N-terminal Fragment

Hierachical Approach on N-terminal Fragment of PTH(1-11)

Abstract

Recent advances in understanding the chemistry of life have shown that peptide-macromolecular interaction constitutes the main physico-chemical mechanisms by which living processes are controlled and modulated. The prevalence and diversity of peptides, along with their participation in crucial physiological functions, has renewed the interest in using peptides and peptide derivatives as therapeutic. In that way, the development of peptide or peptide-mimetic ligands which can target the receptors or the acceptors modulating biological activities is a top priority in biology and medicine. Therefore, establishing systematic structure-based or ligand-based approaches for the design of such ligands has been an important concern [1]. Starting from the hierachical approach proposed by V. Hruby [2], analogues of N-terminal fragment 1-11 paratyroid hormone (PTH) were synthesized with conformational constrains and analyzed by CD, NMR and MD. Recent work by Gardella et al. [3] have shown that analogues of PTH(1-11) fragment with helicity enhancing substitutions yielded potent agonists of PTH(1-34), which is fully active in vitro and in vivo and reproduces all biological responses characteristic of the native intact PTH. In the present work we would like to present our personal approach [4] to investigate the structural role of N-terminal fragment PTH(1-11) at the effect of bioactivity. Starting from the most active modified PTH(1-11), H-Aib-Val-Aib-Glu-Ile-Gln-Leu-Nle-His-Gln-Har-NH2 [3], we synthesized and analysed D-Scan analogues[4] and two series of analogues containing in crucial position aMeVal and aMeNle in order to prepare a new cyclic peptidomimetics of PTH(1-11) .

All analogues were synthesized by SPPS, employing the Fmoc protocol and combining standard coupling methods with specific coupling methods for hindered amino acids [4]. CD studies of the synthesized peptides were performed to compare their conformation with the results of the biological tests. CD provides a facile method to characterize possible overall secondary structural changes induced by modifications in the sequence. The conformational properties of the new analogues were investigated in solution of water and 20% Trifluoroethanol, as in our previous experiments on potentially bioactive PTH-derived peptides [5]. The second step of analysis was NMR measurements performed on all analogues. In fact, a negative difference values in the chemical shift differences of aCH protons (Dd< -0.1ppm) with respect to the corresponding random coil, commonly identify a helical segment. Finally, the use of NOESY and ROESY experiments and molecular dynamics calculations allowed us to connect the biological tests with structural results and address our design of new analogues [4].

References

1. Hruby V. J., Nature Reviews, 2002, 847-958

2. Hruby V. J., Balse P. M., Curr. Med. Chem., 2000, 945-970

3. Gardella T.J., Jüppner H., Trends Endocrin. Met., 2001, 5, 210-217; Shimizu N., Petroni B. D., Khatri A., Gardella T. J., Biochemistry 2003, 42, 2282-2290; Tsomaia N., Pellegrini M., Hyde K., Gardella T. J., Mierke D.F., Biochemistry 2004, 43, 690-699

4. Caporale A., Woznica I., Schievano E., Mammi S., Peggion E., Amino Acids. DOI 10.1007/s00726-009-0337-5; Caporale A., Biondi B., Schievano E., Wittelsberger A., Mammi S., Peggion E., Eur. J. Pharmacol. 2009, 611, 1-7; Fiori N., Caporale A., Schievano E., Mammi S., Geyer A., Tremmel P., Wittelsberger A., Woznica I., Chorev M., Peggion E., J. Pept. Sci., 2007,13(8), 504-12.

5. Barazza A., Wittelsberger A., Fiori N., Schievano E., Mammi S., Toniolo C., Alexander J.M., Rosenblatt M., Peggion E., Chorev M., J. Pept. Res., 2005, 65(1), 23-35

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