Synthesis and activity of dimeric bradykinin antagonists containing diaminodicarboxylic acid bridge residues.

Enhancement of a ligand's interaction with a receptor through presenting the ligand in multimeric form is a topic of general interest. Thus dimerization of single-chain bradykinin antagonist peptides has previously been shown to be beneficial in terms of potency and duration of action. While crosslinking polypeptides at terminal positions using suitable dicarboxylic acids and diamines is comparatively straight-forward synthetically, internal dimerizations are usually achieved through oxidation or double S-alkylations of cysteine residues, resulting in metabolically unfavourable disulphide and thioether cross-links. Using suitably modified standard solid-phase peptide synthesis protocols, dimeric bradykinin antagonist peptides [H-(D-Arg)-Arg-Pro-Hyp-Gly-Phe]2-X-[(D-Phe)-Leu-Arg-OH]2 were synthesized where X corresponds to a L,L-2,7-diaminosuberic or L,L-2,9-diaminosebacic acid residue, respectively. The biological activity of these peptides was comparable to that of conventional dimeric bradykinin antagonists cross-linked through cystine or bis(succinimido)alkyl bridges.

Design of low molecular weight hematoregulatory agents from the structure-activity relationship of a dimeric pentapeptide.

We report herein, a new class of simple hematoregulatory semipeptides, formally derived from the cystine-dimerized peptide pGlu-Glu-Asp-Cys-Lys-OH, where the disulfide bond has been replaced by an isosteric dicarba bridge. The structure-activity relationship (SAR) of a series of analogues incorporating replacements at positions 1 and 2 of peptide 1 led to the design of active conformationally constrained cyclic peptides (12, 13). Ring closure was achieved by cyclization of the N-terminal amino groups at position 2 of peptide 2 using pyrazine-2,3-dicarboxylic acid. Subsequent excision of the putative C-terminal scaffold domain from the active cyclic peptides resulted in the discovery of a new class of low molecular weight hematoregulatory agents exemplified by compound 16. This semipeptide analogue, comprising two D-Ser residues connected via amide bonds to the acid groups of pyrazine-2,3-dicarboxylic acid, had comparable biological activity to the lead peptide 1. The stereochemical requirements for the observed biological activity of these novel compounds were examined. Furthermore, the hematopoietic synergistic activity induced by compound 16 in stromal cell cultures was blocked by an antibody known to neutralize the hematoregulatory effect of 1, indicating a common mechanistic end point. Compounds of the class typified by 16 may form the basis for the development of novel therapeutic agents within the area of immunoregulation.

Structure-activity relationships of novel hematoregulatory peptides.

Hematopoiesis is a lifelong cell renewal process regulated by a family of lineage specific hematopoietic growth factors. Several hematopoietic growth factors such as G-CSF, GM-CSF, and M-CSF have been clinically evaluated for enhancement of host defense in normal and immunocompromised patients and for the treatment of infectious diseases. This paper reports the structure-activity relationships of low molecular weight hematoregulatory peptides based on a nonapeptide (1, SK&F 107647). Like the macromolecular growth factors, these peptides modulate host defense. A molecular target for this class of compounds has not yet been identified. However, the structure-activity relationships established by this study implicate a very specific molecular recognition event that is pivotal for the biological activities of 1 and its analogues.

The production and characterisation of monoclonal antibodies to myc, c-erbB-2 and EFG-receptor using a synthetic peptide approach.

Monoclonal antibodies to myc, c-erbB-2 and epidermal growth factor-receptor (EGF-R) were raised using a synthetic peptide approach. The antibodies were characterised by ELISA, immunoblotting, immunoprecipitation and immunocytochemical procedures against cognate peptide and native proteins. All of the monoclonal antibodies detected peptide-blockable bands of appropriate molecular weight (myc-p62/66 kDa, c-erbB-2-185kDa; EGF-R-150/170 kDa) on immunoblots. The monoclonal antibodies to c-erbB-2 and EGF-R immunostained subpopulations of tumour cells on sections of formalin-fixed, paraffin wax embedded human infiltrating and invasive ductal carcinomas of breast. Intense blood cell staining was observed with the EGF-R antibody. This staining was shown to be peptide blockable and may reveal a true localisation for the EGF-receptor protein, a closely-related (erbB) protein or a degradation product. The monoclonal antibody to a common peptide from the myc protein family was epitope scanned using a modification of the Geysen pin technique. Hexapeptide sequence Ala-Pro-Ser-Glu-Asp-Ile was found to be bound most strongly by the myc monoclonal antibody, and amino acids Pro2 and Glu4 were found to be essential for antibody binding. The use of synthetic peptides for the production of monoclonal antibodies with predetermined specificity, which may be precisely identified using the epitope scanning technique, is discussed.