Structure-activity studies of lGnRH-III through rational amino acid substitution and NMR conformational studies.

Lamprey gonadotropin-releasing hormone type III (lGnRH-III) is an isoform of GnRH isolated from the sea lamprey (Petromyzon marinus) with negligible endocrine activity in mammalian systems. Data concerning the superior direct anticancer activity of lGnRH-III have been published, raising questions on the structure-activity relationship. We synthesized 21 lGnRH-III analogs with rational amino acid substitutions and studied their effect on PC3 and LNCaP prostate cancer cell proliferation. Our results question the importance of the acidic charge of Asp6 for the antiproliferative activity and indicate the significance of the stereochemistry of Trp in positions 3 and 7. Furthermore, conjugation of an acetyl-group to the side chain of Lys8 or side chain cyclization of amino acids 1-8 increased the antiproliferative activity of lGnRH-III demonstrating that the proposed salt bridge between Asp6 and Lys8 is not crucial. Conformational studies of lGnRH-III were performed through NMR spectroscopy, and the solution structure of GnRH-I was solved. In solution, lGnRH-III adopts an extended backbone conformation in contrast to the well-defined ß-turn conformation of GnRH-I.

A peptide corresponding to the C-terminal region of pleiotrophin inhibits angiogenesis in vivo and in vitro.

Pleiotrophin (PTN) is a heparin-binding growth factor that plays a significant role in tumor growth and angiogenesis. We have previously shown that in order for PTN to induce migration of endothelial cells, binding to both α(ν) ß(3) integrin and its receptor protein tyrosine phosphatase beta/zeta (RPTPß/ζ) is required. In the present study we show that a synthetic peptide corresponding to the last 25 amino acids of the C-terminal region of PTN (PTN(112-136) ) inhibited angiogenesis in the in vivo chicken embryo chorioallantoic membrane (CAM) assay and PTN-induced migration and tube formation of human endothelial cells in vitro. PTN(112-136) inhibited binding of PTN to α(ν) ß(3) integrin, and as shown by surface plasmon resonance (SPR) measurements, specifically interacted with the specificity loop of the extracellular domain of ß(3) . Moreover, it abolished PTN-induced FAK Y397 phosphorylation, similarly to the effect of a neutralizing α(ν) ß(3) -selective antibody. PTN(112-136) did not affect binding of PTN to RPTPß/ζ in endothelial cells and induced ß(3) Y773 phosphorylation and ERK1/2 activation to a similar extent with PTN. This effect was inhibited by down-regulation of RPTPß/ζ by siRNA or by c-src inhibition, suggesting that PTN(112-136) may interact with RPTPß/ζ. NMR spectroscopy studies showed that PTN(112-136) was characterized by conformational flexibility and absence of any element of secondary structure at room temperature, although the biologically active peptide segment 123-132 may adopt a defined structure at lower temperature. Collectively, our data suggest that although PTN(112-136) induces some of the signaling pathways triggered by PTN, it inhibits PTN-induced angiogenic activities through inhibition of PTN binding to α(ν) ß(3) integrin.

Enzymatic stability, solution structure, and antiproliferative effect on prostate cancer cells of leuprolide and new gonadotropin-releasing hormone peptide analogs.

Analogs of GnRH, including [DLeu6, desGly1o]-GnRH-NHEt (leuprolide, commercial product), have been widely used in oncology to induce reversible chemical castration. Several studies have provided evidence that, besides their pituitary effects, GnRH analogs may exert direct antiproliferative effects on tumor cells. To study the effect of modifications in positions 4 and 6 of leuprolide on prostate cancer cell proliferation, we synthesized 12 new leuprolide analogs. All GnRH analogs lacked the carboxy-terminal Gly10-amide of GnRH, and an ethylamide residue was added to Pro9. Gly6 was substituted by DLys, Nepsilon-modified DLys, Glu, and DGlu. To improve the enzymatic stability, NMeSer was incorporated in position 4, and the rate of hydrolysis by alpha-chymotrypsin and subtilisin was investigated. Our results demonstrate that this incorporation increases enzymatic stability in all analogs of GnRH, whereas the antiproliferative effect on PC3 and LNCaP prostate cancer cells is similar to that of leuprolide. Conformational studies were performed to elucidate structural changes occurring on substitution of native residues and to study structure-activity relationship for these analogs. The solution models of [DLeu6, desGly10]-GnRH-NHEt (leuprolide), [NMeSer4, DGlu6, desGly10]-GnRH-NHEt, [Glu6, desGly10]-GnRH-NHEt, and [DGIu6, desGly10]-GnRH-NHEt peptides were determined through two-dimensional nuclear magnetic resonance spectroscopy in dimethylsulfoxide. Nuclear magnetic resonance data provide experimental evidence for the U-turn-like structure appeared in all four analogs, which could be characterized as beta-hairpin conformation. The most stable analog [NMeSer4, DGlu6, desGly10]-GnRH-NHEt against proteolytic cleavage forms a second extra backbone turn observed for residues 1-4.

Synthetic peptides as structural maquettes of Angiotensin-I converting enzyme catalytic sites.

