Design and synthesis of highly active and selective α5β1 [alpha-5-beta-1] integrin ligands containing the new IsoDGR binding sequence

In this thesis the rational design and the synthesis of head-to-tail-cyclized peptide libraries containing the new binding motive isoDGR is described. Moreover, insight and basic theories about the reasons for the observed difference in selectivity and activity of the created libraries were discussed. The first approach imitated the sequence of the highly active cyclo(-RGDfV-) and the second mimicked the Gly-flanked NGR loop. However, the synthesized peptides showed only moderate activity or were completely inactive. In addition, it could be shown that the retro-inverso approach also leads to inactivity. However, with the introduction of different aromatic residues (X) next to isoAsp in the cyclic pentapeptides cyclo(-X-isoDGR-G-), a potent candidate was found: compound 27 cyclo(-phg-isoDGR-G-) is both a highly active and a highly selective α5β1 ligand (see Table IV-1). In contrast, if the aromatic residue is located next to Arg, affinity for αvβ3 is gained, especially with compound 76 cyclo(-G-isoDGR-phg-). Head-to-tail-cyclized isoDGR peptides have no free α-amino groups, therefore a direct comparison of the stereochemistry and the influence of the flanking residues is possible. Here, the constrained small cyclic peptides influence the selectivity for αvβ3 or α5β1 integrins, depending on the flanking aromatic substituents. Furthermore, the orientation of the crucial residues has been investigated and their stereochemical control related to the activity could be shown. Moreover, the binding capacity and specificity of compounds 27 and 76 in living cells correlates nicely with the in vitro results. In summary, the discovery of highly active and selective compounds is of interest for the rational design of isoDGR drug conjugates as well as for fusion proteins. Furthermore, these results are essential for the design of proteins using the NGR- isoDGR rearrangement as a controlled switch of binding affinities for different integrin subtypes in in vivo.