Jiang-Hong Gong

N-terminal Peptides of Stromal Cell-derived Factor-1 with CXC Chemokine Receptor 4 Agonist and Antagonist Activities

Peptides corresponding to the N-terminal 9 residues of stromal cell-derived factor-1 (SDF-1) have SDF-1 activity. SDF-1, 1–8, 1–9, 1–9 dimer, and 1–17 induced intracellular calcium and chemotaxis in T lymphocytes and CEM cells and bound to CXC chemokine receptor 4 (CXCR4). The peptides had similar activities to SDF-1 but were less potent. Whereas native SDF-1 had half-maximal chemoattractant activity at 5 nm, the 1–9 dimer required 500 nm and was therefore 100-fold less potent. The 1–17 and a 1–9 monomer analog were 4- and 36-fold, respectively, less potent than the 1–9 dimer. Both the chemotactic and calcium response of the 1–9 dimer was inhibited by an antibody to CXCR4. The basis for the enhanced activity of the dimer form of SDF-1, 1–9 is uncertain, but it could involve an additional fortuitous binding site on the 1–9 peptide in addition to the normal SDF-1, 1–9 site. A 1–9 analog, 1–9[P2G] dimer, was found to be a CXCR4 antagonist. Overall this study shows that the N-terminal peptides are CXCR4 agonists or antagonists, and these could be leads for high affinity ligands.

Structure/Function of Human Herpesvirus-8 Mip-II (1-71) and the Antagonist N-Terminal Segment (1-10)

Kaposis sarcoma‐associated herpesvirus encodes a chemokine called vMIP‐II that has been shown to be a broad range human chemokine receptor antagonist. Two N‐terminal peptides, vMIP‐II(1–10) and vMIP‐II(1–11)dimer (dimerised through Cys11) were synthesised. Both peptides are shown to bind the CXC chemokine receptor 4 (CXCR4). vMIP‐II(1–10) was 1400‐fold less potent than the native protein whilst the vMIP‐II(1–11)dimer was only 180‐fold less potent. In addition, both peptides are CXCR4 antagonists. Through analysis of non‐standard, long mixing time two‐dimensional nuclear Overhauser enhancement spectroscopy experiments, 13C relaxation data and amide chemical shift temperature gradients for the N‐terminus of vMIP‐II, we show that this region populates a turn‐like structure over residues 5–8, both in the presence and absence of the full protein scaffold. This major conformation is likely to be in fast exchange with other conformational states but it has not previously been detected in monomeric chemokine structures. This and other studies [Elisseeva et al. (2000) J. Biol. Chem. 275, 26799–26805] suggest that there may be a link between the structuring of the short N‐terminal chemokine peptides and their ability to bind their receptor.

Antagonists of monocyte chemoattractant protein-1 (MCP-1) identified by modification of functionally critical NH2-terminal residues

Monocyte chemoattractant protein (MCP)-1 analogues were designed to determine the role of the NH2-terminal region in structure and function. The NH2-terminal residue was important for function and receptor binding, as it could not be deleted or extended. However the NH2-terminal pyroglutamate residue of the wild type was not essential as it could be replaced by several other noncyclic amino acids without loss of activity. Residues 7-10 were essential for receptor desensitization, but were not sufficient for function, and the integrity of residues 1-6 were required for functional activity. A peptide corresponding to MCP-1, 1-10 lacked detectable receptor-binding activities, indicating that residues 1-10 are essential for MCP-1 function, but that other residues are also involved. Several truncated analogues, including 8-76, 9-76, and 10-76, desensitized MCP-1-induced Ca2+ induction, but were not significantly active. These analogues were antagonists of MCP-1 activity with the most potent being the 9-76 analogue (IC50 = 20 nM) The 9-76 specifically bound to MCP-1 receptors with a Kd of 8.3 nM, which was three-fold higher than MCP-1 (Kd 2.8 nM). The 9-76 analogue desensitized the Ca2+ response to MCP-1 and MCP- 3, but not to other CC chemokines, suggesting that it is MCP receptor specific. The availability of these compounds will be helpful in evaluating MCP receptor antagonists as anti-inflammatory therapeutics.