C-terminal cyclization of an SDF-1 small peptide analogue dramatically increases receptor affinity and activation of the CXCR4 receptor.

In an effort to improve the activities and bioavailabilities of stromal cell-derived factor-1 (SDF-1, CXCL12) sdf-(1-67)-OH (1), we have prepared a linear peptide analogue [sdf-(1-31)-NH(2) (2)] and two lactam analogues [cyclo(Lys(20)-Glu(24))-sdf-(1-31)-NH(2) (3) and cyclo(Glu(24)-Lys(28))-sdf-(1-31)-NH(2) (4)], consisting of the N-terminal region (amino acids 1-14) joined by a four-glycine linker to the C-terminal region (amino acids 56-67) of 1. Analogues 2 and 4 had eight residues of alpha-helix, as estimated from its circular dichroism (CD) spectra, in contrast to 10 residues in analogue 3. Cyclization of analogue 2 at residues 20 and 24 to give analogue 3 resulted in only a slight change to the theta;(222)/theta;(209) ratio (0.81 to 0.86, where 1.09 is considered a perfect alpha-helix), although an increase in the alpha-helix length of analogue 3 was observed. In contrast, cyclization between residues 24 and 28 by lactamization to give analogue 4 only slightly affected the helical content but clearly resulted in a more classical alpha-helical structure (theta;(222)/theta;(209) = 0.98). Cyclization of the linear analogue 2 enhanced the SDF-1 receptor CXCR4 binding approximately 114-fold, where the IC(50) values derived from (125)I-SDF-1 competitive binding assays with CEM cells were found to be 39.5 +/- 5.9 nM, 28.9 +/- 6.3 microM, 225.8 +/- 11.8 nM, and 254.1 +/- 5.4 nM for analogues 1-4, respectively. Intracellular calcium mobilization ([Ca(2+)](i)) induced after interaction with CXCR4, as measured by EC(50), was significantly reduced in analogue 4 compared to 3, and approached the EC(50) of native SDF-1, indicating a correlation between the degree of alpha-helix and biological activity. Therefore, the biological activity of small peptide SDF-1 analogues is highly dependent on the conformation of its C-terminal region.