analytical reverse phase-high performance liquid chromatography (RP-HPLC) and matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Pharmacological properties of the peptide were evaluated in an ETB preparation (guinea pig lung parenchyma strips) and were identical to those of an S6c standard.

Structure-activity relationships of sarafotoxins: chemical syntheses of chimera peptides of sarafotoxins S6b and S6c.

The three chimera peptides of sarafotoxins S6b (SRTb) and S6c (SRTc), [Thr2]SRTb, [Asn4]SRTb and [Glu9]SRTb, were synthesized chemically. From the comparisons of lethality, vasoconstrictor activity and receptor binding activity of SRTb, SRTa [( Asn13]SRTb), SRTc [( Thr2,Asn4,Glu9,Asn13]SRTb), [Thr2]SRTb, [Asn4]SRTb and [Glu9]SRTb, it appears that the Lys9 to Glu9 substitution greatly diminishes these activities while the Lys4 to Asn4 substitution does not affect them, and the Ser2 to Thr2 substitution or the Tyr13 to Asn13 substitution slightly diminishes these activities. These results suggest that the very low activities of SRTc are caused mainly by the Lys9 to Glu9 substitution, but not by the Ser2 to Thr2 substitution, which was suggested to be responsible for the weak bioactivities of SRTd [( Thr2,Ile19]SRTb).

Functional endothelin-1 receptors in rat astrocytoma C6.

Rat astrocytoma C6 cells have been recently identified as target cells for ET-1, which stimulates inositol lipid turnover in these cells. It is shown here that binding of ET-1 to high-affinity receptors on C6 cells leads to 40-45% inhibition of isoproterenol-induced intracellular cyclic AMP accumulation, as well as to stimulation of inositol lipid turnover, both effects characterized by an absolute requirement of extracellular calcium. Moreover, ET-1, which has been generally reported to have a mitogenic effect on a variety of target cells including primary rat astrocytes, is shown here to stimulate or, alternatively, inhibit DNA synthesis in C6 cells, depending on the subclone considered.

Studies on the disulfide bridges of sarafotoxins. Chemical synthesis of sarafotoxin S6B and its homologue with different disulfide bridges.

Sarafotoxin S6b, a strong vasocontractile peptide with 21 amino acid residues containing two sets of disulfide bridges, was chemically synthesized. The retention time on a reversed-phase HPLC, lethal and vasocontractile activities of natural sarafotoxin S6b agree with those of the synthetic [Cys1-15, Cys3-11]-sarafotoxin S6b. The combination of the disulfide bridges of sarafotoxin S6b is the same as that of endothelin-1, a mammalian vasocontractile peptide, which shows a high degree of sequence homology and shares several common pharmacological properties with sarafotoxins.

Interaction of synthetic sarafotoxin with rat vascular endothelin receptors.

The effects of synthetic analogs of sarafotoxin (STX) S6b, a snake venom peptide with a high sequence homology to the endothelium-derived vasoconstrictor endothelin (ET), on ET receptor binding activity and cytosolic free Ca2+ concentration [( Ca2+]i) were studied in cultured rat vascular smooth muscle cells. Binding studies revealed that [Cys1-15, Cys3-11] STX competed with 125I-ET for the binding to its vascular receptors with lower affinity than that of ET, but was far more effective than [Cys1-11, Cys3-15]STX in inhibiting the binding. [Cys1-15, Cys3-11]STX had a less potent effect on increasing [Ca2+]i than ET, whereas [Cys1-11, Cys3-15]STX was inactive. These data suggest that there may exist heterogenous subpopulations of the vascular ET/STX receptors, and that the proper double cyclic structure of STX is essential for interacting with its putative receptors to induce the [Ca2+]i response.

Chemical synthesis of echistatin, a potent inhibitor of platelet aggregation from Echis carinatus: synthesis and biological activity of selected analogs.

Echistatin, a polypeptide from the venom of the saw-scaled viper, Echis carinatus, containing 49 amino acids and 4 cystine bridges was synthesized by solid-phase methodology in 4% yield. In the final step, air oxidation of the octahydroderivative was found to be optimal at pH 8. The synthetic product was shown to be physically and biologically indistinguishable from native material. It inhibits fibrinogen-dependent platelet aggregation stimulated by ADP with IC50 = 3.3 x 10(-8) M and also prevents aggregation initiated by thrombin, epinephrine, collagen, or platelet-activating factor. Reduction of purified synthetic echistatin to octahydroechistatin with dithiothreitol followed by air oxidation regenerated homogeneous echistatin in quantitative yield. This highly specific refolding strongly suggests that the linear sequence of octahydroechistatin contains all of the information that is required for the proper folding of the peptide. The sequence Arg24-Gly-Asp of echistatin occurs also in adhesive glycoproteins that bind to the platelet fibrinogen receptor--a heterodimeric complex composed of glycoproteins IIb and IIIa. In an effort to evaluate the role of this putative binding site we have synthesized analogs of echistatin with substitution of Arg-24. Replacement with ornithine-24 (Orn-24) resulted in an analog having a platelet aggregation inhibitory activity with IC50 = 1.05 x 10(-7) M. Substitution with Ala-24 gave IC50 = 6.1 x 10(-7) M. The inhibitory activity of the corresponding short sequence analogs Arg-Gly-Asp-Phe (IC50 = 6 x 10(-6) M), Orn-Gly-Asp-Phe (IC50 = 1.3 x 10(-4) M), and Ala-Gly-Asp-Phe (IC50 = 5.0 x 10(-4) M) was also determined. These results suggest that arginine plays a more important role in the binding of the tetrapeptide than in that of echistatin.

Synthesis of endothelin-1 analogues, endothelin-3, and sarafotoxin S6b: structure-activity relationships.

Two disulfide analogues (types A and B) of endothelin-3 (ET-3; formerly, rat ET), sarafotoxin S6b, and apamin, were synthesized to determine their disulfide structures as in the case of endothelin-1 (ET-1; formerly human and porcine ET). The disulfide structures of ET-3 and sarafotoxin S6b were found to be identical with that of ET-1 (type A) but distinct from that of apamin (type B). The vasoconstricting activities of ET-3 and sarafotoxin S6b were about one-60th and one-third that of ET-1, respectively. Such different biological potencies between endothelins and sarafotoxin S6b could be largely attributed to the sequence heterogeneity at the N-terminal portion. ET-1 analogues were also synthesized to clarify the structure-activity relationships. The opening of any disulfide bond in the ET-1 molecule extremely decreased the activity, while oxidation of the Met residue did not alter it. Amidation of the terminal COOH group and extension of the Lys-Arg sequence to the N-terminus led to 16- and 540-fold decreases in activity, respectively. Removal of the C-terminal Trp residue resulted in complete loss of the activity. The other disulfide analogues (type B and C) of ET-1 showed markedly lower activity than the parent molecule (type A). These results indicated the importance of the whole molecule with the proper double cyclic structure for determining its active conformation.