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Integrin ligands containing novel constrained amino acids - Synthesis, structure analysis, and biological evaluation

Final Activity Report Summary - INTEGRIN LIGANDS (Integrin ligands containing novel constrained amino acids - Synthesis, structure analysis, and biological evaluation)

The project focussed on one important class of proteins, the integrins, which are heterodimeric glycoprotein receptors located on the cell surface and are involved in many biological processes. The binding of integrins with their natural ligands is the molecular basis of physiological and pathological processes. Small molecules that are able to interfere with the integrin-natural ligand binding process have pharmacological potential as anti-inflammatory and anticancer agents.

The project aimed at the development of new cyclic peptides containing the recognition sequence arginine-glycine-aspartic acid (RGD), which was essential for the interaction between integrins and their corresponding ligands. More specifically, a new series of small cyclic peptides, which contained novel sterically constrained amino acids, was studied and special focus was placed upon the synthesis and subsequent chemical, biological and functional characterisation of these peptides, in order to perform a systematic investigation of both structural and biological consequences of the discrete amino acid incorporation.

Thus, different cyclic RGD pentapeptides with the basic sequence c-(-Arg-Gly-Asp-Xaa-Yaa-) were synthesised incorporating different amino acids in position Xaa and Yaa. A series of constrained analogues of phenylalanine (Phe) was chosen for position Xaa, while position Yaa was occupied by derivatives of leucine (Leu) or valine (Val). Some of these novel non-proteinogenic amino acids were newly synthesised as part of the project, either by following existing synthetic methologies or by application of new strategies. The linear precursors of the new peptides were synthesised on solid phase; as such cyclisation under pseudo-high dilution, followed by side-chain deprotection, afforded the targeted cyclic peptides.

All new peptides were tested, or were in the process of being tested by the time of the project completion, as ligands of integrins alphaVbeta3 and alpha5beta1 in cell-adhesion assays with the cancer cell lines WM-115 and K-562, respectively, which expressed the corresponding integrin. In these assays, the binding of the natural ligand to the integrin presented by the cells was measured, with simultaneous analysis of the influence of the different RGD peptides on this interaction process. With the exception of one, all peptides that were so far tested were active integrin alphaVbeta3 ligands with good inhibitory activity in the micro- to nanomolar range, comparable to the values obtained for reference peptides.

Furthermore, other cyclic and linear RGD peptides were synthesised for the investigation of the interaction between integrins and the helicobacter protein CagL and mutants thereof. This ongoing project in the host group studied the minimal recognition sequence of the binding of CagL to integrins, as well as the effect that RGD-peptides could have on this interaction.

Structural studies on all the peptides were in progress in the host group as part of this project, by the time of the completion of this report. NMR measurements and molecular dynamics simulations were being used in order to establish the structure of the cyclopeptides in solution and determine the conformational preferences of the novel amino acids that were introduced in the peptides.