SELECTIVE RECOGNITION OF CYCLIC RGD PEPTIDES OF NMR DEFINED CONFORMATION BY ALPHA-II-BETA-3, ALPHA-V-BETA-3, AND ALPHA-5-BETA-1 INTEGRINS

The binding of purified fibrinogen receptor alpha IIb beta 3, vitronec tin receptor alpha V beta 3, and fibronectin receptor alpha 5 beta 1 t o their corresponding ligands in solid-phase binding assays was used t o examine the inhibitory activity of various linear and cyclic penta- and hexapeptides of different conformation containing RGD or RAD seque nces. Cyclic peptides with different defined backbone conformations we re designed by introducing a single D-amino acid or a proline at diffe rent positions in the ring. The data were calibrated for alpha IIb bet a 3 integrin incorporated into a planar lipid bilayer by a physical me thod (total internal reflection fluorescence microscopy) which yielded K-D = 1.7 mu M for a linear RGD peptide and K-D = 0.03 mu M for fibri nogen. With this integrin, three cyclic hexapeptides ([GRGDFL], [ARGDF V], [GRGDFV]) were 2-4-fold more inhibitory than the linear GRGDS pept ide in solid-phase assays and showed similar inhibition as the fibrino gen ligand. Six peptides had the same or a 2-fold lower activity as th e linear reference peptide, and three peptides were up to 7-fold less active. Replacement of Arg or Asp by their stereoisomers or Gly by Ala resulted in a 100-1000-fold reduction in activity. With the two other integrins, a single cyclic pentapeptide [RGDFV] was 10-fold more acti ve (alpha V beta 3) or equal in activity (alpha 5 beta 1) to linear GR GDS, while all of the other cyclic peptides were moderately or distinc tly less active. Changes in the RGD sequence caused a less dramatic re duction in binding strength for alpha V beta 3 and or alpha 5 beta 1 t han for alpha IIb beta 3. inhibitory activity was compared with the di stance between the C-beta atoms of Arg and Asp residues as determined by NMR and indicated that the optimum distance is in the range of 0.75 -0.85 nm for alpha IIb beta 3 and at or below 0.67 nm for alpha V beta 3 and alpha 5 beta 1. This indicates that alpha IIb beta 3 is less se nsitive to variations in the RGD backbone structure and can accommodat e a larger distance than the integrins alpha V beta 3 and alpha 5 beta 1.