The rational design of synthetic peptides is proposed as an efficient strategy for the structural investigation of crucial protein domains difficult to be produced. Only after half a century since the function of ACE was first reported, was its crystal structure solved. The main obstacle to be overcome for the determination of the high resolution structure was the crystallization of the highly hydrophobic transmembrane domain. Following our previous work, synthetic peptides and Zinc(II) metal ions are used to build structural maquettes of the two Zn-catalytic active sites of the ACE somatic isoform. Structural investigations of the synthetic peptides, representing the two different somatic isoform active sites, through circular dichroism and NMR experiments are reported.

In position 7 L- and D-Tic-substituted oxytocin and deamino oxytocin: NMR study and conformational insights.

Incorporation of L- or D-Tic into position 7 of oxytocin (OT) and its deamino analogue ([Mpa(1)]OT) resulted in four analogues, [L-Tic(7)]OT (1), [D-Tic(7)]OT (2), [Mpa(1),L-Tic(7)]OT (3) and [Mpa(1),D-Tic(7)]OT (4). Their biological properties were described by Fragiadaki et al. (Eur J Med Chem 42:799-806, 2007). Their NMR study (NOESY, TOCSY, (1)H-(13)C HSQC spectra) is presented here. Analogues 1, 3 and 4 showed partial agonistic activity, analogue 2 was pure antagonist, suggesting that a cis conformation between residues 6 and 7 of the molecule does not result in antagonistic activity. However, the reduction in agonistic activity of analogues 1, 3 and 4 in comparison to oxytocin is consistent with the reduction of the trans conformation form. Binding affinity for the human oxytocin receptor with IC(50) value of 130, 730, 103, and 380 nM for peptides 1, 2, 3, and 4, respectively, showed lower affinity in the case of D analogues. Deamination slightly increased the affinity. The existence of both cis and trans configurations of the Cys(6)-D-Tic(7) bond is supported by observation of two sets of cross-peaks for (1)H and (13)C nuclei for most of the residues of the peptide not only in NOESY and TOCSY but also in (1)H-(13)C HSQC spectra. The MS and HPLC indicate the presence of a single molecule/peptide, and NMR data thus suggest that this second set of peaks is due to the cis conformation.

NMR structural elucidation of myelin basic protein epitope 83-99 implicated in multiple sclerosis.

Myelin basic protein peptide 83-99 (MBP83-99) is the most immunodominant epitope playing a significant role in the multiple sclerosis (MS), an autoimmune disease of the central nervous system. Many peptide analogues, linear or cyclic have been designed and synthesized based on this segment in order to inhibit the experimental autoimmune encephalomyelitis, the best well-known animal model of MS. In this study, the solution structural motif of MBP(83-99) has been performed using 2D (1)H-NMR spectroscopy in dimethyl sulfoxide. A rather extended conformation, along with the formation of a well defined alpha-helix spanning residues Val(87)-Phe(90) is proposed, as no long-range NOE are presented. Moreover, the residues of MBP peptide that are important for T-cell receptor recognition are solvent exposed. The spatial arrangement of the side chain all over the sequence of our NMR based model exhibits great similarity with the solid state model, while both TCR contacts occupy the same region in space.

Putative bioactive conformations of amide linked cyclic myelin basic protein peptide analogues associated with experimental autoimmune encephalomyelitis.

The solution models of cyclo(87-99) MBP87-99, cyclo(87-99) [Ala91,96] MBP87-99, and cyclo(87-99) [Arg91, Ala96] MBP87-99 have been determined through 2D NMR spectroscopy in DMSO-d6. Chemical shift analysis has been performed in an attempt to elucidate structural changes occurring upon substitution of native residues. NMR-derived geometrical constraints have been used in order to calculate high-resolution conformers of the above peptides. Conformational analysis of the three synthetic analogues show that the bioactivity, or the lack of it, may possibly be due to the distinct local structure observed and the subsequent differences in the overall topology and exposed area after binding with Major Histocompatibility Complex II (MHC II). It is believed that an overall larger solvent accessible area blocks the approach and binding of the T-cell receptor (TCR) on the altered peptide ligand (APL)-MHC complex, whereas more compact structures do not occlude weak interactions with an approaching TCR and can cause Experimental Autoimmune Encephalomyelitis (EAE) antagonism. A pharmacophore model based on the structural data has been generated.

Mapping protein-protein interaction by 13C'-detected heteronuclear NMR spectroscopy.

The copper-mediated protein-protein interaction between yeast Atx1 and Ccc2 has been examined by protonless heteronuclear NMR and compared with the already available (1)H-(15)N HSQC information. The observed chemical shift variations are analyzed with respect to the actual solution structure, available through intermolecular NOEs. The advantage of using the CON-IPAP spectrum with respect to the (1)H-(15)N HSQC resides in the increased number of signals observed, including those of prolines. CBCACO-IPAP experiments allow us to focus on the interaction region and on side-chain carbonyls, while a newly designed CEN-IPAP experiment on side-chains of lysines. An attempt is made to rationalize the chemical shift variations on the basis of the structural data involving the interface between the proteins and the nearby regions. It is here proposed that protonless (13)C direct-detection NMR is a useful complement to (1)H based NMR spectroscopy for monitoring protein-protein and protein-ligand interactions.